Reaction mass of Trisodium tris(D-gluconato(2-)-κO1,κO2)ferrate(3-) and Sodium chloride

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 May - 14 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Trisodium tris(D-gluconato(2-)-κO1,κO2)ferrate(3-) and Sodium chloride

EC Number:

946-138-3

Molecular formula:

C18H30FeNa3O21 + NaCl

IUPAC Name:

Reaction mass of Trisodium tris(D-gluconato(2-)-κO1,κO2)ferrate(3-) and Sodium chloride

Test material form:

solid: particulate/powder

Details on test material:

Appearance Yellow powder Visual
Assay 76.4% NMR
NaCl 19.3% CZE/titration
water 2.7% Karl Fisher titration
pH 9.5 (1% aqueous solution)
Expiry date: November 2017

Specific details on test material used for the study:

Yellow powder
Purity: 76.4%
Batch no: FC-C 11988
Expiry date: 1 November 2017

Test animals / tissue source

Species:

other: chicken eye (slaughter house)

Strain:

other: ROSS, spring chickens

Test system

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

30 mg

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

240 min

Number of animals or in vitro replicates:

3

Details on study design:

Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus using the following procedure: First the eye-lids were carefully removed without damaging the cornea and a small drop of Fluorescein sodium 2.0% w/v (Minims, Chauvin, England) was applied to the corneal surface for a few seconds and subsequently rinsed off with isotonic saline at ambient temperature. Next, the head with the fluorescein-treated cornea was examined with a slit-lamp microscope (Slit-lamp 900 BP, Haag-Streit AG, Liebefeld-Bern, Switzerland) to ensure that the cornea was not damaged. If undamaged (e.g., fluorescein retention and corneal opacity scores of ≤ 0.5), the eye was further dissected from the head without damaging the eye or cornea. Care was taken to remove the eye-ball from the orbit without cutting off the optical nerve too short.
The enucleated eye was placed in a stainless steel clamp with the cornea positioned vertically and 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 isotonic saline from a bent, stainless steel tube, at a target rate of 0.10-0.15 mL/min (peristaltic pump set at speed 5.00, Watson-Marlow 205CA, Rotterdam, the Netherlands). The chambers of the superfusion apparatus as well as the saline were temperature controlled at approximately 32oC (water pump set at 36.4oC; Lauda 103, Germany).
After placing in the superfusion apparatus, the eyes were examined again with the slit-lamp microscope to ensure that they were not damaged. Corneal thickness was measured using the Depth Measuring Attachment No. I for the Haag-Streit slit-lamp microscope, set at 0.095 mm.
Corneal thickness was expressed in instrument units. An accurate measurement was taken at the corneal apex of each eye.
Eyes with a corneal thickness deviating more than 10% of the average corneal thickness of the eyes, eyes showing opacity (score higher than 0.5), or were unacceptably stained with fluores-cein (score higher than 0.5) indicating the cornea to be permeable, or eyes that showed any other signs of damage, were rejected as test eyes and replaced.
Each eye provided its own baseline values for corneal swelling, corneal opacity and fluorescein retention. For that purpose, after an equilibration period of 45-60 minutes, the corneal thickness of the eyes was measured again to determine the zero reference value for corneal swelling calculations.
At time t = 0 (i.e. immediately after the zero reference measurement), the following procedure was applied for each test eye: The clamp holding the test eye was placed on paper tissues outside the chamber with the cornea facing upwards.
Next, the eyes (corneas) were treated with the study substance according to the following scheme:
negative control - saline - 30 uL - 10 sec - 20 ml saline for rinsing (one eye)
positive control - NaOH - 30 mg - 10 sec - 20 ml saline for rinsing (3 eyes)
test substance - 30 mg - - 10 sec - 20 ml saline for rinsing (3 eyes)
After rinsing, each eye in the holder was returned to its chamber.
The eyes were examined at approximately 0, 30, 75, 120, 180 and 240 minutes after treatment, using the criteria and scoring system given in the attachment. Fluorescein retention was only scored at approximately 30 minutes after treatment. All examinations were carried out with the slit-lamp microscope.
After the final examination, the test substance treated eyes, the negative and positive control eyes were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde. The corneas were embedded in paraffin wax, sectioned at ca 4 μm and stained with PAS (Periodic Acid-Schiff). The microscopic slides were subjected to histopathological examination.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 2

Irritation parameter:

fluorescein retention score

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 3

Irritation parameter:

other: Swelling

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Microscopic examination of the corneas treated with the positive control NaOH revealed severe erosion (3/3 corneas), severe necrosis (1/3 corneas) of the epithelium, disorder of stromal fibres (3/3 corneas) and endothelial necrosis (3/3 corneas). Microscopic examination of the corneas treated with Iron trigluconic acid, trisodium salt revealed very slight erosion of the epithelium in all three corneas.

Applicant's summary and conclusion

Interpretation of results:

other: irritating in vitro

Conclusions:

Iron trigluconic acid, trisodium salt caused corneal effects consisting of no or very slight corneal swelling (mean of 1%), slight opacity (mean score of 1.0) and moderate fluorescein retention (mean score of 2.0). Therefore, an in vivo study was also performed.

Executive summary:

Iron trigluconic acid, trisodium salt was evaluated neat for eye irritation potential in the Isolated Chicken Eye (ICE) test. In addition, the test included a negative control (saline) and a positive control (NaOH). Chicken eyes were obtained from slaughter animals used for human consumption. The isolated chicken eyes were exposed to a single application of 30 mg of the test substance for 10 seconds followed by a 20 mL saline rinse. Three main parameters were measured to disclose possible adverse eye effects: corneal thickness (expressed as corneal swelling), corneal opacity and fluorescein retention of damaged epithelial cells. In addition, histopathology of the corneas was performed.

Iron trigluconic acid, trisodium salt caused corneal effects which could lead to a Category 2/2B classification, consisting of no or very slight corneal swelling (mean of 1%), slight opacity (mean score of 1.0) and moderate fluorescein retention (mean score of 2.0). Microscopic examination of the corneas revealed very slight erosion of the epithelium in all three corneas.

The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. Microscopic examination of the cornea did not reveal any abnormalities. The positive control NaOH caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas revealed severe erosion (3/3 corneas), severe necrosis (1/3 corneas) of the epithelium, disorder of stromal fibres (3/3 corneas) and endothelial necrosis (3/3 corneas).

Applying the classification criteria of the ICE, the following irritation classifications can be assigned:

- Category 2B:“Mild irritant/causes eye irritation” (UN-GHS classification);

- Category 2:“Irritating to eyes” (EU-CLP classification).