Iotroxic acid

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-03-13 to 2018-04-19

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 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:

adopted: 09 October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Iotroxic acid

EC Number:

256-917-3

EC Name:

Iotroxic acid

Cas Number:

51022-74-3

Molecular formula:

C22H18I6N2O9

IUPAC Name:

3-{2-[2-(2-{[(3-carboxy-2,4,6-triiodophenyl)carbamoyl]methoxy}ethoxy)ethoxy]acetamido}-2,4,6-triiodobenzoic acid

Test material form:

solid: particulate/powder

Details on test material:

- CAS No.: 51022-74-3
- Batch No.: 856W10A0003
- Aggregate State at RT: solid, powder
- Colour: white to yellowish
- Purity: 99.95 (w/w%)
- Storage Conditions: 2-8 °C, protected from light
- Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: The assay uses isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany
- Number of animals: 3 replicates
- Characteristics of donor animals (e.g. age, sex, weight): No data
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories.
- Time interval prior to initiating testing: Immediately after arrival of the eyes, cornea preparation was initiated.
- indication of any existing defects or lesions in ocular tissue samples: The eyes were carefully examined for defects and any defective eyes were discarded.
- Indication of any antibiotics used: No data

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 20%

VEHICLE
- Concentration (if solution): 0.9%
- Lot/batch no. (if required): 17406416

Duration of treatment / exposure:

4 hours ± 5 minutes at 32 °C

Duration of post- treatment incubation (in vitro):

90 minutes

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated. The eyes were carefully examined for defects and any defective eyes were discarded. The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 ± 1 °C.

NUMBER OF REPLICATES : 3

NEGATIVE CONTROL USED : physiological saline 0.9% NaCl

POSITIVE CONTROL USED : imidazole 20% in physiological saline 0.9% NaCl

APPLICATION DOSE AND EXPOSURE TIME : 750 µL for 4 h ± 5 min

TREATMENT METHOD: Closed chamber method
After the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI. An initial measurement was performed on each of the corneas using the opacitometer. Three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas. The illuminance of each cornea was read and recorded. Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay. The medium was removed from the anterior chamber and replaced with the test item or control. 750 µL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method). After 4 hours ± 5 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an illuminance measurement was performed. Also, each cornea was observed visually and pertinent observations were recorded. After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).

POST-INCUBATION PERIOD: Yes, 90 minutes

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 3

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: see Table 1 under "Any other information on materials and methods incl. tables"

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean of three replicates

Value:

1.41

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Proper conduct of the BCOP was confirmed via a positive response with imidazole and a negative response with 0.9% NaCl. For detailed results please refer to table 2 in box "Any other information on results incl. tables".

Any other information on results incl. tables

Table 2: In Vitro Irritation Score

Cornea No.	Test Item	Corrected Opacity	Corrected OD490 Value	IVIS
1	Negative Control	1.03	0.013	0.94
2		0.60	0.010
3		0.62	0.015
Mean value		0.75	0.013
1	Positive Control	80.24	2.127	120.11
2		77.65	3.057
3		75.51	3.277
Mean value		77.80	2.821
1	Test Item	1.35	-0.002	1.41
2		1.79	0.005
3		1.12	-0.006
Mean value		1.42	-0.001

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions, Iotroxic acid is classified as a non - irritant under the UN GHS "No Category."

Executive summary:

In a primary eye irritation study according to OECD 437, 750 µL of the test substance, Iotroxic acid (Purity 99.95 %) was applied to bovine corneas by closed chamber method for 4 hours. The post incubation period was 90 minutes. The in vitro irritation score for the mean of three replicates was found to be 1.41.The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is valid. The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control. Based on these results, the test item is considered a non – irritant under UN GHS “No Category.”