Iodine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2020-04-17 to 2020-04-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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. Oct. 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Iodine

EC Number:

231-442-4

EC Name:

Iodine

Cas Number:

7553-56-2

Molecular formula:

I2

IUPAC Name:

iodine

Specific details on test material used for the study:

Batch no.: 282 - 101314 / 282 - 101315
Purity: 100 %

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: obtained from the slaughterhouse Müller Fleisch GmbH, Industri-estraße 42, 75217 Birkenfeld, Germany
- Characteristics of donor animals: between 12 and 60 months old
- Storage, temperature and transport conditions of ocular tissue: The eyes were transported 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 7 minutes.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 1035.3, 1003.4, 1003.3 mg
- The test item was ground with a mortar and pestle before use.

Duration of treatment / exposure:

4 hours

Number of animals or in vitro replicates:

three replicates for each treatment group (negative control solution, test item or positive control solution)

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
After having carefully cleaned and sterilised the cornea holders, they were kept in the incubation chamber at 32 ± 1 °C.
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 prewarmed 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. The formation of bubbles was prevented.
After the initial incubation, the medium was completely changed and the baseline opacity for each cornea was recorded. The baseline opacity was measured by placing the cornea holder in an opacitometer and recording the illuminance (unit: LUX).

QUALITY CHECK OF THE ISOLATED CORNEAS
None of the corneas showed an opacity greater than seven opacity units; therefore all corneas were used.

NUMBER OF REPLICATES
Three replicates

NEGATIVE CONTROL USED
HBSS: Hank’s Balanced Salt Solution (HBSS) 10-fold concentrated, diluted in demin. water (1:10), batch no.: T20200417

POSITIVE CONTROL USED
Imidazole solution: 20 % C3H4N2 (CAS-No. 288-32-4), dissolved in HBSS, batch no.: T20200417

APPLICATION DOSE AND EXPOSURE TIME
After removal of the pre-incubation medium (cMEM without phenol red), 750 μL of negative control solution, a defined amount of test item (1003.3 - 1035.3 mg) or 750 μL of positive control solution were applied to each replicate to the epithelial side of the cornea.
Exposure time of the controls and test item on the corneas was 4 hours at 32 ± 1 °C.

TREATMENT METHOD:
Closed chamber method for negative and positive controls; Open chamber-method” for test item

POST-INCUBATION PERIOD: no

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

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Each cornea holder was placed in the opacitometer and the final illuminance value of each cornea was recorded at once.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD492)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The decision criteria as indicated in the TG was used.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

43 539.36

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: it is possible that the IVIS value of the test item is wrongly too high, because the test item color could not be removed

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: At the end of the test it was observed, that all three cornea replicates of the test item were swollen compared to the negative control.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Any other information on results incl. tables

Illuminance Values:

Parameter	Negative Control			Test Item			Positive Control
	1.Rep.	2.Rep.	3.Rep.	1.Rep.	2.Rep.	3.Rep.	1.Rep.	2.Rep.	3.Rep.
(I) Measured values before exposure	1025	1006	1009	1046	1047	1033	1004	1007	999
(I) Measured values after exposure	1024	1010	1023	1*	1*	1*	349	337	346

* Note: due to the intrinsic colored cornea induced by the test item, the LUX value of all three test item replicates was 1.

Opacity Values Test Item and Positive Control:

Parameter	Test Item			Positive Control
	1.Rep.	2.Rep.	3.Rep.	1.Rep.	2.Rep.	3.Rep.
Opacity before exposure	2.25	2.21	2.77	3.99	3.86	4.21
Opacity after exposure	43541.46	43541.46	43541.46	85.46	89.90	86.54
Opacity Difference	43539.21	43539.25	43538.69	81.47	86.04	82.33
Opacity Difference corrected	43539.45	43539.49	43538.93	81.71	86.28	82.57
MeanOpacity Difference corrected	43539.29*			83.52

* Note: the high opacity value of the test item is due to the second opacity measurement. The intrin-sic color of the test item had an influence on the opacity result. The turbidity of the cornea could not be determined correctly; only the infiltrated test item color in the cornea could be measured.

Optical density at 492 nm of Negative Control, Test Item and Positive Control:

Parameter	Negative Control			TestItem			Positive Control
	1.Rep.	2.Rep.	3.Rep.	1.Rep.	2.Rep.	3.Rep.	1.Rep.	2.Rep.	3.Rep.
1.Measurement	0.043	0.041	0.045	0.044	0.047	0.052	1.485	1.040	1.967
2.Measurement	0.043	0.042	0.044	0.043	0.048	0.054	1.474	1.033	1.930
3.Measurement	0.043	0.043	0.044	0.045	0.045	0.054	1.471	1.027	1.919
1.Measurement – blank	0.0067	0.0047	0.0087	0.0077	0.0107	0.0157	1.4487	1.0037	1.9307
2.Measurement – blank	0.0067	0.0057	0.0077	0.0067	0.0117	0.0177	1.4377	0.9967	1.8937
3.Measurement – blank	0.0067	0.0067	0.0077	0.0087	0.0087	0.0177	1.4347	0.9907	1.8827
Mean of each replicate	0.0067	0.0057	0.0080	0.0077	0.0103	0.0170	1.4403	0.9970	1.9023
Mean of the 3 replicates	0.0068			--			--
Corrected	--	--	--	0.0009	0.0036	0.0102	1.4336	0.9902	1.8956
Corrected mean of the 3 replicates	--			0.0049			1.4398

Applicant's summary and conclusion

Interpretation of results:

other: No conclusive result achieved

Conclusions:

Under the conditions of this test, the test item Iodine induced serious eye damage on the cornea of the bovine eye. The calculated mean IVIS was 43539.36.
According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS > 55 induces serious eye damage, that should be classified as UN GHS Category I.
As the intrinsic color of the test item could not be removed from the cornea after the 4 hour incubation, the result of this study must be seen with reservations. It is possible that the IVIS value is wrongly too high and the opacity could not be determined correctly, because of the intrinsic color of the test item that could not be removed from the cornea.
Therefore no conclusive result could be achieved within this study.

Executive summary:

An in vitro study was performed to assess corneal damage potential of Iodine by quantitative measurements of changes in opacity and permeability in a bovine cornea following OECD Guideline 437 and EU Method B.47.

As test system bovine corneas were used. They were collected from slaughtered cattle that were between 12 and 60 months old.

The test item Iodine was brought onto the cornea of a bovine eye which previously had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been determined. The test item was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test item, it could be observed that the whole cornea was coloured by the test item. Afterwards the opacity and permeability values were measured.

The test item was tested neat. No observations were made which might cause doubts concerning the validity of the study.

 

Under the conditions of this test, the test item Iodine induced serious eye damage on the cornea of the bovine eye. The calculated mean IVIS was 43539.36.

According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS > 55 induces serious eye damage, that should be classified as UN GHS Category I.

As the intrinsic color of the test item could not be removed from the cornea after the 4 hour incubation, the result of this study must be seen with reservations. It is possible that the IVIS value is wrongly too high and the opacity could not be determined correctly, because of the intrinsic color of the test item that could not be removed from the cornea.

Therefore no conclusive result could be achieved within this study.