5,5'-diisopropyl-2,2'-dimethylbiphenyl-4,4'-diyl dihypoiodite

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 March 2018 to 27 March 2018

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)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

5,5'-diisopropyl-2,2'-dimethylbiphenyl-4,4'-diyl dihypoiodite

EC Number:

209-007-5

EC Name:

5,5'-diisopropyl-2,2'-dimethylbiphenyl-4,4'-diyl dihypoiodite

Cas Number:

552-22-7

Molecular formula:

C20H24I2O2

IUPAC Name:

4-[4-(iodooxy)-2-methyl-5-(propan-2-yl)phenyl]-5-methyl-2-(propan-2-yl)phenyl hypoiodite

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: yellowish powder
- Storage conditions of test material: at room temperature

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse, where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Storage, temperature and transport conditions of ocular tissue: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularisation by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 296.98 to 318.47 mg

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

90 minutes with sodium fluorecein

Number of animals or in vitro replicates:

3 replicates for each condition (test material, negative control and positive control)

Details on study design:

PREPARATION OF CORNEAS
The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium containing 1 % (v/v) L-glutamine and 1 % (v/v) Foetal Bovine Serum). The isolated corneas were mounted in a corneal holder (one cornea per holder) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1 °C. The corneas were incubated for the minimum of 1 hour at 32 ± 1 °C.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer. The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

TREATMENT METHOD
The medium from the anterior compartment was removed and 750 µL of the negative control and 20 % (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. The test material was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (296.98 to 318.47 mg). The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1 °C.

REMOVAL OF TEST SUBSTANCE
After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red. Possible pH effects of the test material on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.

OPACITY MEASUREMENT
The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test material or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test material or positive control treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

SODIUM FLUORESCEIN
Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated.
The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 5 mg Na-fluorescein/mL cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1 °C.

PERMEABILITY DETERMINATIONS
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 µL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader.
Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.
The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test material was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

DATA INTERPRETATION
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)
Additionally the opacity and permeability values were evaluated independently to determine whether the test material induced irritation through only one of the two endpoints.
The IVIS cut-off values for identifying the test material as inducing serious eye damage (UN GHS Category 1) and test materials not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are as follows:
- IVIS ≤ 3 = Not classified for irritation
- IVIS > 3; ≤ 55 = No prediction can be made
- IVIS > 55 = Category 1, H318: Causes serious eye damage

ACCEPTABILITY OF THE ASSAY
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

-1.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

A summary of the results can be seen in Table 1.

TEST MATERIAL
The corneas treated with the test material showed opacity values ranging from -2.6 to 0.1 and permeability values ranging from -0.015 to 0.027. The corneas were translucent after the 240 minutes of treatment with the test material. No pH effect of the test material was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -2.2 to -0.1 after 240 minutes of treatment with the test material.

CONTROLS
The individual in vitro irritancy scores for the negative controls ranged from 3.2 to 5.9. The individual positive control in vitro irritancy scores ranged from 111 to 146. The corneas treated with the positive control were turbid after the 240 minutes of treatment.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20 % (w/v) Imidazole) was 133 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Any other information on results incl. tables

Table 1: Summary of opacity, permeability and in vitro irritancy scores

Treatment	Mean Opacity	Mean Permeability	Mean In Vitro Irritancy Score
Negative control	4.6	0.021	4.9
Positive control	96	2.515	133
Test material	-1.5	0.002	-1.5

Applicant's summary and conclusion

Interpretation of results:

other: Not irritating to eyes according to EU criteria

Conclusions:

Under the conditions of the study, since the test material induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Executive summary:

The eye irritancy of the test material was investigated in a study which was conducted in accordance with the standardised guideline OECD 437, under GLP conditions.

During the study the test material was applied as supplied for 240 minutes. Negative and positive controls were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

The mean in vitro irritancy score of the positive control (20 % (w/v) Imidazole) was 133 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.  

The negative control gave opacity of 4.6 and permeability 0.021. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.

The corneas treated with the test material showed opacity values ranging from -2.6 to 0.1 and permeability values ranging from -0.015 to 0.027. The corneas were translucent after the 240 minutes of treatment with the test material. No pH effect of the test material was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -2.2 to -0.1 after 240 minutes of treatment with the test material.

In conclusion, since the test material induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.