Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 October 2017 - 13 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study has been performed according to OECD and/or EC guidelines and acc ording to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
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 July 26, 2013
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Identification: 1,4-Bis (4-hydroxy benzoyl) benzene
- Appearance: Off white crystalline powder
- Test item storage: At room temperature
- Stable under storage conditions until: 15 May 2018 (expiry date)
Specific details on test material used for the study:
- Appearance: Off white crystalline powder
- Test item storage: At room temperature
- Stable under storage conditions until: 15 May 2018 (expiry date)

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 (Vitelco, -'s Hertogenbosch, The Netherlands)
- Number of animals: Not specified
- Characteristics of donor animals (e.g. age, sex, weight): Not specified
- Storage, temperature and transport conditions of ocular tissue: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Time interval prior to initiating testing: Not specified

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 337.5 to 362.8 mg
- Concentration (if solution): Undiluted
Duration of treatment / exposure:
240 +/- 10 minutes at 32 +/- 1°C
Number of animals or in vitro replicates:
3 corneas were selected at random for each treatment group
Details on study design:
SELECTION AND PREPARATION OF CORNEAS

The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) 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 (BASF-OP3.0, BASF, Ludwigshafen, Germany). 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.

NUMBER OF REPLICATES

3 corneas were selected at random for each treatment group

NEGATIVE CONTROL USED

physiological saline (Eurovet Animal Health, Bladel, The Netherlands)

POSITIVE CONTROL USED

Imidazole 20% (w/v) (Merck Schuchardt OHG, Germany)

APPLICATION DOSE AND EXPOSURE TIME
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. 1,4-Bis (4-hydroxy benzoyl) benzene was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (337.5 to 362.8 mg). The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test item over the entire cornea. Corneas were incubated in a horizontal position for 240 +/- 10 minutes at 32 +/- 1°C.

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no

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 (Earle’s Minimum Essential Medium Life Technologies). Possible pH effects of the test item 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.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
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 opacity value (measured with the device OP-KIT) was calculated according to:

Opacity = [(I0/I) -0.9894] / 0.0251

With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea.
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 item 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 item 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.

- Corneal permeability:
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) 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 Nafluorescein (Sigma-Aldrich Chemie GmbH, Germany)/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.
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 (TECAN Infinite® M200 Pro Plate 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 item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

- Others (e.g, pertinent visual observations, histopathology): Additionally the opacity and permeability values were evaluated independently to determine whether the test item induced irritation through only one of the two endpoints.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
= mean opacity value + (15 x mean OD490 value)

DECISION CRITERIA:
The IVIS cut-off values for identifying the test items as inducing serious eye damage (UN GHS Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:

In vitro score range: UN GHS:
=< 3 No Category
> 3 ; =< 55 No prediction can be made
> 55 Category 1

ACCEPTABILITY CRITERIA
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.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
1
Value:
0.1
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Remarks:
2.8
Positive controls valid:
yes
Remarks:
150
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: The corneas treated with the positive control were turbid after the 240 minutes of treatment whereas those treated with 1,4-Bis (4-hydroxy benzoyl) benzene appeared clear after treatment.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values if different from the ones specified in the test guideline:
IVIS for the negative control, historical control data: -5.3 - 5.3 (data range obtained by collecting all data over the period of Aug 2014 to Aug 2017).
Opacity for the negative control, historical data: -5.4 - 5.2 (data range obtained by collecting all data over the period of Aug 2014 to Aug 2017).
Permeability for the negative control, historical data: -0.010 - 0.205 (data range obtained by collecting all data over the period of Aug 2014 to Aug 2017).
IVIS for the positive control, historical control data: 86.5 - 211.4 (data range obtained by collecting all data over the period of Aug 2014 to Aug 2017).

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of this study, 1,4-Bis (4-hydroxy benzoyl) benzene induced an IVIS =< 3, therefore, the test substance is not classified for eye irritation or serious eye damage, according to Regulation (EC) No. 1272/2008 (CLP) and to the UN-GHS Regulation.
Executive summary:

The eye hazard potential of the 1,4-Bis (4-hydroxy benzoyl) benzene was evaluated using the Bovine Corneal Opacity and Permeability test (BCOP test), under GLP compliance, according to the OECD Guideline No. 437.

The corneal damage potential of test substance was assessed using fresh bovine corneae. 337.5 to 362.8 mg of test item was directly applied to cornea for 240 minutes at 32 ± 1 °C and corneal opacity was measured. Three corneas were used for each treated series (undiluted test item; negative control: physiological saline; positive control: imidazole). Following the opacity measurement, the permeability of the corneas to sodium fluorescein was evaluated. Two endpoints, corneal opacity and permeability, were measured and combined to give an In Vitro Irritancy Score.

The individual in vitro irritancy scores for the negative controls ranged from 0.8 to 4.7. The individual positive control in vitro irritancy scores ranged from 112 to 199. The corneas treated with the positive control were turbid after the 240 minutes of treatment. The mean in vitro irritancy score of the positive control was 150 and within two standard deviations of the current historical positive control mean (86.5 - 211.4). One of the negative control eyes was excluded from the analysis since the permeability (0.558) was above the historical database (-0.010 - 0.205) which resulted in an IVIS outside the historical data base. Since the other two eyes completely met the criteria and the test item results were not influenced by this result, this does not affect the study outcome (Study Plan deviation). It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

The corneas were slightly translucent after the 240 minutes of treatment with the test item. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from -0.2 to 0.2 after 240 minutes of treatment with 1,4-Bis (4-hydroxy benzoyl) benzene and, the mean IVIS was lower than or equal to 3.

The test substance, 1,4-Bis (4-hydroxy benzoyl) benzene, is not classified for eye irritation or serious eye damage, according to Regulation (EC) No. 1272/2008 (CLP) and to the UN-GHS Regulation.