p-benzoquinone dioxime

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14-02-2018 to 15-02-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

p-benzoquinone dioxime

EC Number:

203-271-5

EC Name:

p-benzoquinone dioxime

Cas Number:

105-11-3

Molecular formula:

C6H6N2O2

IUPAC Name:

N-[(1Z,4Z)-4-(hydroxyimino)cyclohexa-2,5-dien-1-ylidene]hydroxylamine

Test material form:

solid

Details on test material:

- Physical state: Solid
- Storage condition of test material: In refrigerator (2-8°C) container flushed with nitrogen
- Other: Brown powder

Test animals / tissue source

Species:

other: bovine

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse.
- Age at study initiation: not reported
- Weight at study initiation: not reported
- Housing: The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (recognised supplier) 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.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 32 ± 1

Test system

Vehicle:

physiological saline

Remarks:

As the test item is a solid, at 20% w/v suspension in physiological saline was utilised for test item group only

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 microlitres (as a 20% w/v test suspension in physiological saline)
- Concentration (if solution): 20%w/v in physiological saline

Duration of treatment / exposure:

240 ±1 minutes at 32 ± 1ºC.

Duration of post- treatment incubation (in vitro):

Not applicable. According to guideline corneas treated with solids are rinsed thoroughly at the end of the 240 minutes exposure period, but do not require further incubation.

Number of animals or in vitro replicates:

Three (3) per test item, or negative or positive controls, respectively.

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. Following mounting: the anterior and posterior chambers of each BCOP holder were filled with complete Earle’s Minimum Essential Medium (cMEM) and the holders were incubated at 32 ± 1 ºC for a minimum of 1 hour. After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM.

QUALITY CHECK OF THE ISOLATED CORNEAS: The corneas were examined for defects macroscopically. Only corneas with opacity ≥ 7.0 are discarded, in accordance with the guideline.

NUMBER OF REPLICATES: 3 (Triplicate)

NEGATIVE CONTROL USED: physiological saline

SOLVENT CONTROL USED (if applicable): Not applicable.

POSITIVE CONTROL USED: 20% (w/v) Imidazole

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL and 240 ± 10 minutes

TREATMENT METHOD: Closed chamber

POST-INCUBATION PERIOD: No.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At the end of the exposure period the test item and control items were removed from the
anterior chamber and the cornea was rinsed three times with fresh MEM containing phenol red before a final rinse with complete MEM without phenol red. The anterior chamber was refilled with fresh complete MEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed.

- POST-EXPOSURE INCUBATION: Following the final opacity measurement the posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of 5 mg Na-fluorescein/ml cMEM solution Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1°C.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Measured through light transmission through the cornea quantitatively using an opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
- Others (e.g, pertinent visual observations, histopathology): Any other pertinent visual observations would be recorded.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: 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). A test item that induces an In Vitro Irritancy Score >/=55.1 is defined as an ocular corrosive or severe irritant. A test item with an IVIS 3.0 and

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean (n = 3)

Value:

16

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: due to an IVIS > 3 and ≤ 55, no prediction on the classification for eye irritation can be made in the Bovine Corneal Opacity and Permeability test.

Remarks:

Under the GHS prediction model, further testing is required to conclude classification and labelling

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No. The corneas were translucent brown after the 240 minutes of treatment with the test item. No pH effect of the test item was observed on the rinsing medium. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were turbid post treatment.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

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:
1. 20% (w/v) Imidazole was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the current historical control data (HCD) mean. ACTUAL: PC IVIS range of 86.5 to 211.4. Mean = 137.74.
2. Physiological saline solution was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values for bovine corneas treated with the respective negative control of the current historical control data (HCD). When testing solids the negative control limit for IVIS should be 5.3, opacity ≤ 5.2 and for permeability ≤ 0.205. ACTUAL: NC IVIS = 1.1, opacity ≤ 0.5 and permeability ≤ 0.040.

Any other information on results incl. tables

Table 1.0 - Summary of opacity, permeability and in vitro scores

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation Score #1, #2
Negative control	0.5	0.040	1.1
Positive control	121	1.517	144
Test item	15	0.035	16

#1 Calculated using the negative control mean opacity and mean permeability values

#2 Mean in vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

 

Table 2.0 - Opacity score

Treatment	Opacity before treatment	Opacity after treatment	Final Opacity #1	Negative control corrected Final Opacity #2	Mean Final Opacity
Negative control	5.5	6.1	0.7		0.5
	5.1	5.5	0.3
	4.1	4.4	0.4
Positive control	3.9	128.2	124.4	124	121
	4.9	136.7	131.8	131
	4.3	112.4	108.1	108
Test item	4.5	26.1	21.6	21	15
	4.3	22.0	17.7	17
	4.1	11.4	7.3	6.8

#1 Final Opacity = Opacity after treatment – Opacity before treatment.

#2 Negative control corrected Final Opacity = Final opacity – Mean final opacity negative control

#3 Calculations are made without rounding off.

 

Table 3.0 - Permeability score individual values (uncorrected)

Treatment	Dilution factor	OD490 1	OD490 2	OD490 3	Average OD	Final OD		Mean final negative control
Negative control	1	0.011	0.012	0.015	0.013		0.013	0.040
	1	0.077	0.082	0.085	0.081		0.081
	1	0.019	0.025	0.031	0.025		0.025
Positive control	1	1.473	1.472	1.489	1.478		1.478
	6	0.343	0.341	0.347	0.344		2.062
	1	1.321	1.342	1.322	1.328		1.328
Test item	1	0.028	0.029	0.033	0.030		0.030
	1	0.082	0.093	0.080	0.085		0.085
	1	0.108	0.109	0.107	0.108		0.108

#1 Calculations are made without rounding off.

 

Table 4.0 - In vitro irritancy score

Treatment	Final Opacity #1	Final OD4902	In vitro Irritancy Score #2
Negative control	0.7	0.013	0.9
	0.3	0.081	1.6
	0.4	0.025	0.7
Positive control	124	1.438	145
	131	1.824	159
	108	1.289	127
Test item	21	-0.010	21
	17	0.045	18
	6.8	0.068	7.9

#1 Positive control and test item are corrected for the negative control.

#2 In vitro irritancy score (IVIS) = opacity value + (15 x OD490 value).

Applicant's summary and conclusion

Interpretation of results:

other: Under the GHS prediction model, further testing is required to conclude classification and labelling

Remarks:

EU criteria

Conclusions:

Under the conditions of this study, due to an IVIS > 3 and ≤ 55, no prediction on the classification for eye irritation can be made in the Bovine Corneal Opacity and Permeability test.

Executive summary:

The study was performed to OECD TG 437 and EU Method B.47 to assess the irritancy potential of the test item to the eye following exposure to bovine corneas in accordance with GLP. A total of 3 corneas per treatment group were used. A volume of 750 microlitres of the 20%w/v test item suspension was placed the cornea. The negative control group similarly received physiological saline and the positive control group received neat 20% (w/v) Imidazole solution. For each group the corneas were incubated for 240 ± 10 minutes at 32 ± 1°C. After the incubation the solutions were removed and the corneas were washed with MEM with phenol red (Earle’s Minimum Essential Medium) and thereafter with cMEM. Possible pH effects of the test substance on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns. Subsequently the corneas were incubated for 90 ± 5 minutes at 32 ± 1°C with sodium fluorescein. Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated. 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 144.0 and was within two deviations of the historical positive control data mean and within the historical positive control range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The corneas treated with the test item showed opacity values ranging from 6.8 to 21.0 and permeability values ranging from -0.010 to 0.068 and in vitro irritancy scores ranged from 7.9 to 21.0. The test item induced ocular irritation through one endpoint (opacity), resulting in a mean in vitro irritancy score of 16 after 240 minutes of treatment. Under the conditions of this study, due to an IVIS > 3 and ≤ 55, no prediction on the classification for eye irritation can be made in the Bovine Corneal Opacity and Permeability test.