Tetrakis(pentane-2,4-dionato-O,O')zirconium

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Feb 2019 - 19 Feb 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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:

09 October 2017

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Tetrakis(pentane-2,4-dionato-O,O')zirconium

EC Number:

241-510-5

EC Name:

Tetrakis(pentane-2,4-dionato-O,O')zirconium

Cas Number:

17501-44-9

Molecular formula:

C20H28O8Zr

IUPAC Name:

2,4,8,8,10,10,13,15-octamethyl-1λ³,5λ³,7λ³,7λ³,11λ³,11λ³,12λ³,16λ³-octaoxa-6-zirconadispiro[5.5⁶.5⁶.5⁶]henicosa-1,4,7,7,10,10,12,15-octaene-6,6,6,6-tetrakis(ylium)-3,9,9,14-tetraide

Test material form:

solid: particulate/powder

Details on test material:

- Identification: Zirconium (IV) acetylacetonate (Zr-acac)
- Description: White to off-white powder
- Storage conditions: At room temperature

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: young cattle, obtained from the slaughterhouse
- Storage, temperature and transport conditions of ocular tissue: eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Subsequently, eyes were collected and transported in physiological saline in a suitable container under cooled conditions. The isolated corneas were stored in a petri dish with cMEM containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum. The corneas were incubated for the minimum of 1 hour at 32 ± 1 °C.
- The eyes were checked for unacceptable defects and 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 applied: 312.2 to 333.8 mg (complete coverage of the cornea)

NEGATIVE CONTROL:
- Amount applied: 750 µL

POSITIVE CONTROL:
- Amount applied: 750 µL

Duration of treatment / exposure:

240 +/- 10 minutes

Duration of post- treatment incubation (in vitro):

90 +/- 5 minutes in sodium fluorescein

Number of animals or in vitro replicates:

3

Details on study design:

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 item was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (312.2 to 333.8 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
- Number of washing steps after exposure period: 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.

POST-EXPOSURE INCUBATION: 90 ±5 minutes 32 ± 1 °C in sodium-fluorescein for permeability determinations.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacity meter (OP-KIT)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microtiter plate reader (TECAN Infinite® M200 Pro Plate Reader, OD490). OD490 values of less than 1.500 were used in the permeability calculation.
- Other: possible pH effects of the test substance on the corneas were recorded.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
A test substance that induces an IVIS ≤ 3 is not classified for eye irritancy (UN GHS: no category);
A test substance that induces IVIS >55 is defined as a corrosive or severe irritant (UN GHS: category 1);
For a test substance that induces an IVIS >3 and ≤55, no prediction on irritant potency can be made.

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:

mean of 3 replicates

Value:

17

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

RESULTS OF TEST ITEM TREATMENT (details can be found in table 1, 2 and 3 under 'Any other information on results')
- Opacity scores (negative control corrected): 13, 2.7, 6.0
- Permeability scores (negative control corrected): 1.411, 0.468, 0.136
- In Vitro Irritation Scores: 34, 9.7, 8.1

OTHER:
- The corneas were translucent after the 240 minutes of treatment with the test substance.
- No pH effect of the test item was observed on the rinsing medium.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range. Individual in vitro irritancy scores for the negative controls ranged from 0.4 to 1.2. The corneas treated with the negative control item were clear after the 240 minutes of treatment.
- Acceptance criteria met for positive control: yes, the mean IVIS of the positive control was 164 and within two standard deviations of the current historical positive control mean. The corneas treated with the positive control were turbid after the 240 minutes of treatment.

Any other information on results incl. tables

Table 1 Opacity scores

Treatment	Opacity before treatment	Opacity after treatment		Final Opacity^1			Negative control corrected Final Opacity^2	Mean Final Opacity
Negative control	4.1		5.4		1.2				0.7
	3.4		3.5		0.1
	4.3		5.3		0.9
Positive control	4.6		160.2		155.6	155			137
	3.2		113.1		109.9	109
	3.1		149.6		146.5	146
Zirconium (IV) acetylacetonate (ZR acac)	4.0		17.8		13.9	13			7.3
	4.1		7.5		3.5	2.7
	2.7		9.4		6.8	6.0

Calculations are made without rounding off.

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

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

Table 2 Permeability scores (corrected)

Treatment	Dilution factor	Negative control corrected OD490 1^1	Negative control corrected OD490 2^1		Negative control corrected OD490 3^1			Negative control corrected OD490 Average	Negative control corrected final OD490		Average OD
Positive control	1	1.218		1.229		1.230	1.225			1.225		1.845
	6	0.431		0.429		0.430	0.430			2.577
	6	0.289		0.289		0.290	0.289			1.733
Zirconium (IV) acetylacetonate (ZR acac)	1	1.409		1.413		1.412	1.411			1.411		0.672
	1	0.467		0.469		0.469	0.468			0.468
	1	0.136		0.136		0.137	0.136			0.136

Calculations are made without rounding off.

¹  OD490 values corrected for the mean final negative control permeability (0.011).

Table 3 In Vitro Irritancy Score

Treatment	Final Opacity^2	Final OD490^2	In vitro Irritancy Score^1
Negative control	1.2	0.001	1.2
	0.1	0.023	0.4
	0.9	0.010	1.1
Positive control	155	1.225	173
	109	2.577	148
	146	1.733	172
Zirconium (IV) acetylacetonate (ZR acac)	13	1.411	34
	2.7	0.468	9.7
	6.0	0.136	8.1

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Based on an IVIS >3 and ≤55, no prediction for classification of Zirconium (IV) acetylacetonate (ZR acac) can be made.

Executive summary:

The eye hazard potential of Zirconium (IV) acetylacetonate (ZR acac) was assessed in a Bovine Corneal Opacity and Permeability test (BCOP test), performed according to OECD guideline 437 and GLP principles. Three corneas from young cattle were exposed to 312.2 to 333.8 mg (complete coverage) of the test item, next to a negative control group of physiological saline (n=3) and a positive control of 20% (w/v) imidazole (n=3). Duration of treatment was approximately 240 minutes. Results of the negative and positive controls were within the historical data. Therefore, it was shown that the negative control did not induce irritancy on the corneas, the test conditions were adequate and the test systems functioned properly. Since the IVIS of the test item were > 3 ≤ 55 (13, 2.7 and 6.0), no prediction for classification of Zirconium (IV) acetylacetonate (ZR acac) for eye irritation can be made.