Zirconium, acetate lactate oxo ammonium complexes

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-05-03

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:

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:

2017-10-09

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2015-06-05

Test material

Test material information

Constituent 1

Reference substance name:

Zirconium, acetate lactate oxo ammonium complexes

EC Number:

272-702-7

EC Name:

Zirconium, acetate lactate oxo ammonium complexes

Cas Number:

68909-34-2

IUPAC Name:

Zirconium, acetate lactate oxo ammonium complexes

Test material form:

liquid

Details on test material:

State of aggregation: clear liquid, colourless to slightly yellow

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient, tightly closed, store away from incompatible materials (e.g. strong oxidising agents)

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: abattoir A. Moksel AG, Buchloe, Germany
- Storage, temperature and transport conditions of ocular tissue: fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories
- Time interval prior to initiating testing: immediately after arrival of the eyes, cornea preparation was initiated and was used for BCOP testing on the same day.

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): 750 μL of the test substance

Duration of treatment / exposure:

10 minutes

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

Number of bovine corneae per dose:
Test item: triplicates
Negative control: triplicates
Positive control: triplicates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- eyes were examined for defects and any defective eyes were discarded. Eyes with scratches or any kind of opacity were not used.
- tissue surrounding the eyeball was pulled away and the cornea was excised.
- isolated corneas were stored in a petri dish containing HBSS. Before mounting the corneas in corneal holders (BASF, Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with pre-warmed RPMI (without phenol red) containing 1 % FBS and 2 mM L-glutamine (complete RPMI).
- corneas were incubated for one hour at 32 ± 1 °C for equilibration in an air incubator.

QUALITY CHECK OF THE ISOLATED CORNEAS
- after the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI.
- an initial measurement was performed on each of the corneas using the opacitometer.
- three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas.
- the illuminance of each cornea was read and recorded.
- only corneas that had an initial illuminance reading I > I0/1.1651 lux (an equivalent to the opacity threshold of 7 as listed in OECD 437) were used for the assay.

APPLICATION DOSE AND EXPOSURE TIME
- medium was removed from the anterior chamber and replaced with the test item or control.
- 750 μL of the test substance or the control substance was introduced into the anterior chamber (closed-chamber method).
- after 10 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed.

REMOVAL OF TEST SUBSTANCE/CONTROL SUBSTANCES
- epithelium was washed at least three times with MEM (containing phenol red).
- once the medium was free of test substance, the cornea was finally rinsed with complete MEM (without phenol red).

METHODS FOR MEASURED ENDPOINTS:
- anterior chamber was refilled with complete MEM and an illuminance measurement was performed again after 2 hours incubation at 32 ± 1 °C
- each cornea was observed visually and pertinent observations were recorded.
- corneas were visually examined for tissue peeling, residual test chemical and non-uniform opacity patterns and observation were recorded.
- after the illuminance measurement was performed, the permeability was measured.
- the medium was removed from both chambers of the holder.
- posterior chamber was refilled with fresh complete RPMI.
- 1 mL of a 4 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C.
- then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).

Evaluation of the opacity:
- the following formula was used to calculate the opacity, whereas the values a and b are equipment-specific variables empirically determined by the manufacturer:
Opacity = ((I0/I) - b)/ a
with a = 0.025 and b = 0.9894
- value I0 is the illuminance through a holder without cornea, but with windows and liquid. This value is determined by taking the mean for a set of cornea holders and is reevaluated periodically. This I0 value was than calculated to the respective data of the opacitometer and the data according to guideline (opacity < 7). So the initial illuminance could be calculated and corneas below this value were discarded.
- change in opacity for each cornea (test item, positive and negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values of test item treated cornea or positive control were corrected by subtracting from each the average change in opacity observed for the negative-control corneas to obtain the corrected opacity. The mean corrected opacity value for the negtive control and the mean corrected opacity value for the test item and the positive control was calculated by averaging the corrected opacity values of each cornea for a given treatment.

Evaluation of the permeability:
- mean OD490 for the blank cuvettes was calculated.
- mean blank OD490 was subtracted from the OD490 of each cuvette (corrected OD490).
- any dilutions that were made to bring the OD490 values into the linear range of the spectrophotometer (OD490 should be less than 1.500), were taken into account by multiplying the OD490 value of the dilution by the dilution factor.
- final-corrected OD490 of the test article and the positive control were calculated by subtracting the average-corrected OD490 of the negative-control corneas from the corrected OD490 value of each treated cornea:
Final-corrected OD490 = (OD490 – mean blank OD490) – average-corrected negative control OD490
- mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated corneas for that treatment condition.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula was used to determine the in vitro irritation score (IVIS):
IVIS = mean opacity value + (15 x mean permeability OD490 value)
To determine the IVIS of the positive control and the test item, the corrected opacity and OD490 values were used.

ACCEPTABILITY CRITERIA
- the BCOP assay is considered to be valid if the in vitro irritation score obtained with the positive control falls within the two standard deviations of the current historical mean.
- the negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Remarks:

(mean)

Value:

2.09

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Visual Observation after treatment:
None of the 3 corneas treated with Zirconium, acetate lactate oxo ammonium complexes showed any opacity of the tissue.

Measurement after treatment:
Relative to the negative control, the test item caused no increase of corneal opacity and permeability in all 3 corneas.

Acceptance of results:
The test is valid since the IVIS of the positive control falls within two standard deviations of the current historical mean and opacity and permeability of the negative control are less than the respective established upper limits for background opacity and permeability.

Any other information on results incl. tables

Table 1: Opacity

Cornea No.	Test Item	Initial Opacity	Final Opacity	Change of Opacity Value	Corrected Opacity Value
1	Negative Control	0.32	0.32	0.00
2		0.32	0.70	0.38
3		0.52	0.49	-0.03
MV		0.39	0.50	0.11
4	Positive Control	1.65	23.97	22.32	22.21
5		1.22	23.37	22.15	22.04
6		0.90	19.56	18.66	18.54
MV		1.26	22.30	21.04	20.93
7	Test Item	-0.11	1.65	1.76	1.65
8		-0.15	1.01	1.16	1.04
9		-0.57	2.65	3.22	3.11
MV		-0.28	1.77	2.05	1.93
MV = mean value

Table 2: Permeability

Cornea No.	Test Item	OD490	Corrected OD490 Value
1	Negative Control	0.002
2		0.008
3		0.001
MV		0.004
4	Positive Control	1.292	1.288
5		1.472	1.468
6		1.454	1.450
MV		1.406	1.402
7	Test Item	0.013	0.009
8		0.018	0.014
9		0.011	0.007
MV		0.014	0.010
MV = mean value

Table 3:  In vitro irritation score

Cornea No.	Test Item	Corrected Opacity Value	Corrected OD490 Value	IVIS
1	Negative Control	0.00	0.002
2		0.38	0.008
3		-0.03	0.001
MV		0.11	0.004	0.17
4	Positive Control	22.21	1.288
5		22.04	1.468
6		18.54	1.450
MV		20.93	1.402	41.96
7	Test Item	1.65	0.009
8		1.04	0.014
9		3.11	0.007
MV		1.93	0.010	2.09
MV = mean value

Table 4: Historical mean in vitro irritation score of the positive control (ethanol 100%) from February 2015 until May 2018

	IVIS Positive Control - Ethanol 100 %
Mean Value (MV)	48.35
Standard Deviation (SD)	9.56
MV- 2xSD	29.22
MV+2xSD	67.47
Number of Replicates providing Historical Mean: 52
Positive controls are updated after every single experiment or at least every 3 months

Positive controls are updated after every single experiment or at least every 3 months.

Table 5: Historical data on opacity and permeability of the positive control (ethanol 100%) from August 2017 until May 2018

Number of Replicates Providing Historical Mean	Cornea No.	Opacity		Permeability		IVIS
		Change of Opacity Value	Corrected Opacity Value	OD490 Value	Corrected OD490 Value
1	4	37.495	37.899	1.013	1.004	53.36
	5	27.335	27.739	1.147	1.138
	6	30.218	30.622	2.120	2.111
2	4	24.492	23.921	1.442	1.436	49.70
	5	31.754	31.182	1.108	1.102
	6	30.259	29.688	1.755	1.749
3	4	40.460	39.546	1.270	1.266	49.84
	5	28.925	28.010	0.949	0.945
	6	29.064	28.149	1.381	1.377
4	4	22.710	22.292	1.182	1.178	52.16
	5	35.986	35.567	1.780	1.776
	6	24.036	23.618	2.050	2.046
6	4	25.61	24.97	2.025	2.014	63.38
	5	28.89	28.25	2.920	2.909
	6	27.82	27.18	2.405	2.394
7	4	22.66	21.94	1.407	1.399	42.08
	5	22.03	21.31	1.354	1.346
	6	22.31	21.60	1.355	1.347
8	4	31.80	31.03	0.985	0.958	42.99
	5	27.64	26.87	1.166	1.139
	6	27.22	26.45	0.905	0.878
9	4	22.32	22.21	1.292	1.288	41.96
	5	22.15	22.04	1.472	1.468
	6	18.66	18.54	1.454	1.450
Mean Value (MV)		27.577	27.110	1.497	1.488	49.43
Standard Deviation (SD)		5.354	5.364	0.500	0.502	7.26
MV- 2xSD		16.869	16.383	0.497	0.485	34.91
MV+2xSD		38.285	37.837	2.498	2.492	63.95

Table 6:  Historical mean in vitro irritation score of the negative control (NaCl 0.9 %) from February 2015 until May 2018

	IVIS Negative Control - NaCl 0.9 %
Mean Value (MV)	0.80
Standard Deviation (SD)	0.64
MV- 2xSD	-0.47
MV+2xSD	2.08
Number of Replicates providing Historical Mean: 52

Negative controls are updated after every single experiment or at least every 3 months.

Table 7:  Historical data on opacity and permeability of the negative control (NaCl 0.9 %) from August 2017 until May 2018

Replicates Providing Historical Mean	Cornea No.	Opacity	Permeability	IVIS
		Change of Opacity Value	OD490 Value
1	1	-0.57	0.009	-0.27
	2	-0.11	0.013
	3	-0.53	0.004
2	1	-0.25	0.004	0.66
	2	0.80	0.005
	3	1.17	0.008
3	1	0.11	0.004	0.48
	2	0.68	0.005
	3	0.47	0.003
4	1	0.19	0.008	0.18
	2	-0.12	0.005
	3	0.00	0.018
	1	0.11	0.005
5	2	0.74	0.022	0.81
	3	1.06	0.007
6	1	0.26	0.011
	2	1.21	0.004	0.83
	3	0.68	0.008
	1	0.50	0.022
7	2	0.75	0.045	1.18
	3	1.05	0.015
	1	1.09	0.006
8	2	0.60	0.017	1.19
	3	1.47	0.004
	1	0.00	0.002
9	2	0.38	0.008	0.17
	3	-0.03	0.001
Mean Value (MV)		0.43	0.01	0.58
Standard Deviation (SD)		0.55	0.01	0.49
MV- 2xSD		-0.66	-0.01	-0.40
MV+2xSD		1.53	0.03	1.56

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, according to the current OECD 437 study and under the experimental conditions reported, Zirconium, acetate lactate oxo ammonium complexes is not eye irritating (EU CLP/ UN GHS Category 2) and not serious eye damaging (EU CLP/ UN GHS Category 1). The test item should not be classified according to Regulation (EC) No 1272/2008 and subsequent adaptations.