Cerium(3+) neodecanoate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-02-13

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 Ocular Corrosives and Severe Irritants)

Version / remarks:

2017-10-09

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2018-04-26

Test material

Test material information

Constituent 1

Reference substance name:

Cerium(3+) neodecanoate

EC Number:

268-440-8

EC Name:

Cerium(3+) neodecanoate

Cas Number:

68084-49-1

Molecular formula:

C10H19O2.xCe

IUPAC Name:

cerium(3+) tris(2-ethyl-2,5-dimethylhexanoate)

Test material form:

solid

Details on test material:

Appearance: Brown, sticky, solid mass

Specific details on test material used for the study:

Storage Conditions: room temperature, in closed packaging

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: abattoir A. Moksel AG, Buchloe, Germany
- Characteristics of donor animals: between 18 and 32 months
- 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.
- indication of any existing defects or lesions in ocular tissue samples: The eyes were carefully examined for defects and any defective eyes were not used. Eyes with scratches or any kind of opacity were not used.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 750 mg of the test item
Before administration, the test item was heated slowly at 80 ± 2 °C in an incubator until a liquid aggregate state was reached and therefore homogenization was ensured. The amount of approximately 750 mg test item per cornea was transferred into a single vial. Afterwards the test item was cooled down again at room temperature.
Since it was not possible to get a solution with the test item, it was administered directly with a spatula using the open-chamber method and moistened with a drop of physiological saline (0.9% NaCl).

Duration of treatment / exposure:

4 hours ± 5 minutes

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

not required

Number of animals or in vitro replicates:

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 the corneas were mounted in corneal holders (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 mg of the test substance was administered directly with a spatula using the open-chamber method and moistened with a drop of physiological saline (0.9% NaCl) and 750 µL the control substances were introduced into the anterior chamber (closed-chamber method).
- exposure time 4 hours ± 5 minutes at 32 ± 1 °C

REMOVAL OF TEST SUBSTANCE/CONTROL SUBSTANCES
- After incubation either the test substance or the control substance was removed and the epithelium 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 RPMI (without phenol red).

METHODS FOR MEASURED ENDPOINTS:
- anterior chamber was refilled with complete RPMI and an illuminance measurement was performed again.
- 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 medium was removed from both chambers of the holder.
- posterior chamber was refilled with fresh complete RPMI.
- 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C in horizontal position.
- 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 a corrected opacity. The mean corrected opacity value for each treatment 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:

1.21

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

VISUAL OBSERVATION
- all 3 corneas treated with Cerium neodecanoate showed no opacity of the tissue

OPACITY and PERMEABILITY
- Relative to the negative control, the test item caused no increase of corneal opacity and permeability in all 3 corneas

Acceptance of results:
- Acceptance criteria met for negative control: the negative control responses resulted in opacity and permeability values that are less than the respective established upper limits for background opacity and permeability.
- Acceptance criteria met for positive control: the IVIS of the positive control falls within two standard deviations of the current historical mean

Please also refer for results to the field "Any other information on results incl. tables" below

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	2.21	3.16	0.95
2		2.06	2.35	0.29
3		2.31	2.68	0.36
MV		2.19	2.73	0.53
4	Positive Control	2.97	88.54	85.56	85.03
5		3.16	99.17	96.01	95.48
6		2.79	90.25	87.46	86.93
MV		2.97	92.65	89.68	89.15
7	Test Item	2.49	4.04	1.54	1.01
8		1.36	3.16	1.79	1.26
9		1.29	3.50	2.20	1.67
MV		1.72	3.56	1.85	1.31
MV = mean value

Table 2: Permeability

Cornea No.	Test Item	OD490	Corrected OD490 Value
1	Negative Control	0.009
2		0.014
3		0.023
MV		0.015
4	Positive Control	1.378	1.363
5		2.000	1.985
6		2.185	2.170
MV		1.854	1.839
7	Test Item	0.009	-0.006
8		0.010	-0.005
9		0.007	-0.008
MV		0.009	-0.007
MV = mean value

Table 3:  In vitro irritation score

Cornea No.	Test Item	Corrected Opacity Value	Corrected OD490 Value	IVIS
1	Negative Control	0.95	0.009
2		0.29	0.014
3		0.36	0.023
MV		0.53	0.015	0.76
4	Positive Control	85.03	1.363
5		95.48	1.985
6		86.93	2.170
MV		89.15	1.839	116.73
7	Test Item	1.01	-0.006
8		1.26	-0.005
9		1.67	-0.008
MV		1.31	-0.007	1.21
MV = mean value

Table 4: Historical Mean In Vitro Irritation Score of the Positive Control (Imidazole 20%) from February 2015 until February 2019

	IVIS Positive Control - Imidazole 20 %
Mean Value (MV)	122.15
Standard Deviation (SD)	18.00
MV- 2xSD	86.16
MV+2xSD	158.14
Number of Replicates providing Historical Mean: 40

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 (Imidazole 20%) from August 2017 until February 2019

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	122.785	121.861	0.662	0.624	133.420
	5	117.173	116.249	1.220	1.182
	6	102.655	101.731	2.260	2.222
2	4	108.553	106.381	1.473	1.450	123.050
	5	79.491	77.319	2.465	2.442
	6	83.618	81.446	3.065	3.042
3	4	55.644	56.308	2.200	2.189	92.540
	5	71.511	72.175	1.348	1.337
	6	65.148	65.812	2.040	2.029
4	4	68.39	67.72	2.400	2.383	112.690
	5	70.88	70.21	2.810	2.793
	6	94.23	93.56	1.945	1.928
5	4	70.53	70.35	1.296	1.284	102.440
	5	78.68	78.50	1.363	1.351
	6	91.69	91.51	1.840	1.828
6	4	95.54	94.92	1.478	1.467	114.650
	5	83.58	82.96	1.500	1.489
	6	91.11	90.49	2.095	2.084
7	4	89.35	88.86	2.855	2.838	149.420
	5	117.36	116.87	2.215	2.198
	6	130.15	129.66	2.505	2.488
8	4	80.99	80.24	2.140	2.127	120.110
	5	78.40	77.65	3.070	3.057
	6	76.27	75.51	3.290	3.277
9	4	98.03	97.33	2.715	2.710	148.838
	5	77.86	77.15	3.240	3.235
	6	104.46	103.75	5.280	5.275
10	4	69.23	69.21	1.132	1.127	87.870
	5	80.76	80.74	0.912	0.907
	6	62.62	62.60	1.374	1.369
11	4	85.24	84.87	2.015	2.008	109.010
	5	76.27	75.90	2.000	1.993
	6	76.74	76.37	2.000	1.993

12	4	84.33	83.70	1.478	1.464	108.900
	5	77.76	77.13	1.417	1.403
	6	95.73	95.09	1.865	1.851
13	4	55.82	56.12	1.186	1.186	78.980
	5	62.61	62.91	1.461	1.461
	6	57.28	57.58	1.403	1.403
14	4	85.56	85.03	1.378	1.363	116.730
	5	96.01	95.48	2.000	1.985
	6	87.46	86.93	2.185	2.170
	Mean Value (MV)	84.320	83.815	2.026	2.013	114.189
	Standard Deviation (SD)	18.814	18.573	0.916	0.919	20.621
	MV- 2xSD	46.692	46.669	0.194	0.174	72.947
	MV+2xSD	121.948	120.961	3.858	3.852	155.431

Table 6:  Historical Mean In Vitro Irritation Score of the Negative Control (NaCl 0.9 %) from August 2017 until February 2019

	IVIS Negative Control - NaCl 0.9 %
Mean Value (MV)	1.03
Standard Deviation (SD)	0.75
MV- 2xSD	-0.46
MV+2xSD	2.53
Number of Replicates providing Historical Mean: 40

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 February 2019

		Incubation: 240 min
Number of Replicates Providing Historical Mean	Cornae No.	Opacity	Permeability	IVIS
		Change of Opacity Value	OD490 Value
1	1	0.234	0.008	1.49
	2	1.738	0.008
	3	0.800	0.098
2	1	0.978	0.019	2.52
	2	3.920	0.022
	3	1.617	0.028
3	1	-0.149	0.009	-0.50
	2	-0.415	0.015
	3	-1.427	0.009
4	1	0.776	0.008	0.92
	2	0.808	0.022
	3	0.418	0.020
5	1	0.035	0.010	0.35
	2	0.036	0.013
	3	0.466	0.012
6	1	1.030	0.017	0.79
	2	0.190	0.008
	3	0.640	0.009
7	1	0.953	0.009	0.76
	2	0.287	0.014
	3	0.363	0.023
Mean Value (MV)		0.633	0.018	0.904
Standard Deviation (SD)		1.018	0.019	0.937
MV- 2xSD		-1.404	-0.020	-0.970
MV+2xSD		2.670	0.057	2.778

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, Cerium neodecanoate 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.