Cerium(3+) neodecanoate

Endpoint summary

Description of key information

Skin irritation/corrosion: not irritating to skin (OECD 439; GLP)

Eye irritation: not irritating to eye (OECD 437; GLP)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-02-08 to 2019-03-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

2015-07-28

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation Protocol for: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SIT)

Version / remarks:

2017-07-11

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2018-04-26

Specific details on test material used for the study:

Storage Conditions: room temperature, in closed packaging

Test system:

human skin model

Source species:

human

Cell type:

other: normal, human epidermal keratinocytes

Cell source:

other: humans

Source strain:

other: not applicable

Details on animal used as source of test system:

not applicable

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human-derived epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely resembles the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM (EPI-200-SIT; MatTek)
- Tissue lot number: 28680 (pre-test), 28688 (main experiment)

TEST FOR DIRECT MTT REDUCTION
- to check the non-specific MTT-reducing capability of the test item 25 mg of the test item were mixed per 1 mL MTT medium and incubated for 60 min at 37 ± 1 °C in the incubator (5 % CO2, 95 % RH).
- untreated MTT medium was used as control.
- The mixture did not turn blue/purple. Thus, the additional test with freeze-killed tissues and the quantitative corrections were not necessary.

TEST FOR COLOUR INTERFERENCE
- to check the colouring potential of the test item 25 mg of the test item were mixed per 300 µL aqua dest. and per 300 µL isopropanol each in a transparent recipient and incubated at 37 ± 1°C for 60 min (5 % CO2, 95 % RH).
- The mixture of the test item and aqua dest. showed no colouring detectable by unaided eye-assessment.
- The mixture of the test item and isopropanol showed colouring detectable by unaided eye-assessment and absorbed light in the range of 570 ± 30 nm. Hence, the test item was checked for its tissue-colouring potential by performing additional testing with a NSCliving control

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C for 35 ± 1 minutes followed by incubation at room temperature until the 60 ± 1 minute treatment period was completed
- Temperature of post-treatment incubation: 37 ± 1 °C

PRE-INCUBATION:
Upon receipt of the EpiDermTM, the tissues were inspected visually and transferred into 6-well plates containing 0.9 mL assay medium per well. The surface was dried using a sterile cotton tip and the plates were incubated in a humidified incubator at 37 ± 1 °C, 5.0% CO2 and 95 % RH for 60 ± 5 min. Subsequently the tissues were transferred into new wells containing 0.9 mL pre-warmed assay medium per well and were incubated for 20 h, 44 min in a humidified incubator at 37 ± 1 °C, 5.0% CO2 and 95 % RH

INCUBATION
After this pre-incubation the tissues were treated with each dose group in triplicate, starting with the negative control. Start time was recorded with dosing of the first tissue and occurred sequentially for the other tissues, in one-minute intervals. After dosing of all tissues, all plates were transferred to the incubator for 35 ± 1 min. Afterwards all plates were removed from the incubator and placed under the sterile flow at room temperature for the remaining time until the 60 ± 1 min incubation time of the first dosed tissue was over.

REMOVAL OF TEST MATERIAL AND CONTROLS
- after the end of the treatment period the tissues were washed 15 times with DPBS.
- subsequently, the inserts were submerged three times in DPBS and shaken to remove rests of the test item.
- then inserts were rinsed once from the inside and the outside with sterile DPBS.
- inserts were placed in prepared 6-well plates containing pre-warmed fresh assay medium per well.
- plates were post-incubated at 37 ± 1 °C, 5.0% CO2, humidified to 95%, for 22 h, 48 min. Following this incubation the tissues were transferred to new wells containing fresh assay medium and incubated for additional 18 h, 25 min.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL (300 µL/well)
- Incubation time: 3 hours ± 5 minutes
- Extraction of formazan: after the MTT incubation period, the tissues were rinsed three times with DPBS and allowed to dry. The tissues were transferred into 12-well plates and immersed in 2 mL isopropanol, sealed to inhibit evaporation. Extraction was carried out protected from light at room temperature at least for 2 hours with shaking on a plate shaker.
Before using the extracts, the plate had been shaken for at least 15 minutes on a plate shaker and the inserts were pierced with an injection needle. The extract was pipetted up and down 3 times before 2 x 200 µL aliquots per each tissue were transferred into a 96-well plate. OD was measured at 570 nm with a filter band pass of maximum ± 30 nm without reference wavelength in a plate spectrophotometer using isopropanol as a blank.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: tissues pass analysis for tissue viability
- Barrier function: tissues pass analysis for tissue functionality
- Morphology: presence of a functional stratum corneum, a viable basal cell layer, and intermediate spinous and granular layers.
- Contamination: absence of bacteria, yeast, and other fungi (long term antibiotic, antimycotic free culture) as well as absence of HIV1-virus, Hepatitis B virus and Hepatitis C virus
Please also refer to the field "Attached background material" below.

PREDICTION MODEL / DECISION CRITERIA
The mean optical density (OD) of the three negative control tissues was calculated after blank correction. This value corresponded to 100 % tissue viability in the current test. For each individual tissue treated with the test item or the positive control, the individual relative tissue viability is calculated according to the following formula:
Relative viability (%) = [(mean ODtest item / positive control) / ODmean of negative control] * 100
For the test item and the positive control the mean relative viability ± relative standard deviation of the three individual tissues were calculated and used for classification according to the following prediction model:
Irritant potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with DPBS. The test item is identified as requiring classification and labelling according to UN GHS/ EU CLP “Category 2” or “Category 1”, if the tissue viability after exposure and post-incubation is less or equal to 50%. Further testing is required to resolve between UN GHS categories 1 and 2 and decide on the final classification of the test substance. The test substance may be considered as non-irritant to skin in accordance with regulation EC 1272/2008 and UN GHS “No Category” if the tissue viability after exposure and post-treatment incubation is more than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg (39 µL/cm²) of the test item
Due to the stickiness of the test material, a pre-test on one single EpiDerm tissue was performed. Here, it was determined that the test material can be washed off completely off the skin tissue surface. A plastic pin that matches the size of the skin tissue was used as application aid.
Due to homogeneity, the test item was heated to 80°C slowly in a water bath. The amount of approximately 25 mg test item per tissue was transferred into a single vial. Afterwards the test item was cooled down to room temperature before treatment.
Firstly, 25 µL of sterile DPBS were applied to the epidermal surface in order to improve the contact between the solid test item and the epidermis. Afterwards, approximately 25 mg (39 mg/cm2) of the test item were applied on a plastic pin using an application spoon avoiding compression of the test item. Then, the test material treated plastic pin was placed upside down atop the EpiDerm tissue.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL of 5 % SDS solution

Duration of treatment / exposure:

60 ± 1 minute

Duration of post-treatment incubation (if applicable):

approx. 42 hours (43 h, 32 min)

Number of replicates:

triplicates

Irritation / corrosion parameter:

% tissue viability

Value:

92.7

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: The mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0% and killed tissue controls and the quantitative corrections were not necessary.
- Colour interference with MTT: The mixture of 25 mg of the test item per 300 µl isopropanol showed colouring detectable by unaided eye-assessment. Therefore, the absorption of the chemical in isopropanol was measured in the range of 570 ± 30 nm. The mixture absorbed light in the relevant range .
For quantitative correction of results, the non-specific colour of additional viable tissues (NSCliving) was determined by using additional viable tissues without MTT-staining and calculated according to the following formula:
NSCliving [%] = [ODTVT/ODNK]*100 = 0.34%
Mean ODTVT = 0.006
Mean ODNK = 1.755
Although NSCliving was ≤ 5% (0.34%) relative to the negative control of living epidermis, the true MTT metabolic conversion (TODTT) was corrected according to the following formula:
TODTT = ODTM – ODTVT = 1.633 – 0.006 = 1.627
The NSC-corrected mean relative tissue viability (NSCCV) was calculated according to the following formula:
NSCCV [%] = viabilityTM [%] – NSCliving [%] = 93.07 % – 0.34 % = 92.73 %

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (value: 1.798).
- Acceptance criteria met for positive control: mean relative tissue viability (% negative control) of the positive control was ≤ 20% (3.5 %)
- Acceptance criteria met for variability between replicate measurements: standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.5% - 15%).
- The absorbance values were not below historically established boundaries.

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

Result of the Test Item Cerium neodecanoate

Name	Negative Control			Positive Control			Test Item
Replicate Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	1.663	1.848	1.876	0.099	0.098	0.113	1.674	1.583	1.785
	1.671	1.871	1.859	0.099	0.099	0.116	1.686	1.540	1.789
Mean Absolute OD570	1.798****			0.104			1.676
OD570(Blank Corrected)	1.621	1.805	1.833	0.056	0.056	0.071	1.631	1.540	1.743
	1.628	1.828	1.816	0.056	0.056	0.073	1.643	1.497	1.746
Mean OD570of the Duplicates (Blank Corrected)	1.624	1.816	1.825	0.056	0.056	0.072	1.637	1.518	1.744
Total Mean OD570of the 3 Replicate Tissues (Blank Corrected)	1.755*			0.061			1.633
TODTT	-			-			1.627
SD of Mean OD570of the 3 Replicate Tissues (Blank Corrected)	0.113			0.009			0.113
Relative Tissue Viability [%]	92.5	103.5	104.0	3.2	3.2	4.1	93.3	86.5	99.4
Mean Relative Tissue Viability [%]	100.0			3.5**			93.1
Mean Relative Tissue Viability [%] - NSClivingCorrected	-			-			92.7
SD of Relative Tissue Viability [%]***	6.5			0.5			6.4
CV [% Viabilities]	6.5			15.0			6.9

 

^* Blank-corrected mean OD_{570 nm}of the negative control corresponds to 100% absolute tissue viability.

^** Mean relative tissue viability of the three positive control tissues

is ≤ 20%

^*** Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%

^**** Mean absolute OD₅₇₀of the negative control tissues is ≥ 0.8 and ≤ 2.8.

Result of the NSC_livingcontrol

NSCliving	TVT
Tissue	1	2
Absolute OD570	0.047	0.047
	0.049	0.053
OD570(Blank Corrected)	0.004	0.004
	0.006	0.010
Mean OD570of the Duplicates (Blank Corrected)	0.005	0.007
Total Mean OD570of the 2 or 3 Replicate Tissues (Blank Corrected)	0.006
SD of Mean OD570of the Duplicates (Blank Corrected)	0.001
NSCliving[%]	0.3
Relative Tissue Viability [%]	-
Mean Relative Tissue Viability [%]	-
SD of Relative Tissue Viability [%]	-

Historical Data

	Mean Absolute OD570±30nmNC	MeanAbsoluteOD570±30nmPC	Mean Relative Viability [%] PC	SD Viability [%] NC, PC, TI
Mean	1.861	0.114	3.7	4.4
SD	0.247	0.033	1.5	4.1
Range of LCL – UCL	1.367 – 2.355	0.048 – 0.181	0.7 – 6.8	0.0 – 12.5
n	25	25	25	117

LCL:      Lower control limit (95%, mean – 2*SD)

UCL:     Upper control limit (95%, mean + 2*SD)

n:          number of control values

Historical data were generated in 2017.

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the reported conditions of the human skin model test, Cerium neodecanoate is not irritant to the skin (No Category) according to Regulation (EC) No. 1272/2008 and UN GHS.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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

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

Specific details on test material used for the study:

Storage Conditions: room temperature, in closed packaging

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.

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.

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

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

Cerium neodecanoate was not observed to be irritating to the skin in a reliable in vitro skin irritation study according to OECD 439.

Eye irritation:

Cerium neodecanoate was not observed to be irritating to the eye in a reliable in vitro eye irritation study according to OECD 437.

Justification for classification or non-classification

Skin irritation:

Cerium neodecanoate does not possess a skin irritation potential based on an in vitro OECD 439 study. The substance does not require classification as skin irritant according to Regulation (EC) No 1272/2008 and its subsequent adaptations.

Eye irritation:

Cerium neodecanoate does not possess a eye irritation potential based on an in vitro OECD 437 study. The substance does not require classification as eye irritant according to Regulation (EC) No 1272/2008 and its subsequent adaptations.