Cerium(3+) neodecanoate

Skin irritation / corrosion

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015-07-28

Deviations:

no

Test guideline 2

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

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

In vitro test system

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

Results and discussion

In vitro

Results

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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.