Cobalt Nickel Manganese Oxide

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Remarks:

EpiDermTM Model (EPI-200-SIT)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 June 2020 - 28 July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(18 June 2019)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

impurity 1

Reference substance name:

Total unspecified impurities

IUPAC Name:

Total unspecified impurities

Constituent 1

Reference substance name:

Cobalt Nickel Manganese Oxide

Cas Number:

37348-84-8

Molecular formula:

Ni1-x-yMnxCoyOz with Me=Ni+Mn+Co=1 and Ni/Me= 0.45-0.98 Mn/Me= 0.01-0.35 Co/Me= 0.01-0.35 O/Me= 1.00-1.33

IUPAC Name:

Cobalt Nickel Manganese Oxide

Test material form:

solid

Specific details on test material used for the study:

Batch/Lot number: PVX-144A PVX14
Description: Grey Powder
Purity: 100% (7.44% Co ; 6.99% Mn ; 59.67% Ni)
Manufacturer: Umicore
Expiry date: 30 April 2021
Storage conditions: Controlled room temperature (15-25 °C, ≤70% relative humidity). Protected from light and humidity (store in a tightly closed container).
The Certificate of Analysis is attached in Appendix 1 of the study report.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm™ Model (EPI-200-SIT), Source: MatTek, Bratislava, Slovakia, Lot No.:30874, Expiry Date: 19 June 2020

Justification for test system used:

The EpiDermTMhas been validated for irritation testing in an internation l validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD N° 439); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Remarks:

the test item was applied in the original form

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
EpiDerm™ Model (EPI-200-SIT) (Source: MatTek, Bratislava, Slovakia, Lot No.:30874, Expiry Date: 19 June 2020), The preparation of dead epithelium units involved the following procedure performed on living epithelium units (Manufacturer: MatTek, Bratislava, Slovak Republic., Batch No.: 30868).
- Production date:
Fresh = 17 June 2020; for freezing epidermal: 27 May 2020
- Date of initiation of testing:
17 June 2020

EpiDerm™ Model (EPI-200-SIT) (Source: MatTek, Bratislava, Slovakia, Lot No.:30874, Expiry Date: 19 June 2020) units consist of normal, human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis (0.6 cm2). It’s 3D structure consisting of organized and proliferative basal cells, spinous and granular layers, and cornified epidermal layers are mitotically and metabolically active. Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.
The preparation of dead epithelium units involved the following procedure performed on living epithelium units (Manufacturer: MatTek, Bratislava, Slovak Republic., Batch No.: 30868). The process entailed placing the living epithelium units in a 24-well plate and the epidermis units were frozen (approximately -20 °C) overnight. The next day the killed tissues were thawed at room temperature approximately 1 hour and the units were frozen again. This method was performed two times, then the units were kept frozen until use (End of preparation: 28 May 2020, the frozen units are used up to 1 year after freezing). Further use of killed tissues was similar to living tissues.


TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure:
The plates with the test item, negative and positive control treated epidermis units were incubated for 35 minutes (± 1 minute) to the humidified incubator at 37°C with 5 % CO2, in a >95% humidified atmosphere and for the exposure time of 25 minutes (± 0.5 minute) at room temperature (24.6-27.1°C).
- Temperature of post-treatment incubation (if applicable):
day 0: After rinsing, the units were placed into the plate wells with fresh Assay Medium (0.9 mL/well) below them and then incubated for 24 hours (± 2hours) at 37°C in an incubator with 5 % CO2, in a >95 RH % humidified atmosphere.
day 1: At the end of the 24 hours incubation period the units were placed into the plate wells with fresh Assay Medium (0.9 mL/well) below them and then incubated for 18 hours (± 2hours) at 37°C in an incubator with 5 % CO2, in a >95 RH % humidified atmosphere

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:
100 ml DPBS each, 20 tim. Additional rinsing was used (10 times) because the test item stuck the epidermal surface. No change was observed after the additional rinsing.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration:
300 µL MTT medium (1 mg MTT / ml medium)
- Incubation time: 3 hours
- Spectrophotometer:
Thermo Fisher Scientific, Catalogue Number: 240 72800, Serial Number: 0920-14, Date of calibration: 13 August 2018, calibration is valid until August 2020
- Wavelength:
570 nm

NUMBER OF REPLICATE TISSUES:
3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues and killed tissues
- Procedure used to prepare the killed tissues (if applicable):
The process entailed placing the living epithelium units in a 24-well plate and the epidermis units were frozen (approximately -20 °C) overnight. The next day the killed tissues were thawed at room temperature approximately 1 hour and the units were frozen again. This method was performed two times, then the units were kept frozen until use
- N. of replicates : Two additional test item-treated living tissues were used for the non-specific optical density (OD) evaluation. Furthermore, two additional test item-treated killed epidermis and two negative control treated killed epidermis were used. To avoid a possible double correction for colour interference, for non-specific colour in killed tissues, two additional disks were used.
- Method of calculation used:
• Non-Specific MTT reduction calculation (NSMTT%): NSMTT% = [(ODKT- ODKNC) / ODNC] × 100
ODKNC: negative control killed tissues OD ODKT: test item treated killed tissues OD ODNC: negative control OD
If NSMTT % is ≤ 30%, then true MTT metabolic conversion (TODTT) is undertaken as follows:
TODTT = [ODTT – (ODKT – ODKNC)]
ODTT: test item treated viable tissues
• The % relative viability (RV%) for each test item replicate is calculated relative to the
mean negative control:
RV 1% = [TODTT1 / mean ODNC] × 100 RV 2% = [TODTT2 / mean ODNC] × 100 RV 3% = [TODTT3 / mean ODNC] × 100
• The mean value of the 3 individual relative viability % for test item is calculated: Mean Relative Viability % = (RV1% + RV2% + RV3%) / 3
If NSMTT% is > 30% relative to the negative control: additional steps must be undertaken if possible, or the test item must be considered as incompatible with the test.

PREDICTION MODEL / DECISION CRITERIA
The irritation potential of test items can be classified according to the United Nations Globally Harmonized System of Classification and Labelling of Chemicals, and a similar system is used in CLP. In the present study, the irritancy potential of test items is predicted by the mean tissue viability of tissues exposed to the test item. The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean relative viability of three individual tissues after 60 minutes exposure to the test item and 42 hours post incubation is less or equal (≤) to 50% of the mean viability of the negative controls. In case the test chemical is found to be non-corrosive, and shows tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%, the test chemical is considered to be irritant to skin in accordance with UN GHS Category 2. The test item may be considered to be non-irritant to skin in accordance with UN GHS (No Category), if the mean relative viability of three individual tissues after 60 minutes exposure to the test item and 42 hours post incubation is more than (>) 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
approx. 47 mg
- Concentration (if solution):
applied in the original form on 25µl DPBS


NEGATIVE CONTROL
- Amount(s) applied (volume or weight):
30µl
- Concentration (if solution):

POSITIVE CONTROL
- Amount(s) applied (volume or weight):
30µl
- Concentration (if solution):

Additional control
Two additional test item treated viable tissues for colour control.

NSMTT and killed controls
30 μL of negative control (DPBS) or positive control (5% (w/v) SDS solution) or 30 μL of test item were added to each additional control skin unit by using a suitable pipette.

Duration of treatment / exposure:

The plates with the test item, negative and positive control treated epidermis units were incubated for 35 minutes (± 1 minute) to the humidified incubator at 37°C with 5 % CO2, in a >95% humidified atmosphere and for the exposure time of 25 minutes (± 0.5 minute) at room temperature (24.6-27.1°C).

Duration of post-treatment incubation (if applicable):

After rinsing, the units were placed into the plate wells with fresh Assay Medium (0.9 mL/well) below them and then incubated for 24 hours (± 2hours) at 37°C in an incubator with 5 % CO2, in a >95 RH % humidified atmosphere.
At the end of the 24 hours incubation period the units were placed into the plate wells with fresh Assay Medium (0.9 mL/well) below them and then incubated for 18 hours (± 2hours) at 37°C in an incubator with 5 % CO2, in a >95 RH % humidified atmosphere.

Number of replicates:

test item: 3 replicates
negative controls: 3 replicates
positive controls: 3 replicates
As the test item was coloured, two additional test item-treated living tissues were used for the non-specific optical density (OD) evaluation. Furthermore, two additional test item-treated killed epidermis and two negative control treated killed epidermis were used. To avoid a possible double correction for colour interference, for non-specific colour in killed tissues, two additional disks were used.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: mean relative viability

Value:

115.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

Additional controls

As the test item was coloured (grey), two additional test item-treated living tissues were used to determine the non-specific OD value and a non-specific colour percentage (NSCliving%). Results of this non-specific colour determination are shown in Table 1. The mean value for optical density (measured at 570 nm) was 0.011, and the NSCliving% value for the test item was calculated as 0.7%. This value was below 5%, therefore additional data calculation was not necessary.

Table 1: Optical Density (OD) and the Calculated Non Specific Colour % (NSCliving%) of the Additional Control Tissues

Additional data	Optical Density (OD)			NCS% (living)
		Measured	Blank corrected
Treated with pNMCoxide (8:1:1)	1	0.052	0.006
	2	0.062	0.016	0.7
	mean	--	0.011

Notes:
1. Mean blank value was 0.046.
2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

Based on the physical, chemical properties of the test item, NSMTT control was used in this study. Therefore, additional controls and data calculations were necessary to exclude the false estimation of viability. Results of the additional controls on killed epidermis are shown in

Table 2. Based on these observed mean OD (0.000), the calculated NSMTT% was 0.00%.

Table 2: Optical Density (OD) and the calculated Non-Specific MTT Reduction (NSMTT) of the Additional Control Tissues (Killed Epidermis)

Additional control (killed epidermis)	Optical Density (OD)			NSMTT	NSMTT%
		Measured	Blank Corrected
Treated with pNMCoxide (8:1:1)	1 2	0.089 0.085	0.043 0.039	0.000 *-0.004
	mean	--	0.041	0.000	0.0
Treated with DPBS	1 2	0.087 0.091	0.041 0.045
	mean	--	0.043		--

Notes:
1. Mean blank value was 0.046.
2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).
1. * Excluded (negative volume)

Two additional test item-treated killed tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.008, Non- Specific Colour % (NSCkilled%) was calculated as 0.5% (see Table 3).

Table 3: Optical Density (OD) and the calculated Non Specific Colour % (NSCkilled%) of the Additional Control Tissues (Killed Epidermis)

Additional control (killed epidermis)	Optical Density (OD)			NSC% (killed)
		Measured	Blank Corrected
Treated with pNMCoxide (8:1:1)	1 2	0.051 0.057	0.005 0.011	0.5
	mean	--	0.008

Notes:
1. Mean blank value was 0.046.
2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

Viability Results

The results of the OD measured at 570 nm for the test item, positive and negative controls as well as the calculated relative viability percent values (% RV) are presented in Table 4. The OD values for the test item treated skin samples showed 115.8% relative viability compared to the negative control (after adjustment for non-specific MTT reduction).

Table 4: Optical Density (OD) and the Calculated Relative Viability % of the Samples

Substance	Optical Density (OD)			Viability (%RV)	Standard Deviation (SD)
		Measured	Blank corrected
Negative control: DPBS	1 2 3	1.720 1.717 1.673	1.674 1.671 1.627	101.0 100.8 98.1	--  -- --
	mean	--	1.658	100.0	1.6
Positive control: 5% (w/v) SDS solution	1 2 3	0.090 0.079 0.086	0.044 0.033 0.040	2.6 2.0 2.4	-- -- --
	mean	--	0.039	2.3	0.3
Test item:  pNMCoxide (8:1:1)	1 2 3	1.860 2.041 1.996	1.814 1.995 1.950	109.4 120.4 117.7	-- -- --
	mean	--	1.920	115.8	5.7

Notes:
1. Mean blank value was 0.046.
2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

No indication of irritation

Conclusions:

Following exposure of the EpiDerm(TM) Model to the test item, pNMC oxide (8:1:1), the mean relative viability was 115.8% compared to the negative control value. This is above the threshold of 50%, therefore under the condition of this assay the test item was considered to be non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.
In conclusion, in this in vitro EpiDermTM model test with pNMC oxide (8:1:1) (Batch number: PVX-144A PVX14), the results indicate that the test item is non-irritant to skin.

Executive summary:

An in vitro skin irritation test of pNMC oxide (8:1:1) test item was performed in a reconstructed human epidermis model. EpiDerm(TM) Model is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue; CAS number: 298-93-1) assay. The irritation potential of the test item was evaluated according to the OECD No. 439 guideline.

Disks of EpiDerm(TM) Model (three units) were treated with the test item and incubated for 35 minutes 37 degrees Celcius, 5 %CO2 ,>95 RH% humidified atmosphere and 25 minutes at room temperature. Exposure of the test item was terminated by rinsing with Dulbecco’s Phosphate Buffered Saline (DPBS). The epidermis units were then incubated at 37 degrees Celcius for 24 hours in an incubator with 5% CO2, in a >95% humidified atmosphere and 37 degrees Celcius for 18 hours in an incubator with 5% CO2, in a >95% humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 degrees Celcius in an incubator with 5% CO2 protected from light, in a >95% humidified atmosphere. The precipitated formazan crystals were then extracted using extracting solution (MTT-100- EXT) for 2 hours with shaking at room temperature and quantified spectrophotometrically at 570 nm.

The negative control epidermis units were treated with DPBS, whilst the positive control epidermis units were treated with 5% (w/v) Sodium Dodecyl Sulphate (SDS) (three units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test item. The possible MTT interaction potential of the test item was examined using two additional test item treated and two negative control treated killed epidermis units. Furthermore, to avoid a possible double correction for colour interference, a third set of controls (two additional disks) for non-specific colour in killed tissues (NSCkilled) were used. For each treated tissue, the viability was expressed as a percentage relative to the negative control. If the mean relative viability is less orequal (≤) to 50% of the negative control, the test item is considered to be irritant to skin.

Following exposure of the EpiDerm(TM) Model to the test item, pNMC oxide (8:1:1), the mean relative viability was 115.8% compared to the negative control value. This is above the threshold of 50%, therefore under the condition of this assay the test item was considered to be non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EpiDerm(TM) Model test with pNMC oxide (8:1:1), the results indicate that the test item is non-irritant to skin.