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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:
11 June 2020 - 31 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

1
Reference substance name:
Nickel Cobalt Manganese Hydroxide
Cas Number:
189139-63-7
Molecular formula:
Molecular formula: Ni1-x-yMnxCoy(OH)2, Stoechiometry of (Me = Ni + Co + Mn) equals 1, ranges of the elements approx. as: Ni/Me = 0.25 – 0.95 Mn/Me = 0.02 – 0.40 Co/Me = 0.02 – 0.40 OH = 2.00
IUPAC Name:
Nickel Cobalt Manganese Hydroxide
Constituent 1
Chemical structure
Reference substance name:
Manganese dihydroxide
Cas Number:
18933-05-6
Molecular formula:
Mn(OH)2
IUPAC Name:
Manganese dihydroxide
Constituent 2
Chemical structure
Reference substance name:
Cobalt dihydroxide
EC Number:
244-166-4
EC Name:
Cobalt dihydroxide
Cas Number:
21041-93-0
Molecular formula:
CoH2O2
IUPAC Name:
cobalt(2+) dihydroxide
Constituent 3
Chemical structure
Reference substance name:
Nickel dihydroxide
EC Number:
235-008-5
EC Name:
Nickel dihydroxide
Cas Number:
12054-48-7
Molecular formula:
H2NiO2
IUPAC Name:
nickel(2+) dihydroxide
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Batch/Lot number: PVX-089 PVX 14
Description: Dark Grey Powder
Purity: 100% (6.31% Co ; 5.96% Mn ; 50.87% 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 EpiDermTM has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Remarks:
te 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):
58 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
Colour controls: Two test item treated living and killed tissues for colour controls

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:
Two test item treated living and killed tissues for colour controls
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:
Remarks:
mean relative viability
Value:
96.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Validity of the test
Based on the observed colours, the epidermis units were in a suitable condition for use in the assay.
The mean OD value of the three negative control tissues was in the recommended range (1.658). Standard deviation of the viability results for negative control samples was 1.6%.
The positive control treated tissues showed 2.3% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 0.3%.
The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 3.1%.
The mean OD value of the blank samples (acidified isopropanol) was 0.046.
All these parameters met the acceptability criteria, therefore the study was considered to be valid.

The mean OD value of the three negative control tissues should be between 0.8 and 2.8 and the standard deviation value (SD) of the % viability should be ≤ 18.
The acceptable mean percentage viability range for positive controls is 0-20% and the standard deviation value (SD) of the % viability should be ≤ 18.
The SD calculated from individual % tissue viabilities of the three identically treated replicates should be <18.
The mean OD value of the blank samples (acidified isopropanol) should be <0.1.

Historical control data are available
OD data of one replicate tissues and mean viability % at the positive control in this study were lower (OD: 0.033 and viability %: 2.0) than the minimum OD and minimum viability % of the historical control range (OD: 0.038 and viability %: 2.1). OD data of three replicate tissues at the negative control were lower (OD: 1.674, 1.671 and 1.627) than the minimum OD of the historical control range (OD: 1.741). These facts has no impact on the results or integrity of the study since the positive control material showed clear positive result and the acceptable mean percentage viability range for the positive controls is 0-20% and the mean OD value of the three negative control tissues were between 0.8 and 2.8 according to the OECD No. 439.

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 0.007, and the NSCliving% value for the test item was calculated as 0.4%. 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 pNMChydroxide (8:1:1)  1  0.053  0.007  
   2  0.053  0.007

 0.4

   mean  --  0.007  

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 Tablr 2.

Based on these observed mean OD (0.001), the calculated NSMTT% was 0.05%.

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 pNMChydroxide (8:1:1)

 1

2

0.090

0.086 

0.044

0.040

0.001

*-0.0043

 
   mean  --  0.042  0.001  0.05
  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 0.010, Non- Specific Colour % (NSCkilled%) was calculated as 0.6% (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 pNMChydroxide (8:1:1)

 1

2

0.052

0.061

0.006

0.015 

 0.6
   mean  --  0.010  

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 99.6% 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)

 TOD tt
     Measured  Blank corrected      

 Negative control:

DPBS

1

2

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: 

pNMChydroxide (8:1:1)

1

2

1.589

1.673

1.680

1.543

1.627

1.634

93.0

98.1

98.5

--

--

-- 

1.542

1.626

1.633 

   mean  --  1.601  96.6  3.1  1.600

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:
considered non-irritant to skin
Conclusions:
Following exposure of the EpiDerm(TM) Model to the test item, Nickel Cobalt Manganese Hydroxide (8:1:1), the mean relative viability was 96.6% compared to the negative control value (after adjustment for non-specific MTT reduction). 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 Nickel Cobalt Manganese Hydroxide (8:1:1) (Batch number: PVX-089 PVX14), the results indicate that the test item is non-irritant to skin.
Executive summary:

An in vitro skin irritation test of Nickel Cobalt Manganese Hydroxide (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 at 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 18 hours at 37 degrees Celcius 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 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 or equal (≤) 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 hydroxide (8:1:1)), the mean relative viability was 96.6% compared to the negative control value (after adjustment for non-specific MTT reduction). 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 the in vitro EpiDerm(TM) Model with pNMC hydroxide (8:1:1) (Batch Number PVX-089 PVX14), the results indicate that the test item is non-irritant to skin.