Lithium nickel potassium oxide

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation / corrosion, other

Remarks:

Two in vitro assays were carried out for for this in vitro skin irritation and corrosion test strategy: The Skin Corrosion Test (SCT), and Skin Irritation Test (SIT)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

STUDY COMPLETED
09 Apr 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 435 (In Vitro Membrane Barrier Test Method for Skin Corrosion)

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Test guideline 4

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Test guideline 5

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Lithium nickel potassium oxide

Cas Number:

210352-95-7

Molecular formula:

Li.K.Ni.O.H2O

IUPAC Name:

Lithium nickel potassium oxide

Test material form:

solid: particulate/powder

Details on test material:

Lithium nickel potassium oxide (KDLNO)
CAS Number:
210352-95-7
Appearance/Physical state:
Black powder
Storage: Room temperature in the dark

Specific details on test material used for the study:

TEST ITEM
The analyses of the test item (= test substance) were carried out the Competence Center
Analytics, BASF SE, Ludwigshafen, Germany.
Name of test substance: KDLNO
Test substance No.: 17/0258-1
Batch identification: EXP_BASIC_006816
CAS No.: 210352-95-7
Purity: 98%
Identity: Analytically Confirmed
Homogeneity: The test substance was homogeneous by visual inspection.
Storage stability: The product was stable under storage conditions over the study period


ADDITIONAL TEST SUBSTANCE INFORMATION
pH value: Ca. 10 (20% aqueous preparation; determined in the lab prior
to start of the GLP study)
Physical state / color: Solid / black
Storage conditions: Room temperature


Skin Corrosion Test (SCT) and Skin Irritation Test (SIT)
Laminar flow bench: HERAsafe KS 18 (Thermo ELECTRON CORPORATION)
CO2 incubator: Heraeus BBD 6220
Incubation conditions: 37°C ± 1°C, 5% ± 1% CO2, 90% ± 5% relative
humidity
Spectrophotometer: SunriseTM Absorbance Reader
For the determination of the optical density of colored extracts.
Measurement using a filter wavelength 570 nm without reference
filter
EpiDerm™ 200 kit: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia,
containing:
24 EPI-200 tissues (reconstructed epidermis): surface 0.6 cm²
cultured in Millicells® ∅ 1 cm
Tissue for MTTreduction
control:
EPI-200 tissue that is killed by freezing at –20°C
Assay medium: Corrosion test: EPI-100-ASY assay medium
Irritation test: EPI-100-NMM assay medium
MTT diluent: Dulbecco's modified eagle's medium (DMEM)-based
medium used for diluting MTT (MatTek In Vitro Life Science
Laboratories, Bratislava, Slovakia / Sigma, Germany)
Wash buffer: Dulbecco's phosphate-buffered saline (PBS), w/o Ca2+, Mg2+
(MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia and
Biochrom, Germany)
Detection agent: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
(MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia /
Sigma, Germany), 1.0 mg / mL MTT diluent
Extracting agent: Isopropanol p.a.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: EpiDermTM model - normal, human-derived epidermal keratinocytes

Cell source:

other: EpiDermTM tissue kits; Tissue model: EPI-200; Tissue Lot Number: 25844 ; Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Source strain:

other: EpiDermTM tissue kits; Tissue model: EPI-200; Tissue Lot Number: 25844 ; Origin: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Justification for test system used:

The objective was to assess the skin corrosion and irritation potential of the test material.
Using the methods currently available, a single in vitro assay is not sufficient to cover the full
range of skin irritating/corrosion potential including transport classification. Therefore, three in
vitro assays were part of this in vitro skin irritation and corrosion test strategy including transport
classification: The Skin Corrosion Test (SCT), Skin Irritation Test (SIT) and In vitro Membrane
Barrier Test (Corrositex®).

However, in the current case, the results derived with SCT and SIT were sufficient for a final
assessment. Therefore, further testing in Corrositex® was waived.


Skin Corrosion Test (SCT) and Skin Irritation Test (SIT):
The present test is based on the experience that corrosive and irritant chemicals produce
cytotoxicity in human reconstructed epidermis after a short term topical exposure. The test is
designed to predict a skin corrosion or irritation potential of a chemical by using the
three-dimensional human epidermis model EpiDermTM. After application of the test material to
the stratum corneum surface of the EpiDermTM tissue, the induced cytotoxicity (= loss of
viability) is measured by a colorimetric assay. Cytotoxicity is expressed as the reduction of
mitochondrial dehydrogenase activity. The mitochondrial dehydrogenase reduces the yellow-colored water-soluble 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to
the insoluble blue-colored formazan. After isopropanol extraction of the formazan from the
tissues, the optical density of the extract is determined spectrophotometrically. The optical
density of the extracts of tissues treated with the test substance is compared to negative control
values from tissues and expressed as relative tissue viability.

Details on test system:

Three-dimensional human epidermis model (SCT and SIT):
The EpiDermTM model consists of normal, human-derived epidermal keratinocytes, which have
been cultured to form a multi layered, highly differentiated model of the human epidermis.
It consists of organized basal, spinous and granular layers and a multi-layered stratum
corneum containing intercellular lamellar lipid layers arranged in patterns analogous to that
found in vivo. The EpiDermTM tissues (surface 0.6 cm²) are cultured on specially prepared cell
culture inserts (MILLICELLs, 10 mm ∅) and are available commercially as kits (EpiDerm™
200) containing 24 tissues on shipping agarose.

Skin Corrosion Test (SCT) and Skin Irritation Test (SIT)
Negative control (NC): Deionized water (corrosion test); PBS, sterile (irritation test)
Positive control (PC): 8 N potassium hydroxide solution (corrosion test)
5% (w/v) sodium dodecyl sulfate (SDS) in water (irritation test)

MTT reduction control (KC - killed controls):
Deionized water or test substance (corrosion test)
PBS, sterile or test substance (irritation test)

Color control:
The color of a test substance may interfere with the color density produced by metabolic
capacity of the tissue and falsify the test results when residues of the test substance remain
on the tissues after washing and are extracted by isopropanol.
Due to the color of the test substance, a pretest (experimental conduct in accordance with
GLP, but without a GLP status) was performed as follows: the test substance was applied to a
KC tissue, incubated for 1 hour and removed by washing. Thereafter, extraction in isopropanol
was performed, and the OD570 of the extract was determined spectrophotometrically.
Based on the result of the pretest, it was judged that application of color control tissues is not
necessary.

Corrosion test (SCT):
From the day of arrival in the laboratory, tissues were kept in the refrigerator. At least 1 hour,
but not more than 1.5 hours before test substance application, tissues were transferred to
6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C.
The pre-incubation medium was replaced with fresh medium immediately before application.
Two tissues per exposure time (3 minutes at room temperature or 1 hour in the incubator as a
rule) and test group (test material, negative control and positive control; 12 tissues per test)
were used. In addition, two killed control tissues per exposure time were treated with the test
substance and the NC, respectively, to detect direct MTT reduction.
25 μL deionized water was applied first. Thereafter, a bulk volume of ca. 25 μL solid test
material was applied with a sharp spoon and homogeneously distributed with the water.
Control tissues were treated concurrently with 50 μL deionized water (NC, NC KC) or with 50
μL 8 N potassium hydroxide (PC) or test substance (KC). A nylon mesh was placed carefully
onto the tissue surface of NC and NC KC afterwards.
The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after
start of the application treatment. As the test substance could not be fully removed by the
washing procedure carefully wiping with a MEROCEL® sponge (Fritz Ruck Ophthalmologische
Systeme GmbH, Germany) was performed.
Rinsed tissues were kept in 24-well plates (holding plates) at room temperature on assay
medium until all tissues per application time were dosed and rinsed. The assay medium was
then replaced with MTT solution and tissues were incubated for 3 hours.
After incubation, the tissues were washed with PBS to stop the MTT incubation. The formazan
that was produced metabolically by the tissues was extracted by incubation of the tissues in
isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was
determined spectrophotometrically. Blank values were established of 4 microtiter wells filled
with isopropanol for each microtiter plate.

Irritation test (SIT):
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with
0.9 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour, the
pre-incubation medium was replaced with fresh medium and preconditioning continued for
18 ± 3 hours.
Three tissues were treated with the test substance, the PC and the NC, respectively.
In addition, three killed control tissues were used for the test substance and the NC,
respectively, to detect direct MTT reduction.
25 μL sterile PBS was applied first. Thereafter, a bulk volume of ca. 25 μL solid test material
was applied with a sharp spoon and homogeneously distributed together with the fluid.
Control tissues were treated concurrently with 30 μL sterile PBS (NC, NC KC) or with 30 μL
5% SDS (PC) or test substance (KC). A nylon mesh was placed carefully onto the tissue
surface of NC, NC KC and PC afterwards.
The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall
and for 35 minutes in the incubator.
The tissues were washed with sterile PBS to remove residual test material 1 hour after start of
application. As the test substance could not be fully removed by the washing procedure
carefully wiping with a MEROCEL® sponge (Fritz Ruck Ophthalmologische Systeme GmbH,
Germany) was performed. Rinsed tissues were blotted on sterile absorbent paper and
transferred into new 6-well plates pre-filled with 0.9 mL fresh medium. When all tissues were
rinsed, the surface of each tissue was dried carefully with a sterile cotton swab.
Subsequently, the tissues were placed into the incubator at 37°C for 24 ± 2 hours.
After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL
fresh medium and placed into the incubator for an additional 18 ± 2-hour post-incubation
period.
After the post-incubation period, the assay medium was replaced with 0.3 mL MTT solution,
and the tissues were incubated in the incubator for 3 hours.
After incubation, the tissues were washed with PBS to stop the MTT incubation. The formazan
that was produced metabolically by the tissues was extracted by incubation of the tissues in
isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was
determined spectrophotometrically. Blank values were established of 4 microtiter wells filled
with isopropanol for each microtiter plate.

Data evaluation

Principle: The OD570 values determined for the various tissues are
measures of their viability. The quotient of the OD570 of tissues
treated with the test material and the mean OD570 values of the
NC (percent of control) is used for evaluating whether a test
material is corrosive or irritant.

Calculation of individual and mean optical densities:
The individual tissue OD570 is calculated by subtracting the
mean blank value of the respective microtiter plate from the
respective individual tissue OD570 value. The mean OD570 for a
test group of two tissues (corrosion test) or three tissues
(irritation test) treated in the same way is calculated.

Application of measurements using killed control tissues:
In case of direct MTT reduction by the test substance, the OD570
values measured in the freeze-killed control tissues (KC) will be
used to correct the mean OD570 of the tissues treated with the
test substance (mean OD570 KC-corrected). Since killed tissues
might still have a residual enzyme activity that is able to produce
some formazan net OD570, KC is calculated by subtracting the
OD570 KC of the NC from the OD570 KC of the test substance.
In case the net OD570 KC is greater than zero, it is subtracted
from the respective mean OD570 to result in the mean corrected
OD570 KC. The mean corrected OD570 KC represents the
formazan production linked to the tissue viability and therefore
indicates the cytotoxic potency of the test substance.

Tissue viability: The quantification of tissue viability is presented as quotient of
the mean OD570 (or mean corrected OD570 KC if applicable)
divided by the respective OD570 NC value in percent for each
exposure time.

ACCEPTANCE CRITERIA
Skin Corrosion Test (SCT) and Skin Irritation Test (SIT):
In case one of the acceptance criteria below was not met, repetition of the test was considered.

Barrier function and Quality control (QC):
The supplier demonstrates that each batch of the model used
meets the defined production release criteria. MatTek
determines the ET50 value following exposure to Triton X-100
(1%) for each EpiDermTM batch. The ET50 must fall within an
established range based on a historical database of results.
The following acceptability range (upper and lower limit) for
the ET50 is established by the supplier as described in the
cited OECD guidelines.

The following acceptability range (upper and lower limit) for
the ET50 is established by the supplier as described in the
cited OECD guidelines.

Lower acceptance limit: ET50 = 4.0 hours
Upper acceptance limit: ET50 = 8.7 hours

Acceptance criteria for the negative control (NC):
The absolute OD570 of the negative control tissues in the MTT
test is an indicator of tissue viability obtained in the testing
laboratory after the shipping and storing procedure and under
specific conditions of the assay. Tissue viability is acceptable if
the mean OD570 of the NC is ≥ 0.8. The mean OD570 of the NC
should not exceed 2.8.

Acceptance criteria for the positive control (PC):
Corrosion test: 8 N potassium hydroxide solution is used as
positive reference. A tissue viability of ≤ 30% is acceptable for
the 3-minute exposure. Mean viability of the tissues exposed for
1 hour should be <15%.

Irritation test: 5% SDS is used as PC and reflects the sensitivity
of the tissues used in the test conditions. A viability of ≤ 20% is acceptable.

Acceptance criteria for the variability of the tissues (corrosion test):
For every treatment, two tissues are treated in parallel. In the
range of 20% and 100% viability, variability between the
tissues is considered to be acceptable if the coefficient of
variation (CV) of % viability is ≤ 30%.

Acceptance criteria for the variability of the tissues (irritation test):
For every treatment, three tissues are treated in parallel. The
inter-tissue variability is considered to be acceptable if the SD
of % viability is ≤ 18%.

Acceptance criteria for the killed controls (KC):
The OD570 of the tissues for the KC of the NC should be
≤ 0.35. The OD570 value for direct MTT reduction of a test
substance should be ≤ 30% of the OD570 of the NC.

Evaluation of results of SCT
The corrosive potential of the test materials is predicted from the mean relative tissue viabilities
obtained after a 3-minute treatment compared to the negative control tissues treated
concurrently with deionized water. A chemical is considered as "corrosive" if the mean relative tissue viability after the 3-minute treatment with a test material is decreased below 50%.
In addition, materials with a viability of ≥ 50% after the 3-minute treatment are considered as
"corrosive" if the mean relative tissue viability after a 1-hour treatment with a test material is
decreased below 15%.


Evaluation of results of SIT
The irritant potential of the test materials is predicted from the mean relative tissue viabilities
compared to the negative control tissues treated concurrently with sterile PBS. A chemical is
considered as "irritant" if the mean relative tissue viability with a test material is less than or
equal to 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

The potential of the test substance to cause dermal corrosion/irritation was assessed by a
single topical application of ca. 25 μL bulk volume (about 36 mg) undiluted test substance to a
reconstructed three-dimensional, human epidermis model (EpiDerm™).

Duration of treatment / exposure:

For the corrosion test (SCT), two EpiDerm™ tissues were incubated with the test substance
for 3 minutes and 1 hour each. The irritation test (SIT) was performed with three EpiDerm™
tissues, which were incubated with the test substance for 1 hour followed by a 42-hour postincubation
period.

Duration of post-treatment incubation (if applicable):

Skin Irritation Test (SIT):
1 hour followed by a 42-hour post-incubation
period.

Number of replicates:

Skin Corrosion Test (SCT):
2 replicates per test

Skin Irritation Test (SIT):
3 replicates per test

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

>= 88.3 - <= 97.6

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The following results were obtained in the EpiDerm™ skin corrosion/irritation test:
Due to the color of the test substance, it could not be determined whether the test substance
is able to directly reduce MTT. Therefore, an additional MTT reduction control (freeze-killed
control tissues (KC)) was introduced.

Slightly black colored compound residues remained on the test-substance treated tissues
after the washing procedure in both tests. However, this did not interfere with the colorimetric
test as was demonstrated in a pretest prior to start of the GLP study.

Results of the Corrosion test (SCT):
The final relative mean viability of the tissues treated with the test substance determined after
an exposure period of 3 minutes was 97.6%, and it was 88.3% after an exposure period of
1 hour.
Results of the Irritation test (SIT):
The final relative mean viability of the tissues treated with the test substance determined after
an exposure period of 1 hour with an about 42-hour post-incubation was 97.1%.

Based on the results observed and by applying the evaluation criteria
it was concluded that KDLNO does not show a skin irritation potential in the EpiDerm™ in
vitro skin irritation and corrosion test strategy including transport classification under the test
conditions chosen.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results observed and by applying the evaluation criteria
it was concluded that KDLNO does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy including transport classification under the test conditions chosen.

Executive summary:

The following results were obtained in the EpiDerm™ skin corrosion/irritation test:
Due to the color of the test substance, it could not be determined whether the test substance is able to directly reduce MTT. Therefore, an additional MTT reduction control (freeze-killed control tissues (KC)) was introduced.

Slightly black colored compound residues remained on the test-substance treated tissues after the washing procedure in both tests. However, this did not interfere with the colorimetric test as was demonstrated in a pretest prior to start of the GLP study.

Results of the Corrosion test (SCT):
The final relative mean viability of the tissues treated with the test substance determined after an exposure period of 3 minutes was 97.6%, and it was 88.3% after an exposure period of 1 hour.

Results of the Irritation test (SIT):
The final relative mean viability of the tissues treated with the test substance determined after an exposure period of 1 hour with an about 42-hour post-incubation was 97.1%.

Based on the results observed and by applying the evaluation criteria
it was concluded that KDLNO does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy including transport classification under the test conditions chosen.