Lithium nickel potassium oxide

Eye irritation

Administrative data

Endpoint:

eye irritation, other

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

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

Test animals / tissue source

Species:

human

Test system

Vehicle:

unchanged (no vehicle)

Remarks:

The test substance is applied undiluted. Therefore, no preparation of the test substance in a vehicle was performed.

Controls:

yes

yes, concurrent positive control

yes, concurrent negative control

other: MTT reduction control (KC): Deionized water, sterile, or test substance

Amount / concentration applied:

Assessed by a single topical application of ca. 50 μL bulk volume (about 66 mg) undiluted test substance to a reconstructed three-dimensional, human cornea model (EpiOcular™).

Duration of treatment / exposure:

Three test runs were performed. Two EpiOcular™ tissues per test run were incubated with the
test substance for 6 hours followed by a 18-hour post-incubation period.

Duration of post- treatment incubation (in vitro):

Three test runs were performed. Two EpiOcular™ tissues per test run were incubated with the
test substance for 6 hours followed by a 18-hour post-incubation period.

Number of animals or in vitro replicates:

EpiOcular:
2 replicates per test

Details on study design:

EpiOcular:
The EpiOcularTM model (OCL-200) is a three-dimensional, non-keratinized tissue construct
composed of normal human-derived, epidermal keratinocytes used to model the human
corneal epithelium (compare Figure 1). The EpiOcularTM tissues (surface 0.6 cm²) are cultured
on cell culture inserts (MILLICELLs, 10 mm ∅) and are commercially available as kits
(EpiOcular™ 200) containing 24 tissues on shipping agarose.

Tissue model: OCL-200
Tissue Lot Number: 27004 (test run 1), 27009 (test run 2) and 27015 (test run 3)
(Certificates of Analysis see appendix)
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava,
Slovakia


Experimental procedure EpiOcular Test
Direct MTT reduction:
In order to assess the ability of the test material to directly reduce MTT, a pretest (experimental
conduct in accordance with GLP, but without a GLP status) was performed. The test substance
was added to 0.9 mL MTT solution. The mixture was incubated in the dark at about 37°C for
3 hours. A negative control (deionized water) was tested concurrently. If the MTT solution color
or in case of water-insoluble test substances the border to the water-phase turned blue / purple
the test substance was presumed to directly reduce MTT.
The direct reduction of MTT by a test substance interferes with the color density produced by
metabolic capacity of the tissue and would falsify the test results.
In case of direct MTT reduction, two freeze-killed control tissues were treated with the test
article and the negative control each in the same way as described in the following section.
Due to the intense color of the test substance, it was not possible to evaluate whether the
test substance is able to directly reduce MTT. Therefore, freeze-killed control tissues (KC)
were treated with the test article and the negative control.

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 6 hours and removed by washing. Thereafter, extraction in isopropanol
was performed and the OD570 of the extract was spectrophotometrically determined.
Based on the result of the pretest, it was judged that application of color control tissues is not
necessary.


Controls EpiOcular
Negative control (NC): Deionized water, sterile
Positive control (PC): Neat methyl acetate (CAS No.: 79-20-9)
MTT reduction control (KC): Deionized water, sterile, or test substance

ACCEPTANCE CRITERIA
In case one of the acceptance criteria below was not met, repetition of the test was considered.

Acceptance criteria EpiOcular

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 (min) value following
exposure to 100 μL of 0.3% Triton X-100 for each
EpiOcular™ EIT (OCL-200) batch. The ET50 must fall
within a range established 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 Guideline.

Lower acceptance limit: ET50 = 12.2 min
Upper acceptance limit: ET50 = 37.5 min

EpiOcular QC (OCL-200) batch release see Appendix

Acceptance criteria for the NC :
The absolute OD570 of the NC 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 2.5.

Acceptance criteria for the PC: Methyl acetate used as PC usually leads to a tissue
viability of approx. 25%. A viability of < 50% is acceptable.

Acceptance criteria for tissue variability:
Two tissues were treated under the same conditions.
A variability between the two tissues is considered to be
acceptable if the relative difference of the viability is
< 20%.

Acceptance criteria for the KC:
The OD570 of the killed control tissues treated as negative
control should be ≤ 0.35. The value for direct MTT
reduction of a test substance should be ≤ 30% of the NC.


Pre-incubation of the tissues:
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with
1 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour, the
pre-incubation medium was replaced by fresh medium and preconditioning continued in the
incubator at standard culture conditions for 16 – 24 hours.

Pretreatment of the tissues:
After pre-incubation, the tissues were pretreated with 20 μL PBS to wet the tissue surface. The
tissues were incubated at standard culture conditions for 30 minutes.

Application of the test substance:
By using a sharp spoon, a bulk volume of ca. 50 μL test material was applied covering the
whole tissue surface.
Control tissues were concurrently applied with 50 μL sterile deionized water (NC, NC KC) or
with 50 μL methyl acetate (PC) or test substance (KC).
After application, the tissues were placed into the incubator until the total exposure time of
6 hours was completed.

Removal of the test substance and postincubation period:
In order to remove the test substance, the tissues were washed with sterile PBS. For this
purpose, the tissues were immersed and swiveled three times in each of three beakers filled
with PBS. Washed tissues were immediately immersed into 12-well plates pre-filled with
5 mL/well pre-warmed medium (post-soak immersion) to remove residual test substance. 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. After 25 minutes of post-soak immersion, each tissue was dried on absorbent paper
and transferred to fresh 6-well plates filled with 1 mL/well pre-warmed medium.

MTT incubation
After the post-incubation period, the assay medium was replaced by 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 metabolically produced by the tissues was extracted by overnight
incubation of the tissues in isopropanol at room temperature or by incubation for at least
2 hours on a plate shaker. The optical density at a wavelength of 570 nm (OD570) of the extracts
was spectrophotometrically determined. Blank values were established of 4 microtiter wells
filled with isopropanol for each microtiter plate.

Data evaluation EpiOcular:

Principle: The OD570 values determined for the various tissues are
measures of their viability. The ratio 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 was an irritant.

Calculation of individual and mean optical densities:
The OD570 value measured and corrected for each individual
tissue was 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 treated in the same way was 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 KC-corrected OD570). 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
KC-corrected OD570. The mean KC-corrected OD570 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 the ratio of
the mean OD570 (or mean KC-corrected OD570, if applicable)
divided by the respective OD570 NC value in percent.

EpiOcular:
The objective of this in vitro test is to assess the eye irritation potential of the test substance by
using the reconstructed human ocular tissue model EpiOcularTM.
The test is based on the experience that irritant chemicals produce cytotoxicity in human
reconstructed cornea after a short term topical exposure. The test is designed to predict an
eye irritation potential of a chemical by using the three-dimensional, human cornea model
EpiOcularTM. After application of the test material to the surface of the EpiOcularTM 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 spectrophotometrically determined. The optical density of the extracts of the tissues treated
with the test substance is compared to values from negative control tissues and expressed as
relative tissue viability.

Results and discussion

In vitro

Results

Irritation parameter:

other: Tissue Viability

Remarks:

Tissue viability: The quantification of tissue viability is presented as the ratio of the mean OD570 (or mean Killed Control-corrected OD570, if applicable) divided by the respective OD570 Negative Control value in percent.

Value:

>= 64.7 - <= 71.5

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

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.

In all test runs slight to distinct compound residues remained on the test-substance treated
tissues after the washing procedure. 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 1st test run:
In the 1st test run non-concordant replicate measurements of the test-substance treated
viable tissues was obtained (values for single tissues (without KC correction): 80.1% and
49.7%). The value for inter-tissue variability of the viable tissues treated with the test
substance was 30.5% and therefore out of the acceptance range . Thus, a
2nd test run was performed.
Results of the 2nd test run:
After the 2nd test run a borderline result (final relative mean tissue viability 64.7%) was
obtained . Thus, the study was repeated to increase the data base for
evaluation.

Results of the 3rd test run:
The final relative mean viability of the tissues treated with the test substance was 71.5%. All
acceptance criteria were met.

Overall, the results of two valid test runs (relative mean tissue viability of both runs 68.1%) of
the EpiOcular™ eye irritation assay showed, that the test substance does not indicate an eye
irritation potential.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results observed in the EpiOcular Test alone and by applying the evaluation criteria, it was concluded that KDLNO does not show an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.

Executive summary:

The objective was to assess the eye irritating potential of KDLNO. By using the methods currently available a single in vitro assay is not sufficient to cover the full range of eye irritating potential. Therefore, two in vitro assays were part of this in vitro eye irritation test strategy: The Bovine Corneal Opacity and Permeability Test (BCOP Test) and EpiOcular Eye Irritation Test. However, in the current case the results derived with EpiOcular alone were sufficient for a final assessment. Therefore, further testing in BCOP was waived.

The potential of the test substance to cause ocular irritation was assessed by a single topical application of ca. 50 μL bulk volume (about 66 mg) undiluted test substance to a reconstructed three-dimensional, human cornea model (EpiOcular™). Three test runs were performed. Two EpiOcular™ tissues per test run were incubated with the test substance for 6 hours followed by a 18-hour post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The ratio
of the values indicates the relative tissue viability.

Based on the results observed in the EpiOcular Test alone and by applying the evaluation criteria, it was concluded that KDLNO does not show an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.