Alkaline co-precipitation products of soluble cobalt, manganese and nickel salts

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

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)

Version / remarks:

18 June 2019

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.69 Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage

Version / remarks:

31 July 2019

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Alkaline co-precipitation products of soluble cobalt, manganese and nickel salts

Molecular formula:

Co(OH)2(0.005-0.1)Co2O3(0.03-0.3)Mn3O4(0.01-0.1)MnO2(0.02-0.25)Ni(OH)2(0.35-0.91) The stoichiometry of (OH)x+Oy per metal component in the substance cannot be determined unambiguously from solely analytical data, since the composition of the present UVCB substance is highly variable (i.e. complexly mixed and poorly ordered at the atomic level). The composition of the UVCB substance can be estimated based on analytical examination in combination with a calculation that is based on the average oxidation number (NOx) in the sum of the elements Ni, Co, Mn. As a result, the following constituents in concentration ranges as described below can be identified in the UVCB: The sum of all constituents equals 1 and ranges are: • Co(OH)2: 0.005-0.1 • Co2O3: 0.03-0.3 • Mn3O4: 0.01-0.1 • MnO2: 0.02-0.25 • Ni(OH)2: 0.35-0.91

IUPAC Name:

Alkaline co-precipitation products of soluble cobalt, manganese and nickel salts

Test material form:

solid

Specific details on test material used for the study:

Purity: 100 % (UVCB) (for details see analytical report No.: 21L00179)
Homogeneity: The test item was homogeneous by visual inspection.
Storage stability: The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.
Expiry date: April 26, 2023
Storage conditions: Room temperature
Physical state/ color: Solid / black

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

- Model: EpiOcular™ OCL-200
- Source: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
- Certificate of Analysis: provided by supplier
- Lot: 34934

Test system

Vehicle:

unchanged (no vehicle)

Remarks:

Tissues were pretreated with 20 μL PBS in order to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes. Ca. 50 μL (ca. 53 mg) test material was applied covering the whole tissue surface.

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL volume equals 53 mg test item, 50 µL methyl acetate (positive control), 50 µL sterile deionized water (negative control)

Duration of treatment / exposure:

- pre-treatment with 20µL PBS and incubated at standard culture conditions for 30 minutes
- substance treatment: 6 h in the incubator (at 37°C)

Duration of post- treatment incubation (in vitro):

18 h

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 immersed immediately into 12-well plates pre-filled with 5 mL/well pre-warmed medium (post-soak immersion) to remove residual test substance. 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. Subsequently, the tissues were incubated at standard culture conditions for 18 hours (postincubation period).

Number of animals or in vitro replicates:

2

Details on study design:

PRETESTS:
Pretest for direct MTT reduction:
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 order to assess the ability of the test material to reduce MTT directly, a pretest (experimental conduct in accordance with GLP, but without a GLP status) was performed as described below. 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 color of the MTT solution or (in case of water-insoluble test substances) the border to the water phase turned blue / purple, the test substance was presumed to reduce MTT directly. In case of direct MTT reduction, two freeze-killed control tissues (KC) were treated additionally with each the test article and the negative control in the same way.
Based on the result of the pretest, it was judged that application of killed control tissues is not necessary.
Pretest for 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 determined spectrophotometrically.
Based on the result of the pretest, it was judged that application of color control tissues is not
necessary.

STUDY PROCEDURE:
Two tissues were treated with each the test substance, the PC and the NC. In addition, two killed tissues were used for each the test substance and the NC to detect direct MTT reduction.
Pre-incubation of the tissues: 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 with 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 in order to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes.
Application of the test substance: Using a sharp spoon, a bulk volume of ca. 50 μL (ca. 52 mg) test material was applied covering the whole tissue surface. Control tissues were applied concurrently 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 immersed immediately into 12-well plates pre-filled with 5 mL/well pre-warmed medium (post-soak immersion) to remove residual test substance. 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. Subsequently, the tissues were incubated at standard culture conditions for 18 hours (postincubation period).
MTT incubation: 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 metabolically produced 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 spectrophotometrically determined. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.

ACCEPTENCE CRITERIA:
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 2.8.
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 tissues is considered to be acceptable if the relative difference of the viability is < 20%.

Results and discussion

In vitro

Results

Irritation parameter:

mean percent tissue viability 

Run / experiment:

mean of 2 replicates with 6 h exposure each

Value:

74.4

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:

OTHER EFFECTS:
- Visible damage on test system: not specified

DEMONSTRATION OF TECHNICAL PROFICIENCY: profound historical data available

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Any other information on results incl. tables

Individual and mean OD570 values, individual and mean viability values and inter-tissue variability

Exposure period: 6 h
Test substance identification			tissue 1	tissue 2	mean	Inter-tissue variability [%]
NC	viable tissue	mean OD570	1.811	1.953	1.882
		viability [% of NC]	96.2	103.8	100.0	7.5
test substance	viable tissue	mean OD570	1.433	1.368	1.401
		viability [% of NC]	76.2	72.7	74.4	3.5
PC*	viable tissue	mean OD570	0.478	0.840	0.659
		viability [% of NC]	26.1	45.9	36.0	19.8

*The PC tissues were tested in parallel within another test run (No.133). Thus, calculation of viability (% of NC) is based on the NC value (mean OD570: 1.831) of this test run.
Slightly black colored compound residues remained on the tissues treated with the test substance after the washing procedure. However, this did not influence the validity of the study since the test substance was not able to reduce MTT directly.
Further, it was demonstrated in a pretest that the color of the test substance did not interfere with the colorimetric test.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

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

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