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

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: 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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

skin 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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

14 Jun 2021

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 July 2019

GLP compliance:

yes (incl. QA statement)

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 system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm™ 200 kit supplied by MatTek In Vitro Life Science Laboratories (Bratislava, Slovakia)

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPI-200
- Tissue batch number(s): 36107
- Certificate of analysis: provided by supplier

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature for 25 minutes and for 35 minutes in the incubator at 37°C
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The tissues were washed with sterile PBS
- Observable damage in the tissue due to washing: not described

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg / mL MTT diluent (0.3 mL per tissue)
- Incubation time: 3 h
- Spectrophotometer: Sunrise TM Absorbance Reader
- Wavelength: 570 nm
- Filter: no reference filter

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues: EPI-200 tissue that is killed by freezing at –20°C
- N. of replicates : 3
The test substance is not able to reduce MTT directly. Therefore, an additional MTT reduction
control KC (freeze-killed control tissues) was not introduced.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA:
The test chemical is identified as requiring classification and labelling according to UN GHS
(Category 2 or Category 1) if the mean percent tissue viability after exposure and posttreatment
incubation is less than or equal (≤) to 50%.
A single test composed of at least three tissue replicates should be sufficient for a test chemical
when the result is unequivocal. However, in case of borderline results such as non-concordant
replicate measurements and/or mean percent tissue viabilities of 45% - 55%, a second test
should be considered as well as a third one in case of discordant results between the first two
tests.

ACCEPTANCE CRITERIA
- Negative conrol: 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.
- Positive control: 5% SDS is used as PC and reflects the sensitivity of the tissues used in the test conditions. A viability of ≤ 20% is acceptable
- Variability: 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%.

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 μL sterile PBS was applied first. Thereafter, a bulk volume of ca. 25 μL solid test material (ca. 37 mg) was applied with a sharp spoon and homogeneously distributed with the water, so that the surface of the epidermis model was completely covered.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL sterile PBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL 5% SDS

Duration of treatment / exposure:

1 h (25 min at room temperature, 35 min in the incubator at 37°C)

The tissues were washed with sterile PBS to remove residual test material 1 hour after start of
application. 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.

Duration of post-treatment incubation (if applicable):

24 ± 2 h in the incubator (at 37°C)

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.

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of three replicates

Value:

103

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: no
- Direct-MTT reduction: not able to reduce MTT directly; application of killed control tissues is not necessary
- Colour interference with MTT/colorimetric test: 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.

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
- Acceptance criteria met for variability between replicate measurements: yes

Individual and mean OD570 values, individual and mean viability values and standard deviations 

 

Test substance identification			tissue 1	tissue 2	tissue 3	mean	SD
NC	viable tissue	mean OD570	1.517	1.811	1.723	1.684
		viability [% of NC]	90.1	107.5	102.3	100.0	9.0
test substance	viable tissue	mean OD570	1.715	1.833	1.656	1.734
		viability [% of NC]	101.8	108.9	98.3	103.0	15.4
PC	viable tissue	mean OD570	0.046	0.054	0.043	0.048
		viability [% of NC]	2.7	3.2	2.5	2.8	0.4

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.

 

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 a skin irritation potential in the EpiDerm™ in vitro skin irritation test under the test conditions chosen.

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 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

14 June 2021

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.40bis In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method

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

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm™ 200 kit supplied by MatTek In Vitro Life Science Laboratories (Bratislava, Slovakia)

Vehicle:

other: test substance was applied undiluted or minimally moistened with deionized water or PBS

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPI-200
- Tissue batch number(s): 34196
- Certificate of analysis: provided by supplier

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 min at room temperature (experiment 1) or 1 h in the incubator (experiment 2)
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The tissues were washed with sterile PBS
- Observable damage in the tissue due to washing: not described

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg / mL MTT diluent (0.3 mL per tissue)
- Incubation time: 3 h
- Spectrophotometer: Sunrise TM Absorbance Reader
- Wavelength: 570 nm
- Filter: no reference filter

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues (if applicable): EPI-200 tissue that is killed by freezing at –20°C
- N. of replicates : 2
The test substance is not able to reduce MTT directly. Therefore, an additional MTT reduction
control KC (freeze-killed control tissues) was not introduced.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA:
Corrosive potential of the test materials is predicted from the mean relative tissue viabilities
obtained after the 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 the 1-hour treatment with a test material is
decreased below 15%.
A single test composed of at least two tissue replicates should be sufficient for a test chemical
when the result is unequivocal. However, in case of borderline results such as non-concordant
replicate measurements and/or mean percent tissue viabilities of 47% - 53%
(3-minute exposure) and 14% - 17% (1-hour exposure), a second test should be considered
as well as a third one in case of discordant results between the first two tests.

ACCEPTANCE CRITERIA
- Negative conrol: 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.
- Positive control: 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%.
- Variability: 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%.

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 μL deionized water was applied first. Thereafter, a bulk volume of ca. 25 μL solid test material (ca. 37 mg) was applied with a sharp spoon and homogeneously distributed with the water, so that the surface of the epidermis model was completely covered.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL deionized water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL 8 N potassium hydroxide solution

Duration of treatment / exposure:

Experiment 1: 3 min exposure at room temperature
Experiment 2: 1 h exposure in the incubator (37 °C)

The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after
start of the application treatment.

Duration of post-treatment incubation (if applicable):

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.

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

experiment 1: 3 min exposure

Value:

83.4

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

experiment 2: 1 h exposure

Value:

96.7

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: no - Direct-MTT reduction: able to reduce MTT directly - Due to the ability of the test substance to reduce MTT directly, KC tissues were applied in
parallel. However, the results of the KC tissues did not indicate an increased MTT reduction. Thus, the KC was not used for viability calculation.
- Colour interference with MTT: Black 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 there was no increased MTT reduction.

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
- Acceptance criteria met for variability between replicate measurements: yes

Any other information on results incl. tables

Individual and mean OD570 values, individual and mean viability values, standard deviations and coefficient of variation

Exposure period: 3 min
Test substance identification			tissue 1	tissue 2	mean	SD	CV [%]
NC	viable tissue	mean OD570	1.946	2.013	1.980
		viability [% of NC]	98.3	101.7	100.0	2.4	2.4
test substance	viable tissue	mean OD570	1.592	1.709	1.650
		viability [% of NC]	80.4	86.3	83.4	4.2	5.0
PC*	viable tissue	mean OD570	0.290	0.273	0.282
		viability [% of NC]	15.2	14.3	14.8	0.6	4.4

* The PC tissues were tested in parallel within another test run (139). Thus, calculation of viability (% of NC) is based on the NC value (mean OD570: 1.905) 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.

 

Individual and mean OD570 values, individual and mean viability values, standard deviations and coefficient of variation

Exposure period: 1 h
Test substance identification			tissue 1	tissue 2	mean	SD	CV [%]
NC	viable tissue	mean OD570	1.844	1.898	1.871
		viability [% of NC]	98.6	101.4	100.0	2.0	2.0
test substance	viable tissue	mean OD570	1.870	1.747	1.809
		viability [% of NC]	99.9	93.4	96.7	4.6	4.8
PC*	viable tissue	mean OD570	0.096	0.093	0.094
		viability [% of NC]	5.2	5.0	5.1	0.1	2.6

* The PC tissues were tested in parallel within another test run (139). Thus, calculation of viability (% of NC) is based on the NC value (mean OD570: 1.838) 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 a skin corrosion potential in the EpiDerm™ in vitro skin corrosion test under the test conditions chosen.

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