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

Endpoint summary

Description of key information

Skin Irritation Test (OECD 439, GLP): not irritating

Skin Corrosion Test (OECD 431, GLP): not corrosive

Eye Irriation Test (OECD 492, GLP): not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

14 June 2021

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)

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:

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

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

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

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.

 

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.

Reference 2

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.

Reference 3

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 24 Nov 2021 to 26 Nov 2021

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
Expiry date: 28 Feb 2024
pH value: ca. 8 (undiluted test substance moistened with deionized water)
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 able to reduce MTT directly. Therefore, an additional MTT reduction
control KC (freeze-killed control tissues) was introduced. However, the results of the KC tissues
indicate only minimally increased MTT reduction. Thus, the final relative mean viabilities of the
test substance are not substantially affected by the KC corrections.

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%.
- Killed Cointrols (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.

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:

97.5

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/colorimetric test: 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. 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
	KC tissue	mean OD570	0.050	0.052	0.044	0.049
		viability [% of NC]	3.0	3.1	2.6	2.9	0.2
test substance	viable tissue	mean OD570	1.668	1.646	1.608	1.641
		viability [% of NC]	99.1	97.8	95.5	97.5	1.8
	KC tissue	mean OD570 KC NC corrected	-0.010	-0.003	-0.005	-0.006
		viability [% of NC]	-0.6	-0.2	-0.3	-0.3	0.2
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

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.
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.
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.

Reference 4

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 06 Oct 2021 to 07 Oct 2021

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

Version / remarks:

14 June 2021

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)

Specific details on test material used for the study:

Purity: 100% UVCB
Expiry date: 28 Feb 2024
pH value: ca. 8 (undiluted test substance moistened with deionized water)
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:

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 able to reduce MTT directly. Therefore, an additional MTT reduction
control KC (freeze-killed control tissues) was introduced. However, the results of the KC tissues
indicate only minimally increased MTT reduction. Thus, the final relative mean viabilities of the
test substance are not substantially affected by the KC corrections.

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%.%.
- Killed Cointrols (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.

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

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

experiment 1: 3 min exposure

Value:

86.4

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

experiment 2: 1 h exposure

Value:

98.5

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

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
	KC tissue	mean OD570	0.166	0.117	0.142
		viability [% of NC]	8.4	5.9	7.1	1.8	24.5
test substance	viable tissue	mean OD570	1.679	1.742	1.710
		viability [% of NC]	84.8	88.0	86.4	2.3	2.6
	KC tissue	mean OD570 KC NC corrected	-0.060	-0.075	-0.068
		viability [% of NC]	-3.0	-3.8	-3.4	0.5	-15.7
PC	viable tissue	mean OD570	0.290	0.273	0.282
		viability [% of NC]	15.2	14.3	14.8	0.6	4.4

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.
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.
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
	KC tissue	mean OD570	0.058	0.068	0.063
		viability [% of NC]	3.1	3.6	3.4	0.4	11.2
test substance	viable tissue	mean OD570	1.864	1.823	1.843
		viability [% of NC]	99.6	97.4	98.5	1.5	1.6
	KC tissue	mean OD570 KC NC corrected	-0.028	-0.025	-0.026
		viability [% of NC]	-1.5	-1.3	-1.4	0.1	-9.4
PC	viable tissue	mean OD570	0.096	0.093	0.094
		viability [% of NC]	5.2	5.0	5.1	0.1	2.6

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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

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

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)

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

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

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%.

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

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.

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.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 08 Oct 2021 to 10 Mar 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

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)

Specific details on test material used for the study:

Purity: 100% UVCB
Expiry date: 28 Feb 2024
pH value: ca. 8 (undiluted test substance moistened with deionized water)
Physical state / color: solid / black

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

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. 52 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 52 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 as described in section 3.6.
Based on the result of the pretest, it was judged that application of killed control tissues is
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%.
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.

Irritation parameter:

mean percent tissue viability 

Run / experiment:

mean of 2 replicates with 6 h exposure each

Value:

78.6

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:

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

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
	KC tissue	mean OD570	0.027	0.029	0.028
		viability [% of NC]	1.4	1.6	1.5	0.1
test substance	viable tissue	mean OD570	1.509	1.448	1.479
		viability [% of NC]	80.2	76.9	78.6	3.3
	KC tissue	mean OD570 KC NC corrected	-0.010	-0.009	-0.010
		viability [% of NC]	-0.5	-0.5	-0.5	0.1
PC	viable tissue	mean OD570	0.478	0.840	0.659
		viability [% of NC]	26.1	45.9	36.0	19.8

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. The KC was not used for viability calculation.
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 an eye irritation potential in the
EpiOcular™ in vitro eye irritation test under the test conditions chosen.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The following data are available:

• Skin Irritation/Corrosion
  
  
  
  • In vitro: Skin Irritation Test (OECD 439, GLP): not irritating; Skin Corrosion Test (OECD 431, GLP): not corrosive
  
  
  • In vivo: no study available
  
  
  
  


• Eye Irritation/Corrosion
  
  
  
  • In vitro: Eye Irritation Test (OECD 492, GLP): not irritating
  
  
  • In vivo: no study available
  
  
  
  


• Respiratory Irritation/Corrosion
  
  
  
  • In vitro/in vivo: no experimental data available
  
  
  
  

 

In Vitro Skin Irritation/Corrosion Test

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

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 quotient of the values indicates the relative tissue viability.

Since the test substance is a black solid, a pretest was conducted in order to investigate whether the color of the test substance may interfere with the assay. It was demonstrated that the color of the test substance did not interfere with the colorimetric test. However, in order to avoid contamination of the extract solution with the compound residues, the affected tissues treated with the test substance were extracted only from below without piercing.

The following results were obtained in the EpiDerm™ Skin Irritation Test (SIT; OECD 439, GLP):

The mean relative 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 97.5% and 103.0%. The acceptance criteria for the variability of the tissues are met.
Treatment of the tissues with the positive control 5% SDS showed a mean relative viability of 2.8%, reflecting the expected sensitivity of the tissues.
The mean OD570 of the negative control (PBS) fulfills the acceptance criteria and demonstrates the validity of the assay.
The obtained result of the Skin Irritation Test (SIT) did not indicate a skin irritation potential of the test substance.

Based on the prediction model for the test employed, the test substance is considered not irritating under the conditions of the assay.

 

The following results were obtained in the EpiDerm™ Skin Corrosion Test (SCT; OECD 431, GLP):

For the skin corrosion test, two EpiDerm™ tissues were incubated with the test substance for 3 minutes and 1 hour, each. In addition to the test substance, 50 μL per tissue of a negative control (deionized water) and of a positive control (8 N KOH) were applied to two tissues each per exposure period.

The mean relative viability of the tissues treated with the test substance determined after an exposure period of 3 minutes was 86.4% and 83.4%, and it was 98.5% and 96.7% after an exposure period of 1 hour. The acceptance criteria for the variability of the tissues are met.

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.

Application of the positive control 8 N KOH showed a mean relative viability of the tissues of 14.8% (3-minute exposure) and 5.1% (1-hour exposure), reflecting the expected sensitivity of the tissues.

The mean OD570 of the negative control (deionized water) fulfills the acceptance criteria and demonstrates the validity of the assay.

Based on the prediction model for the test employed, the test substance is considered not corrosive under the conditions of the assay.

 

In Vitro Eye Irritation Test (OECD 492, GLP)

The potential of the test substance to cause ocular irritation was assessed by a single topical application of ca. 50 μL bulk volume undiluted test substance to a reconstructed three-dimensional human cornea model (EpiOcular™), so that the surface of the cornea model was completely covered with the test substance.

Two EpiOcular™ tissues were incubated with the test substance for 6 hours followed by an 18-hour post-incubation period. In addition to the test substance, 50 µL per tissue of a negative control (deionized water) and a positive control (methyl acetate) were applied to two tissues each. 

Tissue destruction was determined by measuring the metabolic activity of the tissues 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.

The following results were obtained in the EpiOcular™ eye irritation test:

Since the test substance is a black solid, a pretest was conducted in order to investigate whether the color of the test substance may interfere with the assay. It was demonstrated that the color of the test substance did not interfere with the colorimetric test. However, in order to avoid contamination of the extract solution with the compound residues, the affected tissues treated with the test substance were extracted only from below without piercing.

The mean relative viability of the tissues treated with the test substance was 78.6% and 74.4%. The variability between the results of the tissues is within the acceptance range.

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.

Application of the positive control methyl acetate showed a mean relative viability of the tissues of 36.0%, reflecting the expected sensitivity of the tissues.

The mean OD570 of the negative control (deionized water) fulfills the acceptance criteria and demonstrates the validity of the assay.

Based on the prediction model for the test employed, the test substance is considered not irritating under the conditions of the assay.

Justification for classification or non-classification

Based on substance stecific data and applying the criteria laid down in Regulation (EC) No. 1272/2008, no classification for skin or eye irritation or corrosion is warranted.