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Toxicological information

Skin irritation / corrosion

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Administrative data

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:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Qualifier:
according to
Guideline:
other: OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Zinkphthalocyaninsulfonat
- Lot/batch No.: 176647

Test animals

Species:
other: EpiDerm™ 200 kit
Details on test animals and environmental conditions:
TEST SYSTEM:
- Source: MatTek Corp., Ashland MA, USA

Test system

Controls:
other: historical control values
Amount / concentration applied:
Corrosion test:
From the day of arrival in the laboratory, tissues were kept in the refrigerator. At least 1 hour but not more than 1.5 hours before test-substance application, tissues were transferred to 6- well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. The preincubation medium was replaced with fresh medium immediately before application.
Two tissues per exposure time (3 minutes at room temperature or 1 hour in the incubator, as a rule) and test group (test material, negative control and positive control; 12 tissues per test) were used.
25 μL highly de-ionized water was applied first. Thereafter, a bulk volume of 25 μL of the solid test material was applied with a sharp spoon and homogeneously distributed with the water.
Control tissues were concurrently applied with 50 μL of highly de-ionized water (negative control, NC) or with 50 μL of 8 n potassium hydroxide (positive control, PC).

Irritation test:
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued for 18 ± 3 hours.
Three tissues were treated with the test substance, the PC and NC, respectively.
25 μL sterile PBS was applied first. Thereafter, a bulk volume of 25 μL of the solid test material was applied with a sharp spoon and homogeneously distributed together with the fluid.
Control tissues were concurrently applied with 30 μL of sterile PBS (negative control, NC) or with 30 μL of 5% SDS (positive control, PC). A nylon mesh was placed carefully onto the tissue surface afterwards.
Duration of treatment / exposure:
Corrosion test:
3 minutes or 1 hour

Irritation test:
The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator.
Observation period:
Corrosion test:
3 hours

Irritation test:
The tissues were incubated in the incubator at 37°C for 24 ± 2 hours. After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period.
Details on study design:
Corrosion test:
The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after start of the application treatment. 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 by MTT solution and tissues were incubated for 3 hours.
After incubation, tissues were washed with PBS and the formazan produced by the tissues was extracted with isopropanol over night at room temperature. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

Irritation test:
After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation.
The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol for at least 2 hours at room temperature on a plate shaker. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

Data evaluation

Principle
The OD570 values determined for the various tissues are measures of their viability. The quotient of the OD570 of tissues treated with the test material and the mean OD570 values of the NC (percent of control) is used for evaluating whether or not a test material is corrosive or irritant.

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

Tissue viability
The quantification of tissue viability is presented as the quotient of the mean OD570 divided by the respective OD570 NC value in percent for each exposure time.

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

Assay acceptance criterion for the negative control (NC)
The absolute OD570 of the negative control tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is ≥ 1.0. The mean OD570 of the NC should not exceed 2.5.

Assay acceptance criterion for the positive control (PC)
Corrosion test: Potassium hydroxide as 8.0 normal ready made solution is used as positive reference. A 3-minute treatment with 8.0 n KOH usually reveals a mean relative tissue viability of ~20%. An assay is acceptable if mean relative tissue viability of the 3 min positive control is ≤ 30%.
Irritation test: 5% SDS is used as PC and reflects the sensitivity of the tissues used in the test conditions. A viability of ≤ 20% is acceptable.

Assay acceptance criterion for tissue variability
For every treatment, 2 tissues (corrosion test) or 3 tissues (irritation test) are treated in parallel. The inter-tissue variability is considered to be acceptable if the difference of the OD570 values of the two tissues is ≤ 0.3 (corrosion test) or if the SD of %-viability is ≤ 20 (irritation test).

EVALUATION OF RESULTS
Corrosive potential of the test materials is predicted from the mean relative tissue viabilities obtained after 3 min treatment compared to the negative control tissues concurrently treated with highly de-ionized water. A chemical is considered as "corrosive", if the mean relative tissue viability after 3 min treatment with a test material is decreased below 50%. In addition, those materials with a viability of ≥ 50% after 3 min treatment are considered as "corrosive" if the mean relative tissue viability after 1 hour treatment with a test material is decreased below 15%.

Mean tissue viability
(% of negative control) Prediction
3 min: < 50 corrosive
3 min: ≥ 50 and 1 hour: < 15 corrosive
3 min: ≥ 50 and 1 hour: ≥ 15 non-corrosive

Although the method is not finally validated for categorizing the severity of corrosivity according to certain classification and labeling systems, it is suggested to use the most stringent category for test substances leading to viabilities below 50% after 3 min treatment.

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

Mean tissue viability
(% of negative control) Prediction
≤ 50 irritant
> 50 non-irritant

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other: other: viability [% of NC]
Value:
94
Remarks on result:
other:
Remarks:
Basis: mean. Remarks: corrosion, exposure: 3 min, mean OD570: 1.670. (migrated information)
Irritation / corrosion parameter:
other: other: viability [% of NC]
Value:
104
Remarks on result:
other:
Remarks:
Basis: mean. Remarks: corrosion, exposure: 1 h, mean OD570: 1.651. (migrated information)
Irritation / corrosion parameter:
other: other: viability [% of NC]
Value:
102
Remarks on result:
other:
Remarks:
Basis: mean. Remarks: irritation, mean OD570: 2.024. (migrated information)

In vivo

Irritant / corrosive response data:
In both tests blue discolorations were noticed on all tissues treated with the test substance after washing. However, this did not interfere with the MTT assay as neither direct formazan production nor extraction of the color into the isopropanol was observed.

Based on the observed results and applying the evaluation criteria it was concluded, that Zinkphthalocyaninsulfonat does not show a skin irritation potential in the EpiDerm™ skin corrosion/irritation test under the test conditions chosen.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU