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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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
June 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted study according to GLP
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: Draft OECD guideline - in vitro skin irritation: Reconstructed human Epidermis (RhE) test method
Qualifier:
according to
Guideline:
other: Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.46 “In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test "
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Chemical name: Ethylenediaminetetraacetic acid, manganese disodium complex
Purity: 92.3%
Batch no: CFC 9380
Expiry date: 31 August 2012

Test animals

Species:
other: reconstructed human skin
Details on test animals and environmental conditions:
No test animals used.

Test system

Type of coverage:
other: not applicable
Preparation of test site:
other: not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: negative and positive control
Amount / concentration applied:
Skin tissue was moistened with 5 µl of Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact to the tissue and at least 10 mg of the solid test substance was applied directly on top of the skin tissue. EDTA-MnNa2 was spread to match the size of the tissue
Duration of treatment / exposure:
15 min
Observation period:
42 h
Number of animals:
3 samples each for negative and positive control and for test sample

Negative control: Phosphate buffered saline (PBS, Invitrogen Corporation, Breda, The Netherlands).
Positive control: 5% (aq) Sodium dodecyl sulphate (SDS, Sigma Aldrich, Zwijndrecht, The Netherlands) [CAS Number 151-21-3].
Details on study design:
Tissues: On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 24 hours at 37°C. The level of Maintenance Medium was just beneath the tissue (Figure 1). Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Nice, France.

MTT medium: MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/ml in PBS) diluted (10x) in Assay medium (final concentration
0.3 mg/ml).

Environmental conditions: All incubations, with the exception of the test substance incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 63 - 86%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 37.4 - 37.8°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.

Test for reduction of MTT by the test substance: EDTA-MnNa2 was checked for possible direct MTT reduction before the study was started. To assess the ability of the test substance to reduce MTT, approximately 10 mg of test substance was added to a 12 well plate filled with 2 ml MTT solution (0.3 mg/ml in PBS). The mixture was incubated for approximately 3 hours at 37°C. A negative control, sterile Milli-Q water was tested concurrently.

Application/Treatment of the test substance: The test was performed on a total of 3 tissues per test substance together with negative and positive controls. Ten µl of the undiluted test substance was added into 12-well plates on top of the skin tissues. Three tissues were treated with 10 µl PBS (negative control) and 3 tissues with 10 µl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. After rinsing the cell culture inserts were each dried carefully. The skin tissues were kept in new 12-well plates on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

Cell viability measurement: After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 ml MTT-medium (0.3 mg/ml). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 µl isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the Multiskan Spectrum (Thermo Labsystems). Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: overall irritation score
Value:
72
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 42 h. Max. score: 100.0. Remarks: negative control was set at 100%; test sample 72%, positive control 9%. (migrated information)

In vivo

Irritant / corrosive response data:
The relative mean tissue viability obtained after 15 minutes treatment with EDTA-MnNa2 compared to the negative control tissues was 72%. Since the mean relative tissue viability for EDTA-MnNa2 was above 50% EDTA-MnNa2 is considered to be non-irritant. The positive control had a mean cell viability after 15 minutes exposure of 9%.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: expert judgment
Conclusions:
The relative mean tissue viability obtained after 15 minutes treatment with EDTA-MnNa2 compared to the negative control tissues was 72%. Therefore, EDTA-MnNa2 is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report

Executive summary:

This report describes the ability of EDTA-MnNa2 to induce skin irritation on a human three dimensional epidermal model (EPISKIN Standard model (EPISKIN-SMTM)). The possible skin irritation potential of EDTA-MnNa2 was tested through topical application for 15 minutes. The study procedures described in this report were based on the most recent OECD and EC guidelines. Batch CFC 9380 of EDTA-MnNa2 is an off-white powder. Skin tissue was moistened with 5 µl of Milli-Q water and at least 10 mg of EDTA-MnNa2 was applied directly on top of the skin tissue for 15 minutes. After a 42 hour incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with EDTA-MnNa2 compared to the negative control tissues was 72%. Since the mean relative tissue viability for EDTA-MnNa2 was above 50% after 15 minutes treatment EDTA-MnNa2 is considered to be non-irritant. The positive control had a mean cell viability after 15 minutes exposure of 9%. The absolute mean OD570(optical density at 570 nm) of the negative control tissues was according to the acceptability criteria of the assay. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly. It was concluded that this test is valid and that EDTA-MnNa2 is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.