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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
Study period:
From 26 August 2015 to 31 August 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted by a GLP accredited laboratory using OECD Testing Guideline 439. The study was conducted on the registered substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Method B.46. in vitro skin irritation: Reconstructed Human Epidermis Model Test as described in Commission Regulation (EC) No. 761/2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Cordierite
EC Number:
603-401-4
Cas Number:
1302-88-1
Molecular formula:
Mg2[Al4O3(SiO3)5]
IUPAC Name:
Cordierite
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Identification: Cordierite, sintered, synthetic
CAS Number: 1302-88-1
Physical state: Pale brown powder
Substance type: UVCB
Storage conditions: Room temperature in the dark under nitrogen

Test animals

Species:
other: EPISKIN™ Reconstructed Human Epidermis Model Kit
Strain:
other: EPISKIN™ Reconstructed Human Epidermis Model Kit
Details on test animals or test system and environmental conditions:
EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier : SkinEthic Laboratories, Lyon, France
Date received : 25 August 2015
EpiSkinTM Tissues (0.38cm2) lot number : 15-EKIN-034
Maintenance Medium lot number : 15-MAIN3-034
Assay Medium lot number : 15-ESSC-034

Test system

Type of coverage:
other: The test item was applied topically to the corresponding tissues ensuring uniform covering.
Controls:
other: Triplicate tissues treated with 10 µL of DPBS served as the negative controls and triplicate tissues treated with 10 µL of SDS 5% w/v served as the positive controls.
Amount / concentration applied:
Amount(s) applied (volume or weight with unit): 10 µL
Duration of treatment / exposure:
Exposure: 15 minutes
Post-exposure incubation: 42 hours
Observation period:
Not applicable
Number of animals:
Not applicable
Details on study design:
The negative control item, DPBS, was used as supplied.
The positive control item, SDS, was prepared as a 5% w/v aqueous solution.

Application of Test Item and Rinsing (Day 1)
2 mL of maintenance medium, warmed to approximately 37°C, was pipetted into the second column of 3 wells of the 12-well plate.
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 5 μL of sterile distilled water was topically applied to the epidermal surface in order to improve contact between the test item and the epidermis. Approximately 10 mg (26.3 mg/cm2) of the test item was then applied to the epidermal surface. Triplicate tissues treated with 10 μL of DPBS served as the negative controls and triplicate tissues treated with 10 μL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). After a 7-Minute contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test item). The plates were kept in the biological safety cabinet at room temperature for 15 minutes.
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37°C, 5% CO2 in air for 42 hours.

MTT Loading/Formazan Extraction (Day 3)
Following the 42-Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium.
1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer at -14 to -30 ºC for possible inflammatory mediator determination.
2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37°C, 5% CO2 in air. At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 μL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed.
Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10°C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

Absorbance/Optical Density Measurements (Day 6)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution.
For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 μL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 562 nm (without a reference filter) using the Anthos 2001 microplate reader.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: other: Relative mean viability
Value:
100.5
Remarks on result:
other:
Remarks:
Basis: mean. Time point: 15 minute exposure. Reversibility: other: not applicable. (migrated information)

Any other information on results incl. tables

Mean OD562 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item   OD562 of tissues  Mean OD562 of triplicate tissues  ± SD of OD562  Relative individual tissue viability (%)  Relative mean viability (%)  ± SD of Relative mean viability (%)
Negative Control Item  1.280        1.245        0.061  102.8        100*        4.9
 0.281  102.9
 0.174  94.3
       Positive Control Item  0.080 0.087               0.008  6.4        7.0        0.7
 0.084  6.7
 0.096  7.7
       Test Item  1.272        1.251        0.130  102.2        100.5        10.5
 0.112  89.3
 0.369  110.0

OD = Optical Density

SD = Standard deviation

* = The mean viability of the negative control tissues is set at 100%

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The Relative mean tissue viability calculated as a percentage of the negative control was 100.5% after the 15-minute exposure followed by the 42-hour post-exposure. The test item did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.
Executive summary:

The in vitro skin irritation of the test substance was determined in accordance with the OECD Guideline for Testing of Chemicals 439. The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density was measured at 562 nm.

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The Relative mean tissue viability calculated as a percentage of the negative control was 100.5 after the 15-minute exposure followed by the 42-hour post-exposure. The test item did not meet the criteria for classification according to Regulation (EC) No.1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.