Slags, phosphorus-manufg.

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 February 2010 - 08 February 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test was conducted according to OECD Draft Proposal for a New Guideline: In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method, December 2009, under GLP Standards, and QA.

Justification for data waiving:

other:

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Draft Proposal for a New Guideline: In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method, December 2009

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

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 ", August 2009

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Remarks:

Statement of Compliance

Test material

Test material information

Constituent 1

Reference substance name:

Slags, phosphorus-manufg.

EC Number:

273-732-3

EC Name:

Slags, phosphorus-manufg.

Cas Number:

69012-32-4

Molecular formula:

not applicable due to the multi constituent mineral structure of the substance

IUPAC Name:

aluminium(3+) heptacalcium magnesium(2+) λ²-iron(2+) disodium tris([(trioxidosilyl)oxy]silanetris(olate)) oxosilanebis(olate) hydroxysilanoylolate difluoride

Details on test material:

- Name of test material (as cited in study report): Phosphorous slag
- Physical state: powder
- Analytical purity: confidential
- Lot/batch No.: not indicated
- Stability under test conditions: stable
- Storage condition of test material: At room temperature in the dark

Test animals

Species:

other: not relevant: in vitro test

Strain:

other: Human skin tissue: EPISKIN Standard Model

Details on test animals or test system and environmental conditions:

EPISKIN STANDARD MODEL
- Source: SkinEthic Laboratories, Nice, France
On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 2 hours and 45 minutes at 37°C. The level of Maintenance Medium was just beneath the tissue.

ENVIRONMENTAL CONDITIONS: All incubations, with the exception of the test substance incubation of 15 minutes at room temperature:
- Temperature (°C): 37.0 ± 1.0 (actual range 37.0 - 37.6)
- Humidity (%): 80 - 100 (actual range 65 - 90),
- 5.0 ± 0.5% CO2 in air
- Photoperiod: dark

Test system

Type of coverage:

other: not applicable

Preparation of test site:

other: Skin tissue was moistened with 5 ul of Milli-Q water 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. Phosphorous slag was spread to match the size of the tissue.

Vehicle:

unchanged (no vehicle)

Controls:

other: Phosphate buffered saline (PBS)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): at least 10 mg

POSITIVE CONTROL: 5% (aq) Sodium dodecyl sulphate (SDS) - 10 ul

NEGATIVE CONTROL: Phosphate buffered saline (PBS) - 10 ul

Duration of treatment / exposure:

Exposure: 15 minutes
Incubation: 42 ± 2 hours

Observation period:

Not applicable

Number of animals:

9 skin tissues: negative control, positive control and test item: 3 each

Details on study design:

TEST SITE: EXPOSURE OF THE TISSUES
Skin tissue was moistened with 5 ul of Milli-Q water 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 into 12-well plates. Phosphorous slag was spread to match the size of the tissue.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The tissues were washed with phosphate buffered saline to remove residual test substance
- Time after start of exposure: 15 minutes

SCORING SYSTEM: Determination of the cytotoxic (irritancy) effect is performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment. 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.
For Evaluation Criteria see 'Other information on materials and methods'.

TOOL USED TO ASSESS SCORE: The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the Multiskan Spectrum (absorption/optical density measurement: OD570).

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: other: Absorption (Optical Density - OD570)

Value:

ca. 0.747

Remarks on result:

other:

Remarks:

Basis: mean Triplicate exposures. Max. score: 0.889. Reversibility: other: not applicable. Remarks: SD: ± 0.003; The values are corrected for the non-specific MTT reaction. (migrated information)

Results 2

Irritation / corrosion parameter:

other: other: Tissue viability

Value:

ca. 86

Remarks on result:

other:

Remarks:

Basis: mean. Reversibility: other: not applicable. Remarks: percentage of negative control. (migrated information)

In vivo

Other effects:

Phosphorous slag was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because a color change was observed it was concluded that Phosphorous slag did interact with MTT. In addition to the normal procedure, 3 killed tissues treated with test substance and 3 killed non treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by Phosphorous slag was 13% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test substance treated viable tissues.

Any other information on results incl. tables

The positive control had a mean cell viability of 20% after 15 minutes exposure. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly.

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 obtained after 15 minutes treatment with Phosphorous slag compared to the negative control tissues was 86%. Since the mean relative tissue viability for Phosphorous slag was above 50% after 15 minutes treatment Phosphorous slag is considered to be non-irritant. It is concluded that this test is valid and that Phosphorous slag 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 Phosphorous slag to induce skin irritation on a human three dimensional epidermal model (EPISKIN Standard model (EPISKIN-SM)). The possible skin irritation potential of Phosphorous slag was tested through topical application for 15 minutes. The study procedures described in this report were based on the OECD Draft Proposal for a New Guideline: In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method, December 2009.

Skin tissue was moistened with 5 ul Milli-Q water and at least 10 mg of Phosphorous slag was applied directly on top of the skin tissue. 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. As phosphorous slag directly interacted with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), 3 killed test substance treated tissues and 3 killed non treated tissues were used for the cytotoxicity evaluation with MTT, in addition to the normal procedure.

The non-specific reduction of MTT by Phosphorous slag was 13% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test substance treated viable tissues. 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 Phosphorous slag compared to the negative control tissues was 86%.

Since the mean relative tissue viability for Phosphorous slag was above 50% after 15 minutes treatment Phosphorous slag is considered to be non-irritant. The positive control had a mean cell viability after 15 minutes exposure of 20%. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly.

Finally, it is concluded that this test is valid and that Phosphorous slag is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.