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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:
2014-04-22 to 2014-07-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Aluminium nitride Grade C
- CAS No.: 24304-00-5
- Physical state: solid
- color: white
- pH 8 (50 g/l at 20°C)
- Storage condition of test material: at room temperature, protected from light

Test animals

Species:
other: human epidermis model: EpiSkin
Details on test animals and environmental conditions:
Test system:
The test was carried out with the reconstituted three-dimensional human skin model EPISKIN-SM™ (SkinEthic). This skin model consists of normal (non-cancerous), adult human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Test system

Vehicle:
water
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg (26.3 mg/cm²) + 5 µl Aqua dest.

PREPARATION OF THE TEST ITEM:
Firstly, 5 μL distilled water (aqua dest.) were applied by a pipette to the epidermal surface in order to improve further contact between the powder and the epidermis. The water was gently spread with the pipette. Afterwards, approximately 10 mg (26.3 mg/cm²) of the powder were applied to the epidermis surface.

CONTROLS:
Controls were set up in parallel to the test item cultures in order to confirm the validity of the test.
Negative control: Phosphate Buffered Saline (PBS; Gibco, Cat. No. 14040-091, Lot No. 1528370).
Positive control: 5% sodium dodecyl sulfate (SDS; AppliChem, Art.-No. A7249,0250, CAS No.: 151-21-3, Lot No. 1X002858) in Aqua dest.
Duration of treatment / exposure:
15 ± 0.5 min.
Details on study design:
EXPERIMENTAL PROCEDURE
Upon receipt of the EPISKIN-SMTM, the tissues were transferred into 12-well plates containing 2 mL prewarmed maintenance medium per well. The 12-well plates were incubated in a humidified incubator at 37 ± 1 °C, 5.0% CO2 for at least 24 h.
After this pre-incubation the tissues were treated in triplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. Then the tissues were incubated at room temperature for 15 ± 0.5 min. Afterwards, the tissues were washed with PBS to remove any residual test item. Excess PBS was removed by blotting bottom with blotting paper. The inserts were placed in a prepared 12-well plate containing 2 mL prewarmed fresh maintenance medium and post-incubated at 37 ± 1 °C, 5.0% CO2 for 42 ± 1 h.
After this incubation period the plates were placed for 15 ± 2 min. on a plate shaker. Then the inserts were transferred in a prepared 12-well plate containing 2 mL prewarmed MTT medium and further incubated for 3 h ± 5 min. at 37 ± 1 °C, 5.0% CO2.
After the 3 h MTT incubation period the tissues were placed on blotting paper to dry the tissues. Afterwards a total biopsy of the epidermis by using the special biopsy punch was performed and the epidermis was separated from the collagen matrix with the aid of forceps. Both parts (epidermis and collagen matrix) were transferred into suitable tubes and 500 μL of acidic isopropanol were added. Extraction was carried out protected from light over the weekend at 2 - 8°C.
At the end of the formazan extraction period the tubes were mixed by vortexing until solution colour became homogeneous.
If any visible cell/tissue fragments were in suspension, the tubes were centrifuged at 500 rpm to eliminate the fragments and avoid further possible interference with the absorbance readings.
Per each tissue 2 x 200 μL aliquots of the extract were transferred into a 96-well plate and OD was measured at 550 nm without reference wavelength in a plate spectrophotometer.

SCORING SYSTEM:
Irritant potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with PBS. The test item is considered to be irritant to skin in accordance with regulation EC 1272/2008 and UN GHS Category 2 (17), if the tissue viability after 15 min of exposure and 42 h of post-incubation is less or equal to 50%. The test substance may be considered as non-irritant to skin in accordance with UN GHS No Category if the tissue viability after exposure and post-treatment incubation is higher than 50%.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other: other: mean relative tissue viability
Value:
104.3
Remarks on result:
other:
Remarks:
Basis: mean triplicates. Time point: 15 min. Max. score: 100.0. Remarks: max score: 100% viability obtained by the negative control. (migrated information)
Irritation / corrosion parameter:
other: other: mean relative tissue viability
Value:
25.6
Remarks on result:
other:
Remarks:
Basis: mean triplicates. Time point: 15 min. Max. score: 100.0. Remarks: positive control; max score: 100% viability obtained by the negative control. (migrated information)

Any other information on results incl. tables

Pre-experiment:

The mixture of 10 mg test item per 2 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple.

The mixture of 10 mg of the test item per 90 μl aqua dest. showed no colouring detectable by unaided eye-assessment.

Results of the main experiment:

Table 1: Experimental Results

Name

Negative Control

Positive Control

Test Item

Tissue

1

2

3

1

2

3

1

2

3

Absolute OD550

0.780

0.761

0.727

0.260

0.158

0.288

0.751

0.946

0.774

0.805

0.801

0.780

0.249

0.154

0.277

0.735

0.878

0.759

OD550(blanc corrected)

0.737

0.717

0.684

0.217

0.115

0.245

0.707

0.903

0.731

0.762

0.758

0.736

0.206

0.111

0.233

0.692

0.835

0.716

Mean OD550of the duplicates (blanc corrected)

0.749

0.738

0.710

0.211

0.113

0.239

0.700

0.869

0.724

Total mean OD550of 3 replicate tissues (blanc corrected)

0.732*

0.188

0.764

SD OD550

0.029

0.060

0.085

relative tissue viability [%]

102.3

100.7

97.0

28.8

15.4

32.7

96.6

118.6

98.8

Mean relative tissue viability [%]

100.0

25.6**

104.3

SD tissue viability [%]***

2.7

9.1

12.5

*             corrected mean OD550of the negative control corresponds to 100% absolute tissue viability.

**          mean relative tissue viability of the 3 positive control is ≤ 40%

***        The standard deviation (SD) obtained from the three tested tissues is ≤ 18%

 

Table 2: Quality Criteria

 

value

Cut off

pass / fail

mean OD550 nmblank

0.043

< 0.1

pass

mean absolute OD550 nmNC

0.775

0.6 ≤ NC ≤ 1.5

pass

mean % viability PC

25.6

≤ 40%

pass

SD of viability

2.7 – 12.5%

≤ 18%

pass

 

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In this study under the given conditions the test item showed no irritant effects in an validated in vitro system (EPISKIN). The test item is classified as ‘non-irritant’ in accordance with UN GHS ‘No Category’.
Executive summary:

The potential for the test item to induce skin irritation was tested by using the three dimensional human skin model EpiSkin-SMTM (SkinEthic) comprising a reconstructed human epidermis with functional stratum corneum. Ten (10) mg Aluminium nitride grade C was applied directly atop the EpiSkin-SMTM tissue for 15 min. followed by 42 h post incubation period and immediate determination of cytotoxic effects via MTT reduction assay.

Irritant potential from the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with PBS.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was ≥ 50% (104.3%) after 15 min treatment and 42 h post incubation.

The controls confirmed the validity of the study. The mean OD550 of the six blank values was < 0.1. The mean absolute OD550 of the three negative control tissues was ≥ 0.6 and ≤ 1.5. The mean relative tissue variability (% negative control) of the positive control was ≤ 40% (25.6%).

In this study under the given conditions the test item showed no irritant effects. The test item is classified as ‘non-irritant’ in accordance with UN GHS ‘No Category’.