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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:
4 September 2012 - 15 October 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

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:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Lanthanum trihydroxide
EC Number:
238-510-2
EC Name:
Lanthanum trihydroxide
Cas Number:
14507-19-8
Molecular formula:
H3LaO3
IUPAC Name:
lanthanum(3+) trihydroxide
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Description: Yellow powder
Storage Conditions: Room temperature in the dark

Test animals

Species:
other: in vitro Reconstructed Human Epidermis (RHE) Model
Details on test animals or test system and environmental conditions:
The EPISKIN model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

EPISKIN Model Kit
Supplier: SkinEthic Laboratories, Nice, France

Test system

Type of coverage:
other: not applicable
Preparation of test site:
other: not applicable
Vehicle:
unchanged (no vehicle)
Controls:
other: in vitro concurrent positive and negative controls were used
Amount / concentration applied:
10 mg
Duration of treatment / exposure:
A treatment period of 15 minutes was followed by a post-exposure incubation period of 42 hours.
Observation period:
Not applicable
Number of animals:
Not applicable
Details on study design:
MATERIAL PREPARATION
-Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca++ and Mg++ was used as the negative control.
-Sodium Dodecyl Sulphate (SDS) 5 % w/v aqueous dilution was used as the positive control.
-A 3 mg/mL MTT stock solution was prepared in DPBS. The stock solution was diluted to 0.3 mg/mL with assay medium when required.
-A 0.04 N concentration of hydrochloric acid in isopropanol was prepared when required.

PRE-TEST
Assessment of Direct Test Material Reduction of MTT

MTT dye metabolism, cell viability assay:
The MTT assay, a colourimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells.
One limitation of the assay is possible interference of the test material with MTT. A test material may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test material is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test material present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.

Test for Direct MTT Reduction:
The test material was checked for the ability to directly reduce MTT according to the following procedure:
10 mg of the test material was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5 % CO₂ in air for 3 hours. Untreated MTT solution was used as a control.
If the MTT solution containing the test material turns blue, it is presumed to have reduced the MTT.
The test material was shown not to directly reduce MTT.

PRE-INCUBATION (Day 0: Tissue Arrival):
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test material and each control material. The tissues were incubated at 37 °C, 5 % CO₂ in air overnight.

MAIN TEST
APPLICATION OF TEST MATERIAL 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 material for an exposure period of 15 minutes. It was applied topically to the corresponding tissues, ensuring uniform covering. Approximately 10 mg of the test material was applied to the epidermis surface (which had been previously moistened with 5 µL of sterile distilled water to improve the contact between the solid test material and the epidermis). 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 material, the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre). After 7 minutes of contact time, the SDS solution was re-spread with a pipette tip to maintain the distribution for the remainder of the contact period. The plate(s) were kept in a 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 material. 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 homogenise the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30 °C 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 plate(s). 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 EPISKIN biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 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 an homogenous coloured solution.
For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a pre-labelled 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 540 nm (without a reference filter) using the Anthos 2001 microplate reader.

INTERPRETATION OF RESULTS
-Quantitative MTT Assessment (percentage tissue viability)
For the test material, the relative mean tissue viabilities obtained after the 15 minute exposure period followed by the 42 hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way:
Relative mean viability (%) = (mean OD540 of test material / mean OD540 of negative control) x 100

Classification of irritation potential is based upon relative mean tissue viability according to the following:

Criteria for in vitro interpretation Classification
Relative Mean Tissue Viability is ≤50 % Irritant (I) R38
Relative Mean Tissue Viability is >50 % Non-Irritant (NI)

The results were evaluated according to EU labelling regulations Commission Directive 2001/59/EC for classification and labelling of dangerous items.

QUALITY CRITERIA
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:

-Positive Control:
The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was ≤40 % relative to the negative control treated tissues, and the standard deviation value of the percentage viability is ≤18 %.

-Negative Control:
The assay establishes the acceptance criterion for an acceptable test if the mean OD540 for the negative control treated tissues was ≥0.6, and the standard deviation value of the percentage viability is ≤18 %.

-Test Material:
The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤18 %.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Test material (mean)
Value:
116.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Negative control (mean)
Value:
100
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Positive control (mean)
Value:
7.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The individual and mean OD540 values, standard deviations and tissue viabilities for the test material, negative and positive controls are given in Table 1.
The relative mean viability of the test material treated tissues was 116.5 % after a 15 minute exposure period.

Any other information on results incl. tables

Direct MTT Reduction

An assessment found that the test material was not able to directly reduce MTT.

Quality Criteria

- The relative mean tissue viability for the positive control treated tissues was 7.8 % relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.3 %. The positive control acceptance criterion was therefore satisfied.

- The mean OD540 for the negative control treated tissues was 0.753 and the standard deviation value of the percentage viability was 4.8 %. The negative control acceptance criterion was therefore satisfied.

- The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 13.1 %. The test material acceptance criterion was therefore satisfied.

 

Table 1: Mean OD540 Values and Percentage Viabilities for the Negative and Positive Controls and the Test Material

Material

OD540 of Tissues

Mean OD540 of Triplicate Tissues

± SD of OD540

Relative Individual Tissue Viability (%)

Relative Mean Viability (%)

± SD of Relative Mean Viability (%)

Negative Control

0.773

 

0.753

 

0.036

102.7

 

100*

 

4.8

0.711

94.4

0.774

102.8

Positive Control

0.070

 

0.058

 

0.010

9.3

 

7.8

 

1.3

0.054

7.2

0.051

6.8

Test

Material

0.977

 

0.877

 

0.099

129.7

 

116.5

 

13.1

0.875

116.2

0.779

103.5

*The mean viability of the control group is set at 100 %

SD = Standard deviation

Applicant's summary and conclusion

Interpretation of results:
other: not classified according to EU criteria
Conclusions:
Under the conditions of this study, the test material was considered to be a non-irritant and requires no classification in accordance with EU criteria.
Executive summary:

The skin irritation potential of the test material was evaluated in vitro using the EPISKIN reconstructed human epidermis model in accordance with the standardised guidelines OECD 439 and EU Method B.46 under GLP conditions.

Triplicate tissues were treated with the test material for an exposure period of 15 minutes. At the end of the exposure period, each tissue was rinsed before being incubated for 42 hours, after which each tissue was taken for MTT loading. After MTT loading, a total biopsy of each epidermis was made and formazan crystals were extracted out of the MTT-loaded tissues. Duplicate tissues treated with Dulbecco’s Phosphate Buffered Saline with Ca++ and Mg++ served as the negative control and duplicate tissues treated with 5 % w/v aqueous Sodium Dodecyl Sulphate served as the positive control. The optical density of all treated tissues was measured at 540 nm.

The relative mean viability of the test material treated tissues was 116.5 % after the 15 minute exposure period.

Under the conditions of this study, the test material was considered to be a non-irritant and requires no classification in accordance with EU criteria.