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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:
12 - 18 Jun 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
Aluminium dihydrogen triphosphate
EC Number:
237-714-9
EC Name:
Aluminium dihydrogen triphosphate
Cas Number:
13939-25-8
Molecular formula:
AlH2O10P3
IUPAC Name:
aluminium dihydrogen triphosphate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Aluminium dihydrogen triphosphate
- Physical state: off white powder
- Analytical purity: assay min. 95.0% (based on min. 16.5% Al2O3 and min. 72.5% P2O5)
- Purity test date: 15.06.2012
- Lot/batch No.: MV 500
- Storage condition of test material: at room temperature in the dark

Test animals

Species:
human
Strain:
other: EPISKIN™ reconstructed human epidermis model
Details on test animals or test system and environmental conditions:
TEST SKIN MODEL
- Source: SkinEthic Laboratories, Lyon, France
- Description: 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 of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
ADAPTATION TO CELL CULTURE CONDITIONS
2 mL of maintenance medium, warmed to appr. 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 item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.
INCUBATION CONDITIONS (Incubator)
- Temperature (°C): 37
- CO2 gas concentration (%): 5

Test system

Type of coverage:
other: open in vitro system
Preparation of test site:
other: intact reconstructed human epidermis
Vehicle:
unchanged (no vehicle)
Controls:
other: concurrent control tissues treated with Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca2+ and Mg2+ served as negative controls, positive controls were exposed to 5% SDS
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 10 mg
- Other: The epidermis surface had previously been moistened with 5 μl of sterile distilled water to improve contact between the solid test item and the epidermis.
NEGATIVE CONTROL
- Negative control: Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca2+ and Mg2+
- Amount(s) applied: 10 µL
POSITIVE CONTROL
- Positive control substance: Sodium Dodecyl Sulphate (SDS), 5% w/v
- Amount(s) applied: 10 µL
Duration of treatment / exposure:
15 min
Observation period:
Not applicable. Post-treatment incubation period: 42 h
Number of animals:
Not applicable.The test was performed in triplicates for each treatment and control group.
Details on study design:
TEST SITE
- Area of exposure: ca. 0.38 cm²
- % coverage: 100%

REMOVAL OF TEST SUBSTANCE
- Washing: At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing Dulbecco's Phosphate Buffered Saline (DPBS) with Ca2+ and Mg2+. 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 37ºC, 5% CO2 in air for 42 h.
- Time after start of exposure: 15 min

CELL VIABILITY MEASUREMENTS
- Method: colourimetric MTT reduction assay
- Principles of method: Cell viability is measured by enzymatic 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. The formazan product is extracted from the tissue with acidified isopropanol and the optical density of the extracts is measured at 540 nm using acidified isopropanol as blank.
- Time after exposure: 42 h (MTT loading/Formazan extraction) and on Day 6 (Absorbance/Optical density measurements)

INTERPRETATION OF RESULTS
For the test item, the relative mean tissue viabilities obtained after the 15 min exposure period followed by the 42 h post-exposure incubation period were compared to the mean of the negative control tissues (n = 3). The relative mean viabilities were calculated as follows:

Relative mean viability (%) = (mean OD540 of test item / mean OD540 of negative control) x 100

Classification of irritation potential was based upon relative mean tissue viability following the 15 min exposure period followed by the 42 h post-exposure incubation period according to the following criteria:

Relative mean tissue viability ≤ 50%: Irritant (Category 2 according to Regulation (EC) No. 1272/2008 (CLP) and UN-GHS)
Relative mean tissue viability > 50%: Non-irritant (Not classified according to Regulation (EC) No. 1272/2008 (CLP) and UN-GHS)

QUALITY CRITERIA
The results of the assay were 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 item: 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 tisues is ≤ 18%.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
other: other: relative cell viability (%)
Value:
100
Remarks on result:
other:
Remarks:
Basis: mean value negative control. Time point: 15 min + 42 h post-exposure. Reversibility: other: reversibility not applicable. Remarks: standard deviation: 6.2%. (migrated information)
Irritation / corrosion parameter:
other: other: relative cell viability (%)
Value:
8.6
Remarks on result:
other:
Remarks:
Basis: mean value positive control. Time point: 15 min + 42 h post-exposure. Reversibility: other: reversibility not applicable. Remarks: standard deviation: 1.0%. (migrated information)
Irritation / corrosion parameter:
other: other: relative cell viability (%)
Value:
128.1
Remarks on result:
other:
Remarks:
Basis: mean value test material. Time point: 15 min + 42 h post-exposure. Reversibility: other: reversibility not applicable. Remarks: standard deviation: 4.5%. (migrated information)

In vivo

Irritant / corrosive response data:
The mean OD540 values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in Table 1. The mean viabilities and standard deviations of the test item and positive control, relative to the negative control are also given in Table 1.
The relative mean viability of the test item treated tissues was 128.1% after a 15-minute exposure period.
Other effects:
The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.

Any other information on results incl. tables

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 8.6% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.0%. The positive control acceptance criterion was therefore satisfied.

The mean OD540 for the negative control treated tissues was 0.659 and the standard deviation value of the percentage viability was 6.2%. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 4.5%. The test item acceptance criterion was therefore satisfied.

Table1 : Mean OD540 Values and Percentage Viabilities for the Negative Control Material, Positive Control Material and Test Material

Material

OD540of tissues

Mean OD540of triplicate tissues

±SDof OD540

Relative individual tissue viability (%)

Relative mean viability (%)

± SD of Relative mean viability (%)

Negative Control Material¤

0.666

0.659

0.041

101.1

100*

6.2

0.696

105.6

0.615

93.3

Positive Control Material¤

0.059

0.056

0.006

9.0

8.6

1.0

0.061

9.3

0.049

7.4

Test Material

0.836

0.844

0.030

126.9

128.1

4.5

0.819

124.3

0.877

133.1


SD=    Standard deviation

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

¤ = Control group shared with Harlan Laboratories Ltd Project numbers 41200853, 41200860,41200861, 41200866, 41200871, 41200880 and 41201543


Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information in vitro Criteria used for interpretation of results: EU
Conclusions:
The relative mean viability of the test item treated tissues was 128.1% after a 15-minute exposure period. Under the conditions of this study, the test item is thus considered to be not irritating in vitro. Therefore, the test item does not meet the criteria for classification according to Regulation (EC) No 1272/2008 (CLP) or the Globally Harmonized System (GHS).