Arsenic

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-06-16 to 2011-06-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (Original Guideline adopted July 22. 2010)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ECVAM international validation study on in vitro tests for acute skin irritation: Report on the validity of the EpiSkin TM and EpiDerm assays and on the Skin Integrity Function Test (Altern Lab Anim. 2007 Dec; 35 (6):559 - 601).

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: "Commission Regulation (EC) No. 440/2008 B 46.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-03-30

Test material

Test material information

Constituent 1

Reference substance name:

Arsenic

EC Number:

231-148-6

EC Name:

Arsenic

Cas Number:

7440-38-2

Molecular formula:

As

IUPAC Name:

arsenic

Details on test material:

- Name of test material (as cited in study report): Arsenic metal, powder (particle size < 0.2 mm, purity > 99.99%)
- Physical state: powder
- Storage condition of test material: at room temperature, under N2 atmosphere
- Particle size distribution: D10 = 18.24 µm; D50 = 93.65 µm; D90 = 176.67 µm
- Particle size: < 0.2 mm

Test animals

Details on test animals or test system and environmental conditions:

Not applicable - Since this is an in vitro study there is no information on test animals.

Test system

Vehicle:

water

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approximately 10 mg of the neat test item were applied to each of triplicate tissues (approximately 26.32 mg/cm^2)

VEHICLE
- Amount(s) applied (volume or weight with unit): the test item tissues were wetted with 15 µl of deionised water.

Duration of treatment / exposure:

15± 1 min

Observation period:

not applicable

Number of animals:

not applicable

Details on study design:

CELL CULTURE
EpiSkin TM kits (Lot No.: 11-EKIN-024) are purchased from SkinEthic Laboratories (06000 Nice, France). The EpiSkin TM tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis.
It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiSkin TM tissues (surface 0.38 cm^2) are cultured on specially prepared cell culture inserts.
EpiSkin TM tissues were shipped with ice packs on medium-supplemented agarose gels in a 12-well plate. On day of experiment EpiSkin TM tissues were transferred to 12-well plates with maintenance medium.

TREATMENT:
The negative control (deionised water (produced in-house, lot no. 02.05.11; 10 µL were applied to each of triplicate tissues) and positive control (5% sodium lauryl sulphate (lot no. 1353471 51508322; Fluka, Sigma-Aldrich, 89555 Steinheim, Germany) solution in deionised water, prepared freshly prior to the performance of the experiment; 10 µL were applied to each of triplicate tissues), and the test item were added into the insert atop the concerning EpiSkin TM triplicate tissues. Additionally, the test item tissues were wetted with 15 µl of deionised water. The plates were placed into the incubator for 15± 1 min at 37 ± 1.5 °C, 5 ± 0.5 % CO2.
After the end of the treatment interval the inserts were removed immediately from the plate. Using a wash bottle the tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for 42 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2.

MTT ASSAY:
Cell viability is measured by dehydrogenase conversion of MTT [(3-4, 5-dimethyl thiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide], in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues (Faller, C., Bracher, M., Dami, N., Roguet, R., 2002. Predictive ability of reconstructed human epidermis equivalents for assessment of skin irritation of cosmetics. Toxicology in vitro 16 (5), 557-552.).The percent reduction of cell viablity in comparison of untreated negative controls is used to predict skin irritation potential (see OECD TG 439).
After the treatment procedure was completed for all tissues of each time point cell culture inserts were transferred from the holding plates to plates containing 2 mL assay medium containing 0.3 mg/mL MTT per well. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5 % CO2) MTT solution was aspirated from the wells and the wells were rinsed three times with PBS. Tissues samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol / 2 N HCl 49:1 (v/v)) into each vial. The tissue samples were completely covered by isopropanol. The vials were sealed to inhibit isopropanol evaporation. The formazan salt was extracted for about 45 hours in the refrigerator.
Per each tissue sample 2 X 200 µL aliquots of the formazan blue solution were transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany) with 570 nm ± 1 nm filter. Mean values were calculated from the 2 wells per tissue sample.

EVALUATION OF RESULTS:
The mean OD of the three negative control tissues was calculated. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula:
Relative viability (%) = [OD test item/ OD negative control] * 100
For the test item and the positive control the mean relative viability +/- standard deviation of the three individual tissues are calculated and used for classification according to the following prediction model:
For the current test, an irritation potential of a test item according to EU classification R38 (according to directive 67/548/EEC), H315 (according to regulation (EC) 1272/2008) is recommended if the mean relative tissue viablity of three individual tissues is reduced below 50 % of the negative control.

ACCEPTABILITY OF THE ASSAY:
The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD of the three tissues is ≥0.6 till ≤ 1.5.
An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is ≤ 40%.
The standard deviations in between tissues of the same treatment group should be ≤ 18%.

The data of the quality control (determined by SkinEthic Laboratories, 06000 Nice, France) of the respecitve EpiSkin TM lot is mentioned below under "Attached background material" (the acceptance limit of the IC50 should be between 1.0 and 3.0 mg/mL after 18 hours treatment with SLS).

TEST FOR DIRECT MTT REDUCTION.
For correct interpretation of results it was necessary to assess the ability of the test item to directly reduce MTT. To test for this ability approximately 24 mg of the test item were added to 1 mL of MTT solution and the mixture was incubated in the dark at room temperature for 60 minutes. Untreated MTT medium was used as control. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 8.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

After treatment with the test item arsenic metal, powder (particle size < 0.2 mm, purity > 99.99 %) the mean relative absorbance value decreased to 8.8 %. This value is below the treshold for irritancy of ≤ 50 %. Therefore, the test item is considered to possess an irritant potential.

Any other information on results incl. tables

Results after treatment with arsenic metal, powder (particle size < 0.2 mm, purity > 99.99 %) and controls

 

Dose group	Treatment Interval	Absorbance 570 nm Tissue 1*	Absor-bance 570 nm Tissue 2*	Absorbance 570 nm Tissue 3*	Mean Absorbance of 3 Tissues	Relative Absorbance [%] Tissue 1, 2 + 3**	Standard Deviation [%]	Rel. Absorbance [% of Negative Control]***
Negative Control	15 min	0.738	0.751	0.758	0.749	98.6 100.3 101.2	1.3	100.0
Positive Control	15 min	0.248	0.263	0.201	0.237	33.1 35.2 26.8	4.4	31.7
Test item	15 min	0.065	0.050	0.083	0.066	8.1 6.6 11.1	2.3	8.8

* Mean of two replicate wells after blank correction

** relative absorbance per tissue [rounded values]: [100 x (absorbance tissue)]/ (mean absorbance negative control)

*** relative absorbance per treatment group [rounded values]: [100 x (mean absorbance test item)]/(mean absorbance negative control)

Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

Historical data:

Positive Control		Negative Control
Number of Studies	122	Number of Studies	122
Period	July 2007 - May 2011	Period	July 2007 - May 2011
Mean Viability	17.8 %	Mean OD	1.051
Standard Deviation	10.7 %	Standard Deviation	0.185
Range of Viabilities	0.7 % - 39.7 %	Range of ODs	0.59 – 1.68

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

After treatment with the test item arsenic metal, powder (particle size < 0.2 mm, purity > 99.99 %) the mean relative absorbance value decreased to 8.8 %. This value is below the treshold for irritancy of ≤ 50 %. Therefore, the test item is considered to possess an irritant potential.
The test item should be classified and labeled as skin irritant according to regulation (EC) No.: 1272/2008.