Arsenic

Endpoint summary

Description of key information

Arsenic metal powder (particle size < 0.2 mm, purity > 99.99%) was tested in vitro for its corrosive and/or irritating properties on skin according to OECD TG 431 and 439, respectively. The substance proved to be irritating but not corrosive.

In an in vitro eye irritation study according to OECD TG 437, arsenic metal was not corrosive to the eye, but proved to be mildly irritating. In vivo (OECD TG 405), the substance caused irreversible effects in the eyes of the tested rabbits.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

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

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

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

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.

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.

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.

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

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.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-07-12 to 2011-07-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 431: In vitro skin corrosion: Human Skin Model Test (Original guideline adopted 2004-04-13)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-03-30

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.

Vehicle:

water

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): about 25 mg of the test material were applied to the tissues and wetted with 25 μL deionised water. The test item was spread to cover the surface of the tissue.

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 μL deionised water

Duration of treatment / exposure:

Exposure periods of 3 minutes and 1 hour

Observation period:

not applicable

Number of animals:

not applicable

Details on study design:

CELL CULTURE:
EST-1000™kits (Lot No.: EST 110627-001) were purchased from CellSystems® Biotechnologievertrieb GmbH (53842 Troisdorf, Germany). The EST-1000™ tissue consisted of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consisted 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 EST-1000™ tissues (surface 0.6 cm²) were cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm ∅).
EST-1000™ kits were shipped at 4 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt EST-1000™ tissues were kept in the refrigerator at 2 - 8 °C in the refrigerator until prior to use. At least one hour before starting the assay, tissues were transferred to 6-well plates with maintenance medium, which is immediately replaced before the test is started.
The Human Skin Model supplier ensures and demonstrated that each batch of the Human Skin Models used met defined production release criteria, e.g. viability, barrier function, no bacterial and mycoplasma contamination and morphology. These data are attached below (please refer to "Attached background material").

PRE-WARMING OF EST-1000TM TISSUES:
At least one hour before dosing the EST-1000™ tissues were removed from the refrigerator. Under sterile conditions using sterile forceps, the inserts were transferred into 6-well plates containing the pre-warmed medium. Two 24-well plates were prepared as holding plates, each well containing 1 mL maintenance medium per well. The holding plates were pre-warmed in an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) until use.

EXPOSURE:
Duplicate EST-1000™ tissues were exposed to the test item, positive control (potassium hydroxide as 8.0 N ready-made solution (Sigma 82024 Taufkirchen, lot no.096K6091); volume 50 µL) or negative control (deionised water (produced in-house, lot no. 140611); volume 50 µL) for each of two different exposure periods.
After the pre-incubation of the EST-1000™ tissues was completed (1 hour 30 minutes for the 1 hour exposure and 1 hours 53 minutes for the 3 minutes exposure) the medium in each well was replaced by 1.0 mL fresh medium per well. The negative control was added to the surface of duplicate EST-1000™ tissues. Subsequently, the remaining tissues were exposed to the test item and the positive control in the same manner. The 6-well plates were then placed into an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2).
At the end of the exposure period the tissues were removed from the 6-well plate and gently rinsed using a wash bottle containing PBS to remove any residual test material. Excess PBS was removed by gently shaking the tissue insert and blotting the lower surface with blotting paper.

MTT ASSAY:
After the exposure procedure was completed for all tissues of each time point, the cell culture inserts were transferred from the holding plates to the plates containing 300 µL of MTT solution (MTT solution with a final concentration of 1 mg/mL). After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) the MTT solution was aspirated from the wells and the wells were rinsed three times with PBS. The inserts were transferred into new 24-well plates. The inserts were immersed in extractant solution by gently pipetting 2 mL of extractant solution (isopropanol) into each insert ensuring that the tissue was completely covered. The 24-well plate was sealed to minimise isopropanol evaporation. The formazan salt was extracted for 19 hours and 40 minutes.
After the extraction period the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken, and the insert was discarded. 24-well plates were then placed on a shaker for approx. 15 minutes until the solution was homogeneous in colour.
3 x 200 μL aliquots of the blue formazan solution were transferred from each tissue into a 96-well flat bottom microtiter plate. The optical density (OD) was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany) at 570 nm (OD570) without reference filter. The mean values were calculated for each set of 3 wells per tissue insert.

TEST FOR DIRECT MTT REDUCTION BY TEST ITEM:
To check MTT reducing capability a solution of MTT in DMEM (1.0 mg/mL) was prepared and each approx. 50mg of the test item were added to 1 mL
MTT medium. If the mixture turned blue/purple after about 1 hour at room temperature, the test material would have been presumed to have reduced the MTT.

INTERPRETATION OF RESULTS:
The mean OD value obtained for the duplicate tissues per test item were used to calculate the percent viability relative to the negative control, which was arbitrarily set at 100%.
According to EU CLP and DSD:
The test item is considered to be corrosive to skin:
(1) if the viability after 3 minutes exposure is less than 50%, or
(2) if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is less than 15%.
The test item is considered to be non-corrosive to skin:
(1) if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

ACCEPTABILITY OF THE ASSAY:
The absolute OD570 of the negative control tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability met the acceptance criterion if the mean OD570 of the two tissues in both treatment intervals was ≥ 0.8.
The assay met the acceptance criterion if mean relative tissue viability of the positive control was ≤ 30%.

Irritation / corrosion parameter:

other: other: relative viability (%)

Value:

90.8

Remarks on result:

other:

Remarks:

Basis: mean. Time point: after 3 minute treatment. Reversibility: no data. (migrated information)

Irritation / corrosion parameter:

other: other: relative viability (%)

Value:

91.6

Remarks on result:

other:

Remarks:

Basis: mean. Time point: after 1 hour treatment. Reversibility: no data. (migrated information)

Other effects / acceptance of results:

After exposure to the test item arsenic metal, powder (particle size < 0.2 mm, purity >99.99 %) the relative absorbance values decreased slightly to 90.8% after 3 minutes and to 91.6% after 60 minutes treatment. Both values exceeded the threshold of 50% for the 3 minutes exposure and 15% for the 1 hour exposure. Therefore, the test item was considered to be non corrosive.

Results

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

Dose group	Exposure interval	Absorbance 570 nm tissue 1*	Absorbance 570 nm tissue 2*	Mean absorbance of 2 tissues	Rel. absorbance (% of negative control)**
Negative control	3 min	1.252	1.281	1.267	100.0
Positive control	3 min	0.158	0.077	0.118	9.3
Arsenic metal, powder (particle size < 0.2 mm, purity > 99.99 %)	3 min	1.217	1.084	1.151	90.8
Negative control	1 hour	1.319	1.144	1.232	100.0
Positive control	1 hour	0.014	0.017	0.015	1.2
Arsenic metal, powder (particle size < 0.2 mm, purity > 99.99 %)	1 hour	1.116	1.142	1.129	91.6

* Mean of three replicate wells after blank correction

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

The optical evaluation of the MTT-reducing capacity of the test item after one hour incubation with MTT-reagent did not show evidence of a blue colour and thereby was not considered to be an MTT reducer.

Historical data:

3 minutes treatment:

Positive control		Negative control
Number of studies	59	Number of studies	59
Period	March 2009 – May 2010	Period	March 2009 – May 2010
Mean viability	12.0 %	Mean OD	1.278
Standard deviation	7.06 %	Standard deviation	0.241
Range of viabilities	33 % - 5 %	Range of ODs	0.9 – 2.0

60 minutes treatment:

Positive control		Negative control
Number of studies	59	Number of studies	59
Period	March 2009 – May 2010	Period	March 2009 – May 2010
Mean viability	2.19 %	Mean OD	1.235
Standard deviation	3.43 %	Standard deviation	0.233
Range of viabilities	16% - 0.5 %	Range of ODs	0.8 – 1.8

Interpretation of results:

GHS criteria not met

Conclusions:

The test item arsenic metal, powder (particle size < 0.2 mm, purity > 99.99 %) was non corrosive to skin according to the classification criteria of Regulation (EC) 1272/2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-06-27 to 2011-07-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

2002-04-24

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Age at study initiation: 15 weeks
- Weight at study initiation: 2835 g
- Housing: individually in stainless steel cages
- Diet (ad libitum): pelleted standard Teklad Global High Fiber Rabbit Diet 2031C (batch no. 80/10, Provimi Kliba AG, 4303 Kaiseraugst / Switzerland); a piece of wood (batch no. 122201, imported by Indulab AG, Gams / Switzerland from ABEDD® - LAB & VET GmbH, 1160 Vienna / Austria) and a haystick 4642 (batch no. 37/10, Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) were also provided for environmental enrichment.
- Water (ad libitum): community tap water from Itingen
- Acclimation period: 5 days; under laboratory conditions after health examination. Only animals without any visual signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 17-23 °C
- Relative humidity: 30-70% (values above 70% were transiently observed during room cleaning)
- Air changes: 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12 (music played during the daytime light period.)

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): the test item was placed as a weight of 0.1 g/animal in the conjunctival sac of the left eye after gently pulling the lower lid away from the eyeball. The lid was then gently held together for about one second to prevent a loss of test item. The right eye remained untreated and served as the control.

Duration of treatment / exposure:

24 hours

Observation period (in vivo):

at approximately 1, 24, 48 and 72 hours as well as 7, 10, 14, 17 and 21 days after test item instillation

Number of animals or in vitro replicates:

1 male rabbit
(as it was suspected that the test item might produce irritancy, a single animal was treated first and the treatment of two further animals was delayed. Due to severe signs of irritation or corrosion observed in the first animal and for animal welfare reasons, further animals were not instilled with the test item.)

Details on study design:

The eyes of the animals were examined one day prior to test item administration. Only animals with no signs of ocular injury or irritation were used in the test.

REMOVAL OF TEST SUBSTANCE
- Washing: the eye of the test animal was rinsed with 0.9% saline solution after 24 hours after instillation of the test item, since residues of the solid test item had not been removed from the eye of the test animal by physiological mechanisms at the first observation time point of 1 hour after treatment. After 2, 3, 4, 7 and 10 days after instillation of the test item, a washing out with saline was used in order to remove ocular discharge for evaluation of eye irritation.

SCORING SYSTEM: according to the Draize scale
Scleral reddening and ocular discharge were also assessed (scale for assessment can be seen in the field "Any other information on materials and methods incl. tables" below).

TOOL USED TO ASSESS SCORE:
Eye examinations were made with a Varta Cliptrix diagnostic-lamp (Roth AG, 4153 Reinach / Switzerland).

OBSERVATIONS:
- Viability / Mortality: daily from acclimatization of the animals to the end of the observation period.
- Clinical Signs (systemic): daily from acclimatization of the animals to the end of the observation period.
- Body Weights: at start of acclimatization, on the day of application and at the end of the observation period.
- Pathology: no necropsy was performed on the animal sacrificed at termination of observation.
The animal was sacrificed by intravenous injection of 1.0 mL/kg body weight of a solution of 162 mg/mL sodium pentobarbitone into the ear vein and discarded.

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

not fully reversible within: 21 days

Remarks on result:

other: During the 1-hour and 24 hour observation the corneal opacity could not be scored due to swelling of the conjunctivae.

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

not fully reversible within: 21 days

Remarks on result:

other: During the 1-hour and 24 hour observation the iris light reflex could not be scored due to swelling of the conjunctivae.

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

3

Max. score:

3

Reversibility:

not fully reversible within: 21 days

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

4

Max. score:

4

Reversibility:

not fully reversible within: 21 days

Irritant / corrosive response data:

The instillation of Arsenic Metal, Powder <0.2 mm, >99.99% into the eye of the male rabbit resulted in corneal opacity (grade 2: easily discernible translucent area with details of iris obscured) in an involved corneal area greater than three quarters and in markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperemia or injection of the iris with iris still reacting to light (iris light reflex, grade 1) and in diffuse beefy red conjunctivae (grade 3) and in chemosis (swelling with lids more than half-closed, grade 4)[description of mean values across the scoring times 24, 48 and 72 hours after instillation of the test item].
The sclera was not accessible due to swelling on day 1 and the sclera, iris and cornea were also not accessible on day 2. Marked and moderate redness of the sclera was noted from day 10 onwards. Additionally, slight, moderate or marked discharge, test item remnants and/or mucus were noted from day 2 onwards.
The eye irritation scores increased after 72 hours (during days 7 to 17) for corneal opacity and iris light reflex, and slightly decreased after 10 or 14 days for reddening of the conjunctivae and chemosis.
The effects were not reversible after 21 days.
The marked signs of eye irritation were considered not to result from mechanical effects of the test item on the eye, since test item remnants were removed by rinsing the eye with saline, since severity grades and nature of findings such as corneal opacity, iritis or chemosis increased even after eye rinsing and since findings were not reversible during 21 days.

Other effects:

- Viability / Mortality: no intercurrent death occurred during the course of the study.
- Clinical Signs: no clinical signs were recorded throughout the entire observation period.
- Body Weights: the body weight was within the range commonly recorded for this strain and age.
- Pathology: no necropsy was performed at the end of the study.

Interpretation of results:

Category 1 (irreversible effects on the eye)

Conclusions:

Arsenic metal powder <0.2 mm, >99.99% is considered to cause irreversible effects on the eyes.
According to the criteria specified by Directive 67/548/EEC and subsequent regulations, the test item is classified as risk of serious damage to eyes (R41).
According to the EC Regulation No. 1272/2008 and subsequent regulations, the test item is classified as Category 1.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

other: validated "in vitro" test method

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-05-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 437: Bovine Corneal Opacity and Permeability Test method for Identifying Ocular Corrosives and Severe Irritants (07 September 2009)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The Bovine Corneal Opacity and Permeability Assay (BCOP) was performed according to the INVITTOX (UK) protocol no. 98 "The Bovine Corneal Opacity and Permeability Assay", 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-03-30

Details on test animals or tissues and environmental conditions:

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

Vehicle:

physiological saline

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): the anterior compartment received the test item at a volume of 0.75 mL each on the surface of the corneae

Duration of treatment / exposure:

The incubation time lasted 240 minutes (± 5 minutes).

Observation period (in vivo):

not applicable

Number of animals or in vitro replicates:

not applicable

Details on study design:

PREPARATION OF THE INCUBATION MEDIA
The medium MEM (Minimum Essential Medium), supplemented with sodium bicarbonate and L-glutamine was prepared one day prior to the start of the assay, and stored in a refrigerator (2 - 8 °C).
Immediately before starting the test, MEM was supplemented with 1% fetal calf serum (FCS). Afterwards, it was called cMEM (complete medium).
The OECD foresees the use of EMEM (Eagle’s minimal essential medium) which is in composition and osmolarity equivalent to the cMEM, thus cMEM could be used without restriction.

EXPERIMENTAL PERFORMANCE
COLLECTION OF BOVINE EYES
Freshly isolated bovine eyes from at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were transported to the laboratory in Hank’s BSS supplemented with streptomycin / penicillin at ambient temperature. The corneae were isolated on the same day after delivery of the eyes, inserted in pre-cooled preservation medium composed of Medium 199 supplemented with L-glutamine, Na-bicarbonate and Taurine, and stored in the refrigerator at 2 – 8 °C until the following day. Shortly before use, Dextran was added to the medium.

PREPARATION OF CORNEAE
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneae used in the experiment were collected in Hank’s BSS supplemented with streptomycin / penicillin and checked finally with a view box for defects listed above.
Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OEDC guideline 437, annex III, that consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. After the anterior part of the holder was positioned on top of the cornea and fixed in place with screws, both compartments of the holder were filled with complete medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure no air bubbles were present within the compartments.
For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.
At the end of the incubation period, the complete medium was removed from both compartments and replaced with fresh cMEM, and the basal opacity was determined (t0).

OUTLINE OF STUDY
The corneae were distributed as follows:
Group 1: negative control (number of corneae: 3)
Group 2: positive control (number of corneae: 3)
Group 3: test item (number of corneae: 3)
Fresh cMEM was placed in the posterior compartment, while the anterior compartment received the test item or negative control (saline (produced in-house, lot no. 12.4.11)) or positive control (10 % (w/v) benzalkonium chloride (Sigma, 89555 Steinheim, Germany, lot no. 036K0208) in 0.9 % (w/v) NaCl in deionised water (saline, produced in house, lot no. 12.4.11)) at a volume of 0.75 mL each on the surface of the corneae and was incubated at 32 ± 1 °C in the water-bath in a horizontal position.
Prior to the application the test item was suspended in saline (20% (w/v)).
The incubation time lasted 240 minutes (± 5 minutes).
After the test item or control items, respectively, were rinsed off from the application side with saline, fresh cMEM was added in both compartments and opacity was measured (t240)(OP_KIT opacitometer (Electro Design, 63-Riom France)).
In the second step of the assay, permeability of the cornea was determined (spectrophotometer Versamax® (Molecualr Devices, 85737 Ismaning, Germany)). Fresh complete medium was added to the posterior compartment and 1 mL of a Na-fluorescein solution, 0.5 % (w/v) dissolved in HBSS (Hank’s buffered salt solution), was placed in the anterior compartment. Corneae were incubated again in a horizontal position for an additional 90 minutes at 32 ± 1 °C in the water-bath. The optical density of an aliquot of the mixed complete medium from the posterior chamber was measured spectrophotometrically at 490 nm (OD490).

PROCEDURES
OPACITY MEASUREMENT
The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. The opacitometer was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
After recording the basal opacity of all corneae, the values of all corneae were noted. Any cornea deviating by more than 3 units, also –3 units, was discarded.
Complete medium was completely removed from the anterior compartment and replaced by the test item, positive or negative control. The anterior compartment was plugged. The cornea was turned to a horizontal position and slightly rotated to ensure uniform covering of the cornea with the test item and was incubated in a water-bath at 32 ± 1 °C for 240 minutes. Afterwards, the opacity was measured again (t240).

PERMEABILITY DETERMINATION
Following to the opacity readings, the permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the complete medium was removed from the anterior compartment and replaced by 1 mL of a 0.5% (w/v) fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C. Complete medium from the posterior compartment was removed with a 5 mL-syringe, well mixed and the optical density at 490 nm (OD490) was determined with a spectrophotometer.

CRITERIA FOR DETERMINATION OF A VALID TEST
The test was acceptable if the positive control caused an at least moderate effect and the negative control did not cause any effect (Irritation Score ≤ 3) on the corneae.

EVALUATION OF RESULTS
OPACITY
The change of opacity value of each treated cornea or positive and negative control corneae was calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea.
The average change in opacity of the negative control corneae was calculated and this value was subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

PERMEABILITY
The corrected OD490 value of each cornea treated with positive control and test item was calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

IN VITRO IRRITATION SCORE CALCULATION
The following formula was used to determine the in vitro irritation score of the negative control:
In vitro Irritation Score = opacity value + (15 x OD490 value)
The following formula was used to determine the in vitro irritation score of the positive control and the test item:
In vitro Irritation Score = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The in vitro irritation score was calculated for each individual treatment and positive control cornea. The mean in vitro score irritation value of each treated group was calculated from the individual in vitro irritation score values.
Depending on the score obtained, the test item was classified into one of the following categories as can be seen in table 1 shown in "Any other information on materials and methods incl. tables" below.

Irritation parameter:

in vitro irritation score

Value:

11.6

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The in vitro irritation score was calculated as follows: In vitro Irritation Score = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)

TABLE OF RESULTS

Results after 240 minutes incubation time

Table 2

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		In vitro Score	Mean in vitro Score	Proposed in vitro Irritation Scale
		Mean		Mean
Negative Control	-1	-0.33	0.079	0.076	0.19	0.81	Non eye irritant
	-1		0.058		-0.13
	1		0.091		2.37
Positive control	217.33*		0.071*		218.40	219.56	Severe eye irritant
	211.33*		0.411*		217.50
	215.33*		0.496*		222.77
Test item	12.33*		0.348*		17.55	11.60	Mild eye irritant
	9.33*		0.018*		9.60
	7.33*		0.020*		7.63

*corrected values, i.e. after substraction of mean value of the negative control

HISTORICAL DATA

Table 3.

	Positive Control	Negative Control
Mean in vitro Score	139.73	1.86
Standard Deviation	31.64	0.74

Values of 36 studies with solid test items performed in 2008 to 2010

Interpretation of results:

other: not corrosive, but mild eye irritant

Remarks:

Criteria used for interpretation of results: other: INVITTOX (UK) protocol no. 98

Conclusions:

The test item arsenic metal, powder (particle size < 0.2 mm, purity > 99.99 %) is not corrosive to the eye. Corresponding to the INVITTOX (UK) protocol no. 98 classification arsenic metal, powder (particle size < 0.2 mm, purity > 99.99 %) is mild eye irritant.
According to the EC-Regulation 1272/2008 and subsequent regulations, the test item is not corrosive to the eye.
According to the EC-Commission directive 67/548/EEC and its subsequent amendments, the test substance is not corrosive to the eye.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of in vitro and/or in vivo studies, the following hazard classification is proposed for arsenic metal powder, according to Regulation (EC) No. 1272/2008: Skin Irrit. 2 - H315 (Causes skin irritation) and Eye Damage 1 - H318 (Causes serious eye damage​).

From the data available, there is no indication that arsenic metal massive should be classified for skin or eye irritation according to Regulation (EC) No. 1272/2008.