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Description of key information

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-12-12 to 2012-12-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 2010-07-22
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
, 2009
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: In vitro EpiDerm TM Skin irritation Test (EPI-200-SIT) for use with MatTek Corporation's Reconstructed Human Epidermal Model EpiDerm (EPI-200), Rev. 1/19/2010
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:
other: Dulbecco's Phosphate Buffered Saline (DPBS)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approximately 25 to 28 mg of the test item were each applied to the tissues, wetted with the vehicle
and spread to match the surface of the tissues.

VEHICLE
- Amount(s) applied (volume or weight with unit): 50 μL of Dulbecco's Phosphate Buffered Saline
Duration of treatment / exposure:
60 minutes
Observation period:
not applicable
Number of animals:
not applicable
Details on study design:
CELL CULTURE
Epi-200 SIT kits (Lot No.: 16864, Kit B) and MTT-100 assays diluent were purchased from MatTek Corporation (82105 Bratislava, Slovakia). The EpiDerm™ 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 EpiDerm™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm ∅).
EpiDerm™ tissues were shipped at 4 °C on medium-supplemented agarose gels in a 24-well plate and reached the laboratory on December 11, 2012. On day of receipt the preincubation phase of the EpiDerm™ tissues started.

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 28 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:
Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.

TREATMENT
- Pre-warming of EpiDerm™ Tissues: one day prior to the performance, the plastic bag containing the 24-well plate with epidermal tissues was opened under sterile conditions. Under an airflow using forceps, the gauze was removed and the inserts were taken out. Any remaining agarose that adhered to the outer sides of the inserts was removed by gentle blotting on the sterile filter paper or gauze, and the tissues were placed in the empty, sterile 6-well plate. Prior to the exposure with the test item and with the controls the EpiDerm™ tissues were inspected for quality: If necessary, it was taken care, that
1. air bubbles between agarose and insert were not > 30% of the total surface,
2. liquid on top of the insert was removed with steriles cotton tips,
3. if again moisture is observed on top of the inserts after the pre-incubation or in case of visible defects the respective skin models were discarded.
0.9 mL of the assay medium (20 – 25 °C) was pipetted into each well of sterile 6-well plates. The inserts with the EpiDerm™ tissues were placed in the upper wells, and were pre-incubated for 60 minutes in the incubator (37 ± 1.5 °C, 5 ± 1% CO2, 95 ± 5% RH). Following, the inserts were transferred from upper wells into the lower wells of the 6-well plates, and, the pre-incubation was continued for further about 23 hours (37 ± 1.5 °C, 5 ± 1% CO2, 95 ± 5% RH).
- After pre-incubation of EpiDerm™ tissues was completed, the negative control (DPBS (MatTek, lot no. 071212 MHD); volume: 30 µL) and positive control (5% SLS (MatTek, lot no. 052212 MHB)) solution in deionised water, prepared freshly prior to the performance of the experiment) and the test item were added into the insert atop the corresponding EpiDerm™ triplicate tissues. The 6-well plates were placed into the incubator for 35 minutes at 37 ± 1.5 °C, 5 ± 0.5 % CO2, 95 ± 5% RH. Thereafter, plates were removed from the incubator and placed into the sterile hood until the period of 60 minutes was completed.
After the end of the treatment interval the inserts were removed immediately from the 6-well plate and tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in DPBS at least three times. Afterwards the inserts were once again rinsed with DPBS from the inside and the outside. Excess DPBS was removed by gently shaking the inserts and blotting the bottom with sterile blotting paper. The tissues were then transferred into new 6-well plates with 0.9 mL of fresh assay medium in the upper row. The surface of the tissues was carefully dried using sterile cotton tipped swap. Tissues were incubated for 22.5 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2.
New 6-well plates (or lower row of the same plates) were filled with 0.9 mL of fresh assay medium, and the inserts were transferred to the new plates. The wells were incubated for another 19 hours post-incubation at 37 ± 1.5 °C, 5 ± 0.5 % CO2.

MTT ASSAY
Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethylthiazole 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 viability in comparison of untreated negative controls is used to predict skin irritation potential (see OECD TG 439) and is used for the purpose of classification as irritating or non-irritating according to chemicals law (EU CLP, UN GHS).
After the 42-hours incubation period was completed for all tissues and exposure groups, culture inserts were transferred from the holding plates to the MTT-plates containing 300 µL MTT solution. After a 3-hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO2), the MTT solution was aspirated from the wells, and the wells were rinsed three times with DPBS. Inserts were transferred onto new 24-well plates. The inserts were immersed into extractant solution by gently pipetting 2 mL extractant solution (isopropanol) in each insert. The level rose above the upper edge of the insert, thus tissues were completely covered from both sides. The 24-well plate was sealed to inhibit the isopropanol evaporation. The formazan salt was extracted for about 69 hours at room temperature.
After the extraction period was completed, the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken. Afterwards the insert was discarded. The 24-well plates were placed on a shaker for 15 minutes until the solution was homogeneous in colour.
Per each tissue, 3 × 200 μL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate from the 15 minutes exposure. Optical density was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany) with a 570 ± 1 nm filter. Mean values were calculated from the 3 wells per tissue.

EVALUATION OF RESULTS
The mean optical density (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 formulas:
Relative viability (%) = (OD test item/ OD mean of negative control) X 100
For the test item and the positive control the mean relative viability ± standard deviation of the three individual tissues was 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 viability of three individual tissues is reduced below 50% of the negative control.

ACCEPTABILITY OF THE ASSAY
- Criterion 1 (negative control): 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 OD570 of the negative control tissues is ≥ 1.0 and ≤ 2.5.
- Criterion 2 (positive control): an assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is ≤ 20%.
- Criterion 3: Standard deviation: the relative SD of the viability (= relative absorbance) of 3 identical replicates should be < 18%.
OD values should not be below historically established boundaries.
Irritation / corrosion parameter:
other: other: relative viability (%)
Value:
104.2
Remarks on result:
other:
Remarks:
Basis: mean. Time point: after 60 min. incubation. Reversibility: no data. (migrated information)
Irritant / corrosive response data:
Compared to the relative absorbance value of the negative control the mean relative absorbance value was not reduced (104.2%; threshold for irritancy of ≤ 50%) after exposure of the test item silver bromide to the skin tissues. Therefore, the test item is not considered to possess an irritant potential.

HISTORICAL DATA

Positive Control

Negative Control

Number of Studies

67

Number of Studies

67

Period

May 2010 – November 2012

Period

May 2010 – November 2012

Mean Viability

6.6%

Mean Absorption

1.717

Standard Deviation

2.1%

Standard Deviation

0.274

Range of Viabilities

4% - 9.4%

Range of absorption

1.423 – 2.651

Table 1: Results after treatment with silver bromide and the controls

Dose group

Treatment interval

Absorbance
570 nm
Tissue 1*

 

Absorbance
570 nm
Tissue 2*

 

Absorbance
570 nm
Tissue 3*

 

Mean Absor-bance
of 3 Tissues

 

Mean Rel. Absorbance

[% of Negative Control]**

 

Negative control

60 min

2.568

2.348

2.114

2.344

100.0

Positive control

60 min

0.083

0.063

0.063

0.070

3.0

Test item

60 min

2.655

2.413

2.255

2.441

104.2

* Mean of three replicate wells after blank correction

** relative absorbance per treatment group [rounded values]: 100 x (meanabsorbancetestitem)/(mean absorbancenegative control)

- after treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 1.0 and ≤ 2.5 for the 60 minutes treatment interval thus showing the quality of the tissues.

- treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 3.0% thus ensuring the validity of the test system.

- the relative standard deviations between the % variabilities of the test item, the positive and negative controls were 9.7%, 16.8%, and 8.3%, respectively (below the threshold given in the "OECD Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”of 18%), thus ensuring the validity of the study.

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

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item silver bromide is not irritant to skin.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-01-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
adopted 2009-09-07
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
, 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997
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 an in vitro study there is no information on test animals.
Vehicle:
other: 0.9% (w/v) NaCl in deionised water
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
Prior to the test the test item was crushed in a mortar with a pistil to improve its consistency. A 20% (w/v) suspension of the test item in the vehicle was prepared using ultrasonic technique.
Duration of treatment / exposure:
240 minutes
Observation period (in vivo):
not applicable
Number of animals or in vitro replicates:
not applicable
Details on study design:
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.
Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD 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 basal opacity was determined (t0). Each cornea with a value of the basal opacity > 7 was discarded and not used in the test.

OUTLINE OF STUDY
The anterior compartment received the test item or negative control (0.9% (w/v) NaCl in deionised water (produced in-house, lot no. 12.12.12)) or positive control (10% (w/v) Benzalkonium chloride (Sigma, 89555 Steinheim, Germany, lot no. 036K0208) in 0.9% (w/v) NaCl in deionised water (produced in-house, lot no. 12.12.12)) at a volume of 0.75 mL each on the surface of the corneae. Since the test item could not be suspended homogeneously, it was taken care that each 0.75 mL of the prepared stock suspension was distributed evenly to the corneae. The test item, positive control and negative control were tested in triplicate.
The anterior compartment was plugged. The corneae were turned into a horizontal position and slightly rotated to ensure uniform covering of the corneae with the test or control items and were incubated in a water-bath in horizontal position at 32 ± 1 °C for 240 minutes.
After the incubation, the test item or control items, respectively, were rinsed off from the application side with 0.9% (w/v) NaCl in deionised water, fresh cMEM was added into the anterior compartment and opacity was measured (t240).
In the second step of the assay, permeability of the cornea was determined. 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, replacing the cMEM. 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).

CRITERIA FOR DETERMINATION OF A VALID TEST
The test was acceptable if the in vitro irritation score of the positive control was ≥ 30 and the in vitro irritation score of the negative control was ≤ 3.

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 irritation 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 the following category according to OECD guideline 437 (table 1 in the field "Any other information on materials and methods incl. tables" below).
Irritation parameter:
other: in vitro irritation score
Basis:
mean
Time point:
other: 240 minutes
Score:
3.03
Irritant / corrosive response data:
Relative to the negative control, exposure of the test item silver bromide to the corneae did not cause any increase of the corneal permeability. Only a very slight increase of the opacity values occurred. The calculated mean in vitro irritation score was 3.03 (threshold for corrosivity / severe irritancy: ≥ 55.1). According to OECD guideline 437 the test item is not classified as corrosive / severe irritant to the eye.

Table 1: Results after 240 minutes incubation time

Test group

Opacity value = Difference (t240 – t0) of opacity

Permeability at 490 nm (OD490)

In vitro irritation score

Mean in vitro irritation score

Proposed in vitro irritation scale

 

 

Mean

 

Mean

 

 

 

Negative control

1

 

0.33

0.048

 

0.046

1.72

 

1.02

Non corrosive / non severe irritant

0

0.044

0.66

0

0.046

0.69

Positive control

147.67*

0.003*

147.71

 

155.24

Corrosive / severe irritant

162.67*

0.022*

163.00

154.67*

0.023*

155.01

Silver bromide

3.67*

0.023*

4.01

3.03

Non corrosive / non severe irritant

3.67*

0.020*

3.97

0.67*

0.030*

1.12

*corrected values

- With the negative control (0.9% (w/v) NaCl in deionised water) neither an increase of opacity nor permeability of the corneae could be observed (mean in vitro irritation score 1.02).

- The positive control (10% (w/v) Benzalkonium chloride in 0.9% (w/v) NaCl in deionised water) induced clear opacity of the corneae (mean in vitro irritation score 155.24) corresponding to a classification as corrosive /severe irritant to the eye (CLP/EPA/GHS (Cat 1)).

Table 2: Historical data

 

Positive control

Negative control

Mean in vitro Irritation Score

176.71

1.78

Standard Deviation

42.65

0.75

 Range of in vitro irritation scores 99.4 - 292.3  0.41 - 2.99

Values of 138 studies with solid test items performed until November 2012

Interpretation of results:
other: not corrosive / not severely irritating to the eye
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
In conclusion, according to the current study and under the experimental conditions reported, the test item silver bromide is not corrosive / not severely irritating to the eye (CLP/EPA/GHS (Cat 1)).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for selection of skin irritation / corrosion endpoint:
AgBr was not irritating/corrosive to skin in an in-vitro test system. An in-vivo study (OECD 404) to confirm this results in-vivo is ongoing (February/March 2013) and will be included in a dossier update.

Justification for selection of eye irritation endpoint:
AgBr was not irritating/corrosive to eye in an ex-vivo test system (BCOP). An in-vivo study (OECD 405) to confirm this results in-vivo is ongoing (February/March 2013) and will be included in an dossier update.

Justification for classification or non-classification

Based on in-vitro (skin) and ex-vivo (eye) test results, AgBr was not irritating or corrosive towards skin/eye. Further studies to confirm these results and the non-classification in-vivo are ongoing (February/March 2013) and will be included in a dossier update.