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Skin irritation / corrosion

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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
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study reliable without restrictions

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 2010-07-22
according to guideline
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
, 2009
according to 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
GLP compliance:
yes (incl. QA statement)
signed 2009-03-30

Test material

Constituent 1
Chemical structure
Reference substance name:
Silver bromide
EC Number:
EC Name:
Silver bromide
Cas Number:
Molecular formula:
silver bromide
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): silver bromide
- Storage condition of test material: at room temperature, protected from light

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

other: Dulbecco's Phosphate Buffered Saline (DPBS)
Amount / concentration applied:
- 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.

- 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:
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.

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

- 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.

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.

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.

- 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.

Results and discussion

In vitro

Irritation / corrosion parameter:
other: other: relative viability (%)
Remarks on result:
Basis: mean. Time point: after 60 min. incubation. Reversibility: no data. (migrated information)

In vivo

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.

Any other information on results incl. tables


Positive Control

Negative Control

Number of Studies


Number of Studies



May 2010 – November 2012


May 2010 – November 2012

Mean Viability


Mean Absorption


Standard Deviation


Standard Deviation


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

570 nm
Tissue 1*


570 nm
Tissue 2*


570 nm
Tissue 3*


Mean Absor-bance
of 3 Tissues


Mean Rel. Absorbance

[% of Negative Control]**


Negative control

60 min






Positive control

60 min






Test item

60 min






* 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.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Migrated information Criteria used for interpretation of results: EU
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.