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Diss Factsheets

Administrative data

skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-11-11 to 2014-11-17
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

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 2013-07-26
according to guideline
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
adopted 2009
according to guideline
other: In Vitro EpiDermTM 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 2013-04-11

Test material

Constituent 1
Reference substance name:
Constituent 2
Reference substance name:
EC Number:
Constituent 3
Chemical structure
Reference substance name:
Cas Number:
Molecular formula:
Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): 4,4‘ –sulfonyldiphthalic acid dianhydride
- Molecular formula: C16H6O8S
- Molecular weight: 358.28 g/mol
- Physical state: solid powder, beige, odourless
- Storage condition of test material: at room temperature, protected from light and moisture

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
Amount / concentration applied:
- Amount(s) applied (volume or weight with unit):approximately 25 to 26.5 (~ 43 mg/cm²) of the tets item were applied to the tissues wetted with 25 µL vehicle

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.: 19695, Kit B) and MTT-100 assays diluent were purchased from MatTek Corporation (82105 Bratislava, Slovakia).
- EpiDerm™ tissue (surface 0.6 cm²) 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.

1) Colour interference:
- prior to the treatment the colour interference potential of the the test item was evaluated by spectral analysis of the test item mixed with water.
- ~25 mg of the test item was mixed with 300 µL of deionised water.
- test item suspension was incubated for 60 minutes at 37 ± 1.5°C (5 ± 0.5% CO2)
2) Direct MTT Reduction:
- approximately ~25 mg of the test item were added to 1 mL of MTT solution (MTT diluted with DMEM (resulting: 1 mg/mL)) and the mixture was incubated in the dark at 37 ± 1.5°C (5 ± 0.5% CO2) 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.

- one day prior to the performance, the tissues were placed in the sterile 6-well plate.
- prior to the exposure of the test item and of the controls the EpiDerm™ tissues were inspected for quality.
- 0.9 mL of the assay medium (20 - 25°C) was pipetted into each well of sterile 6-well plates and the inserts with the EpiDerm™ tissues were pre-incubated for a total duration of about 24.5 hours (37 ± 1.5 °C, 5 ± 1% CO2, 95 ± 5% RH).
- after the pre-incubation of EpiDerm™ tissues, medium was replaced by 0.9 mL of fresh medium per well in the 6-well plates.
- the negative control (Dulbecco's Phosphate Buffered Saline (DPBS) (30 μL)) and positive control (5% sodium lauryl sulphate (SLS) solution in deionised water (30 μL)) and the test item were added into the insert atop the corresponding EpiDerm™ triplicate tissues.
- treatment time was 60 minutes in total. Within this period the 6-well plates were placed in the incubator for 35 minutes at 37 ± 1.5°C, 5 ± 0.5% CO2. In the remaining period the plates were placed in a sterile bench at room temperature until the end of treatment.
- after the end of the treatment interval the inserts were removed from the plate and tissues were rinsed with DPBS in order to remove any residual test material.
- tissues were transferred into new 6-well plates with 0.9 mL of fresh assay medium and tissues were incubated for approximately 23 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2.
- after incubation the inserts were transferred into new 6-wells plates containing fresh medium. Thereafter tissues were incubated for another 18.5 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2.
- complete incubation time was about 42 hours.

- cell viability is measured by dehydrogenase conversion of MTT, in cell mitochondria, into a blue formazan salt.
- after the 42-hours incubation period, culture inserts were transferred from the holding plates to plates containing 300 µL of 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 with DPBS. Inserts were transferred onto new 24-well plates.
- the inserts were immersed into extractant solution (isopropanol).
- the formazan salt was extracted for about 69 hours at room temperature.
- after the extraction period, the inserts were pierced to allow the extract to run into the well from which the insert was taken.
- 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 (OD) was read in a microplate reader (Versamax® Molecular Devices, Softmax Pro, version 4.7.1) with a 570 ± 1 nm filter.
- mean values were calculated from the 3 wells per tissue.

- mean OD of the three negative control tissues was calculated.
- 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 (%) = [(meanOD test item/positive control)/ ODmean of negative control] x 100
- for the test item and the positive control the mean relative viability ± rel. standard deviation of the three individual tissues was calculated and used for classification.
- 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.

Results and discussion

In vitro

Irritation / corrosion parameter:
other: other: relative viability(%)
Remarks on result:
Basis: mean. Time point: after 60 minutes 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 reduced to 88.9% after exposure of the skin tissues to the test item. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

Any other information on results incl. tables


Positive control

Negative control [OD570]

Mean viability


Mean absorption


Rel. standard deviation


Rel. standard deviation


Range of viabilities

2.9 – 11.3%

Range of viabilities

1.397 – 2.651

Mean absorption



 Rel. standard deviation


 Range of absorbance

 0.030 - 0.194

Data of 125 studies performed from November 2011 until May 2014.

Results after treatment with the test item and the controls

Dose Group


Treatment Interval

Absorbance 570 nm Tissue 1*

Absorbance 570 nm Tissue 2*

Absorbance 570 nm Tissue 3*

Mean Absorbance of 3 Tissues

Mean Rel. Absorbance

[% of Negative Control]**

Negative control

60 minutes






Positive control

60 minutes






Test item

60 minutes






* Mean of three replicate wells after blank correction

** Relative absorbance per treatment group [rounded values]: [100 X (mean absorbance test item / positive control)] / (mean absorbance negative control)

- the optical pre-experiment (colour interference pre-experiment) to investigate the test item's colour change potential in water did not led to a change in colour. The OD570 of the test item suspension in water was 0.185.This value was too low to have an influence on the outcome of the study. Consequently, an additional control with viable tissues without MTT application was not necessary to be performed.

- the optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour. Conseqeuntly, an additional control with freeze-killed tissues was not necessary to be performed.

- after treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 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 4.6% thus ensuring the validity of the test system.

- the rel. standard deviations between the % variabilities of the test item, the positive and negative controls were below 11% (threshold of the "OECD Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: 18%), thus ensuring the validity of the study.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Migrated information Criteria used for interpretation of results: EU
The test item is not an irritant to the skin.
According to the Directive 67/548/EEC and its subsequent amendments, the test substance is not irritating to the skin.
According to the Regulation (EC) No 1272/2008 and subsequent regulations, the test item is not classified as irritating to the skin.