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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-07-03 to 2013-07-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
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:
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.
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
, 2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2013-04-11

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Details on test animals and environmental conditions:
Not applicable - Since this is an in vitro study there is no information on test animals.

Test system

Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL of the undiluted test item were applied to each of triplicate tissues
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.: 18346) 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.
Historical data and the quality certificate of the supplier of the test kit demonstrated the robustness of the test system or rather of the test kit.

TEST FOR DIRECT MTT REDUCTION BY THE TEST ITEM
- approximately 30 μL 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.

TREATMENT:
- 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 was pipetted into each well of 6-well plates and the inserts with the EpiDerm™ tissues were pre-incubated for a total duration of about 19 hours (37 ± 1.5 °C, 5 ± 1% CO2, 95 ± 5% RH).
- after the pre-incubation of EpiDerm™ tissues, 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.
- the 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 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 nearly 24 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 about 17 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2.
- complete incubation time was approximately 43.5 hours.

CELL VIABILITY TEST:
- cell viability is measured by dehydrogenase conversion of MTT, in cell mitochondria, into a blue formazan salt.
- after the 43.5-hours incubation period, culture inserts were transferred from the holding plates 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) in each insert.
- the formazan salt was extracted for about 69.5 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.
- per each tissue, 3 × 200 μL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate
- 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:
- 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 (%) = [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.
- 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

Results
Irritation / corrosion parameter:
other:
Value:
15.4
Remarks on result:
other:
Remarks:
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 decreased to 15.4% after exposure of the test item to the skin tissues. This value is below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential.

Any other information on results incl. tables

HISTORICAL DATA

Positive control

Negative control

Mean viability

5.7%

Mean absorption

1.858

Standard deviation

2.2%

Standard deviation

0.309

Range of viabilities

2.6 – 9.4%

Range of viabilities

1.423 – 2.651

Data of 102 studies performed from November 2011 until July 2013.

Result 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

 1.821

 1.815

 1.890

 1.842

 100.0

Positive control

60 minutes

 0.122

 0.107

 0.128

 0.119

 6.4

Test item

60 minutes

 0.267

 0.274

 0.313

 0.284

 15.4

* 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)

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

- 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 6.4% thus ensuring the validity of the test system.

- The standard deviations between the % variabilities of the test item, the positive and negative controls were below 10% (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:
Category 2 (irritant)
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item is an irritant to the skin.
According to the Directive 67/548/EEC and its subsequent amendments, the test substance is irritating to the skin (Xi; R38).
According to the Regulation (EC) No 1272/2008 and subsequent regulations, the test item is classified as irritating to the skin (Category 2, H315).