[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experiment start date - 12 November 2009; Experiment completion date - 13 November 2009; Study completion date - 01 December 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 431 (In vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Details on test material:

- Substance type: reactive dyestuff
- Physical state: orange powder
- Analytical purity: 69.9% of all colored components
- Lot/batch No.: TZ 5891 / BOP 02-09
- Expiration date of the lot/batch: 2014-07-31
- Storage condition of test material: At room temperature at about 20 °C

Specific details on test material used for the study:

Identification: FAT 40851/A TE
Batch Number: TZ 5891 / BOP 02-09
Purity: 69.9% all coloured components
Appearance: Orange powder
Expiry Date: July 31, 2014
Storage Conditions: At room temperature at about 20 °C

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

water

Remarks:

Deionised water

Details on test system:

EST-1000 kits were purchased from CellSystems® Biotechnologievertrieb GmbH (53562 St. Katharinen; 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 (November 10, 2009) EST-1000 tissues were kept in the refrigerator at 2 - 8 °C until November 12, 2009 prior to use. At least one hour before starting the assay, tissues were transferred to 6-well plates with assay medium, which is immediately replaced before the test is started.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The test material was not crushed or ground in a mortar and a pestle since this did not improve the consistency. About 25 mg of the test material were wetted with 50 μL deionised water. The test item was spread to cover the surface of the tissue. For the positive and negative controls, a volume of 50 μL was dosed per tissue.

Duration of treatment / exposure:

3 minutes and 1 hour.

Number of replicates:

Two

Test animals

Species:

human

Test system

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): 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.
- Time after start of exposure: after 3 min and 1h exposure

SCORING SYSTEM: MTT assay
After the exposure procedure, the cell culture inserts were incubated for 3 hours with MTT solution. Subsequently, 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 and extracted with isopropanol for 17 hours 35 minutes.
Aliquots of the blue formazan solution were transferred from each tissue into a 96-well flat bottom microtiter plate and optical density (OD) was read at 570 nm (OD570).
The mean values were calculated for each set of 3 wells per tissue insert.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean Experiment I & II - 3 min

Value:

93.9

Negative controls validity:

valid

Remarks:

100% tissue viability

Positive controls validity:

valid

Remarks:

5.7% tissue viability

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean Experiment I & II - 1 hour

Value:

88.8

Negative controls validity:

valid

Remarks:

100% tissue viability

Positive controls validity:

valid

Remarks:

4.6% tissue viability

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The optical evaluation of the MTT-reducing capacity of the test item after a 1 hour incubation with MTT-reagent did not show evidence of a blue colour and thereby was not considered to be an MTT reducer.

Any other information on results incl. tables

After exposure to the test item test substance the relative absorbance values were irrelevantly decreased to 93.9% after 3 minutes. After the 1 hour exposure relative absorbance values were reduced to 88.8%. Both values did not exceed the threshold for corrosivity of 50% for the 3 minutes exposure and 15% for the 1 hour exposure.

Therefore, the test item was not considered to be corrosive.

The optical evaluation of the MTT-reducing capacity of the test item after a 1 hour incubation with MTT-reagent did not show evidence of a blue colour and thereby was not considered to be an MTT reducer.

Table 1: Results after treatment with test substance

Dose group	Exposure time	OD570		Mean absorbance	Rel. absorbence [% of negative control]
		Tissue 1 *	Tissue 2 *
Negative control	3 min	1.869	1.484	1.676	100.0
Positive control	3 min	0.089	0.100	0.095	5.7
test substance	3 min	1.635	1.514	1.574	93.9
Negative control	1 h	1.696	1.939	1.817	100.0
Positive control	1 h	0.083	0.085	0.084	4.6
test substance	1 h	1.451	1.778	1.614	88.8

* Mean of three replicate wells after blank correction

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was non corrosive to skin.

Executive summary:

This in vitro study was performed according to OECD Guideline 431 and to EU Method B.40 under GLP to assess the corrosive potential of test substance by means of the Human Skin Model Test.

 

Independent duplicate tissues of the human skin model EST-1000 were exposed to either the test item, the negative control or the positive control for 3 minutes and 1 hour, respectively. About 25 mg of the test item were applied to each tissue, wetted with 50 μL deionised water, and spread evenly over the surface of the tissue. A volume of 50 μL of either the negative control (deionised water) or the positive control (8.0 N KOH) was applied to each tissue. Viability of the cells were determined by MTT assay by measuring the absorbance at 570 nm (OD₅₇₀) and expressed as relative absorbance in % of negative control.

 

After exposure to the negative control the absorbance values exceeded the required acceptability criterion of mean OD₅₇₀ ≥0.8 for both treatment intervals thereby confirming the acceptable quality of the tissues. Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period and for the 1 hour exposure period thus confirming the validity of the test system.

After exposure to the test item, the relative absorbance values were decreased to 93.9% after 3 minutes. After the 1 hour exposure relative absorbance values were reduced to 88.8%. Both values did not exceed the threshold for corrosivity of 50% for the 3 minutes exposure and 15% for the 1 hour exposure. Therefore, the test item was not considered to be corrosive.

 

In conclusion, it can be stated that in this study and under the reported experimental conditions, the test item test substance was non corrosive to skin.

According to the referred classification (Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008), test substance does not have to be classified with respect to skin corrosion.