Di(µ-2,2´,2´´-nitrilotris(ethanol)-diperchlorato)dinatrium

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 12th to 13th, 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted April 13th, 2004.

Deviations:

no

Principles of method if other than guideline:

SOP 118 008 20, edition 1, dated 29 February 2008.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

480-340-8

EC Name:

-

Cas Number:

156157-97-0

Molecular formula:

C12H30Cl2N2Na2O14

IUPAC Name:

Di(µ-2,2´,2´´-nitrilotris(ethanol)-diperchlorato)dinatrium

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: normal keratinocytes

Source strain:

other: not applicable

Vehicle:

water

Details on test system:

HUMAN SKIN MODEL
- Model used: commercially Epi-200-Kit. EpiDerm tissue consisting of normal, human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers and a multi -layered stratum corneum containiing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues are cultured on specially prepared cell culture inserts.
- Origin: produced from MakTek Corporation, Ashland, USA.
- Tissue batch number: 10529.
- Delivery date: June 11th, 2008.
- Date of initiation of testing: June 12th, 2008.

PREPARATION: on the day of the experiment, the MTT concentrate was thawed. The concentrate was diluted with the MTT solvent and the solution was stored at 4 °C in the dark. The PBS concentrate was diluted with deionised water 1:10. The pH was adjusted to pH 7.0. The tissue plate was brought out of the fridge one hour before the treatment. The assay medium was warmed in the water bath at 37 °C.

PRE-INCUBATION: four 6-well-plates were prepared with 0.9 ml assay medium in each well. The inserts containing the tissues were transferred to the wells using sterile forceps and the 6-well-plates were set into the incubatior at 37 °C and 5 % CO2 for one hour. For each experiment, one 24-well-plate was prepared as holding plate. 12 wells of each plate were filled with 300 μl assay medium, the other 12 with 300 μl MTT medium. One additional plate was left empty. The plates were stored in the incubator.

APPLICATION OF TEST SUBSTANCE AND CONTROL SUBSTANCES: for each experiment (three minutes and one hour) two 6-well plates were used. Afte pre-incubation, the assay medium was replaced by fresh assay medium. Two wells were used as negative control, two wells were used as positive controls and two other wells for testing the test material. The solid test item was ground, and 25 mg test item were applied to the skin model after which the test item was moistened with 25 μl H2O.

REMOVAL OF TEST MATERIAL AND CONTROLS: after the respective incubation time, the inserts were removed from the plates using sterile forceps. The inserts were thoroughly rinsed with PBS, blotted with sterile cellulose tissue and set into the respective holding plate, using the wells containing assay medium. Afte transfer of all inserts, they were immediately moved to the wells containing MTT solution, blotting the bottom with cellulose tissue again before setting the insert into the MTT well.

TISSUE VIABILITY MEASUREMENT AFTER TREATMENT / EXPOSURE: the tissues were incubated with MTT medium for three hours. After this time, the MTT medium was aspirated and replaced by PBS buffer. This was then aspirated too and replaced several times. At last, each insert was thoroughly dried and set into the empty pre-warmed 24-well-plate. Into each well, 2 ml isopropanole were pipetted, taking care to reach the upper rim of the insert. The plate was then covered with parafilm and left to stand overnight at room temperature. On the next day, the inserts in which formazan had been produced overnight were pierced with an injection needle, taking care that all colour was extracted. The inserts were then discarded and the plate was gently shaken for 15 minutes in order to achieve homogenisation. From each well, three replicates with 200 μl solution each were pipetted into a 96-well-plate which was read in a plate spectral photometer at 570 nm.

EVALUATION: the photometric absorption of the negative controls was considered as 100 %. For the mean of the three replicates of test item and positive control, formazane production was calculated as % photometric absorption compared with the negative control.

DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the formazane production after 3 minutes exposure is less than 50 %, or if the formazane production after 3 minutes exposure is greater than 50 % and the viability after 1 hour exposure is less than 15 %.
- The test substance is considered to be non-corrosive to skin if the formazane production after 3 minutes exposure is greater than 50 % and the formazane production after 1 hour exposure is greater than 15 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 mg

VEHICLE: 25 μl deionised water was used to moisten the test item applied in the skin model.

NEGATIVE CONTROL
- Amount applied: 50 μl.

POSITIVE CONTROL
- Amount applied: 50 μl.

Duration of treatment / exposure:

three minutes and one hour.

Number of replicates:

two replicates per negative, positive control and test item.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

formazane production

Run / experiment:

three minutes

Value:

103.8

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

formazane production

Run / experiment:

one hour

Value:

74.3

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS
- Acceptance criteria met for negative control: negative control was within the normal range (O.D > 0.8) showing an O.D of 1.377.
- Acceptance criteria met for positive control: positive control showed a clear corrosive effect, with value of three minute-experiment being 0.521 and value of one-hour experiment being 0.314.

Any other information on results incl. tables

The absorption values of negative control, test item and positive control are given in the table below.

Table: absorption values.

Negative control		Test item		Positive control
Tissue 1	Tissue 2	Tissue 1	Tissue 2	Tissue 1	Tissue 2
1.635	1.387	1.553	1.526	0.442	0.521	3 min
1.653	1.337	1.625	1.521	0.509	0.54
1.728	1.33	1.595	1.597	0.538	0.573
1.197	1.585	1.07	0.966	0.303	0.293	1 hour
1.175	1.574	1.018	1.025	0.305	0.305
1.14	1.589	1.031	1.03	0.332	0.345
Mean		Mean		Mean
1.512		1.57		0.521		3 min
1.377		1.023		0.314		1 hour

For the test item and positive control the percentage values of formazan production were calculated in comparison with the negative control and are presented in the following table.

Table: % formazane production.

Test item	Positive control
103.80%	34.40%	3 min
74.30%	22.80%	1 hour

Applicant's summary and conclusion

Interpretation of results:

other: classified as irrtant to the skin according to the CLP Regulation (EC) No.1272/2008

Conclusions:

The substance is considered as not corrosive, but as a skin irritant.

Executive summary:

The corrosion potential of the substance to the skin was assessed in-vitro according to the OECD Guideline 431. The substance was applied topically to a three-dimensinal human skin model, comprising a reconstituted epidermis with a functional stratum cornneum. Corrosivity was identified by the ability of the substance to produce a decrease in cell viability quantified by the formazane production. For this reason, the tissues, after the removal of the substance, were incubated with MTT medium and the optical density of the extracted formazan was measured at 570 nm. Negative and positive controls run in parallel; the % photometric absorption of test item and positive control was calculated by comparison with the negative control.

Relative absorbance were increased to 103 % after three minutes and were decreased to 74.3 % after one hour treatment with the substance. These values are laying above the threshold for corrosivity.

However, taking in consideration the corrosion potential of the substance to eye, the substance should be considered as a skin irritant.

The substance is not considered to be corrosive to the skin but as a skin irritant.