Tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD TG 431

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

The experiment was carried out on a reconstructed human epidermis EST-1000 (CeIiSystems, St. Katharinen, Germany).

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate

EC Number:

266-442-3

EC Name:

Tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate

Cas Number:

66669-53-2

Molecular formula:

C7H7Na4O9P

IUPAC Name:

tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate

Details on test material:

Content: 90.2%

In vitro test system

Test system:

other: Reconstructed human epidermis EST-1000 (CellSystems, St. Katharinen, Germany).

Source species:

other: Reconstructed human epidermis EST-1000 (CellSystems, St. Katharinen, Germany).

Cell type:

other: Reconstructed human epidermis EST-1000 (CellSystems, St. Katharinen, Germany).

Cell source:

other: Reconstructed human epidermis EST-1000 (CellSystems, St. Katharinen, Germany).

Source strain:

other: Reconstructed human epidermis EST-1000 (CellSystems, St. Katharinen, Germany).

Vehicle:

unchanged (no vehicle)

Amount/concentration applied:

Application of the test material and incubation
For testing of chemical induced corrosivity the EST-1000 inserts were exposed to 25 mg of the test item (plus 50µl 0.9% NaCl to moisten and ensure good contact with the skin) for 3min. (RT) and 60 min. in the incubator (3 inserts per period of incubation time), respectively. 0.9% NaCl (50µl) treated epidermal models were used as negative controls (determination in triplicates).

Duration of treatment / exposure:

Application of the test material and incubation
For testing of chemical induced corrosivity the EST-1000 inserts were exposed to 25 mg of the test item (plus 50µl 0.9% NaCl to moisten and ensure good contact with the skin) for 3min. (RT) and 60 min. in the incubator (3 inserts per period of incubation time), respectively. 0.9% NaCl (50µl) treated epidermal models were used as negative controls (determination in triplicates).

Test animals

Species:

other: not applicable - in-vitro 3D skin corrosion test

Strain:

other: not applicable - in-vitro 3D skin corrosion test

Test system

Type of coverage:

other: not applicable - in-vitro 3D skin corrosion test

Preparation of test site:

other: not applicable - in-vitro 3D skin corrosion test

Vehicle:

other: not applicable - in-vitro 3D skin corrosion test

Controls:

other: not applicable - in-vitro 3D skin corrosion test

Duration of treatment / exposure:

After an exposure period of 3 or 60 minutes, followed by a post-treatment incubation period of about 3 hours, the cell viability was measured

Observation period:

not applicable - in-vitro 3D skin corrosion test

Number of animals:

not applicable - in-vitro 3D skin corrosion test

Details on study design:

Determination of cell viability (MTT)
After the incubation period the inserts were washed carefully in PBS and MTT reduction was performed. For viability testing the inserts were placed in new 24 well plates containing 300 µl of MTT solution (37°C, 1mg/ml in MTT-assay medium, delivered by Cell Systems®). The tissues were incubated for about 3 hours under cell culture conditions (5% C 2, 37°C, max humidity). The extraction of blue formazan was performed in Isopropanol (24 weil plates, 2ml/insert) on a vertical shaker (at least 60 min.). For determination of cell viability the absorption of the Isopropanol extracts were measured in duplicates at 570 nm in an automatie reader (EL808, BioTek; 96 well format, 200 µl).

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: cell viability

Run / experiment:

mean

Value:

105.05

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 3 min

Remarks:

score = 105.05; max. score =100, non corrosive - scores in {%] viability

Results 2

Irritation / corrosion parameter:

other: cell viability

Run / experiment:

mean

Value:

106.25

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 60 min

Remarks:

score = 106.25; max score = 100, non corrosive - scores in [%] viability

Any other information on results incl. tables

Compound       [%] Cell viability 3 min       [%] Cell viability 60 min      Classification*

Bayhibit S                   105.05                                    106.25             Non-corrosive                   

Negative control          100.00                                    100.00              Negative control

*: Classification was done in accordance with the existing guideline and

internationally accepted protocols, i.e. evaluation of LD50 values after 3 min. and/or less than 15% viability after a 60 min. incubation period.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The results show that no corrosive property of the test item was determined by the assay used.

Executive summary:

The experiment was carried out on a reconstructed human epidermis EST-1000 (CellSystems, St. Katharinen, Germany) for detection of topically applied skin corrosives with the test item tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate.

Corrosive skin effects of substances are defined as irreversible damage of skin; namely, visible necrosis through the epidermis and into the dermis, following the application of a test substance for up to four hours. In vivo corrosive reactions are typified by ulcers, bleeding, and bloody scabs.

A 100% concentration was tested on the skin/epidermal equivalents in triplets. For the determination of time related cytotoxic effects the incubation periods were 3 min. and 60 min., respectively. By this the study, was conducted in accordance with the OECD 431 guideline as well as with an EC guideline (amending Council Directive 67/548/EEC, B.40 Skin corrosion) using the test item concentrations and incubation periods recommended there. These tests are also related to the revised OECD 404 guideline "Acute Dermal Irritation/Corrosion".

To check the reliability of this test procedure a blinded positive/negative control study is conducted at regular intervals in the lab. This reliability check is always performed by two technicians, two laboratories in parallel using the same batches of the control test items.

The test item was applied at a 100% concentration, i.e. 25 mg per insert. (plus 50 µl 0.9% NaCl to moisten and ensure good contact with the skin)

The MTT (Methylthiazoletetrazolium) method has determined the following values of viability after 3 min. or after 60 min. of incubation: 105% and 106% (rounded), respectively. Thus, the results show that no corrosive property of the test item was determined by the assay used.