Reaction products of tetraethylenepentamine and maleic anhydride

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-04-08 to 2014-04-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test material form:

liquid: viscous

Details on test material:

- Name of test material (as cited in study report): XTA 823- Substance type: yellow slightly viscous liquid- Physical state: liquid- Analytical purity: substance is a UVCB (49.8%)- Lot/batch No.: BLW060113- Expiration date of the lot/batch: 2014-12-30- Stability under test conditions: no data- Storage condition of test material: at room temperature in the dark- other:- Production date: 2014-02-30

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Test animals

Species:

other: reconstructed human epidermis

Strain:

other: not applicable

Details on test animals or test system and environmental conditions:

EPISKIN(TM) Reconstructed Human Epidermis Model Kit- Source: SkinEthic Laboratories, Lyon, France- Date received: 2014-04-08- EpiSkin(TM) Tissues (0.38cm²) lot number: 14-EKIN-012- Maintenance Medium lot number: 14-MAIN3-014- Assay Medium lot number: 14-ESSC-013Pre-incubation (day 0: tissue arrival)- Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert: tissues satisfactory: yes; temperature indicator color satisfactory: yes; agar medium color satisfactory: yes- 2 ml of maintenance medium, warmed to approximately 37°C, was pipetted into the 2 wells of the first column of a pre-labeled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (2 units per plate). A different 12-well plate was used for each test item, control and time point. The tissues were incubated at 37°C, 5% CO2 in air overnight.- After 24 hours the medium underneath the tissues was refreshed an the tissues were returned to the incubator for approximately 24 hours further.

Test system

Type of coverage:

other: not applicable

Preparation of test site:

other: not applicable

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL- Amount(s) applied (volume or weight with unit): 50 µl - Concentration (if solution): used as supplied (undiluted)NEGATIVE CONTROL : 0.9% sodium chloride solution- Amount(s) applied (volume or weight with unit): 50 µl- Batch: 300999 104- Purity: 0.9%- Expiry date: 2015-01-01- Storage conditions: at room temperature- Concentration: used as suppliedPOSITIVE CONTROL: glacial acetic acid- Amount(s) applied (volume or weight with unit): 50 µl- Batch: 114957- Purity: >99.7%- Expiry date: 2014-11-08- Storage conditions: at room temperature- Concentration: used as supplied

Duration of treatment / exposure:

240 minutes

Observation period:

2 days

Details on study design:

PRE-TEST PROCEDURETest for direct MTT reduction (day 0)- 50 µl of the test item was added to 2.0 ml of a 0.3 mg/ml MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37°C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control..MAIN STUDYApplication of test item and rinsing (day 1)- 2.0 ml of assay medium, warmed to approximately 37°C, was pipetted into 2 wells of the second and third columns of the 12-well plate. The tissues were transferred into the second column.- Duplicate tissues were treated with the test item for exposure periods of 3, 60 and 240 minutes. Duplicate tissues were treated with the positive and negative control items for an exposure period of 240 minutes. 50 µl of the test item was applied topically to the corresponding tissus ensuring uniform coverage of the tissues. Duplicate tissues, treated with 50 µl of 0.9% w/v sodium chloride solution, served as negative controls and duplicate tissues, treated with glacial acetic acid served as positive controls. - The treated tissues were kept in a biological safety cabinet at room temperature for the appropriate exposure period.- At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing Dulbecco's phosphate buffered saline (PBS) with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. Each rinsed tissue was placed into to the third column of the 12-well plate until all tissues were rinsed. - 2.0 ml of a 0.3 mg/ml MTT solution freshly prepared in assay medium, was pipetted into 2 wells of the fourth column of each 12-well plate. The tissues were transferred into the MTT filled wells. The tissues were incubated for approximately 3 hours at 37°C, 5% CO2 in air. At the end of the 3-hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was taken using the EPISKIN(TM) biopsy punch. The epidermis was carefully separated from tje collagen matrix using forceps and both parts (epidermis and collagen matrix) were placed into labeled 1.5 ml micro tubes containing 500 µl of acidified isopropanol. Each tube was plugged, mixed thoroughly and stored overnight at room temperature, protected from light, to extract formazan crystals out of the MTT-loaded tissus.Asorbance/optical density measurements (day 2)- At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution.- For each tissue, duplicate 200 µl samples were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 µl of acidified isopropanol alone was added to the two wells designated as 'blanks'. The optical density was measured (quantitative viability analysis) at 562 nm (wihtout a reference filter) using the Anthos 2001 microplate reader.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

after 240 min exposure

Value:

108.1

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

after 60 minutes exposure

Value:

143.9

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

after 3 minutes exposure

Value:

129.1

In vivo

Other effects:

Direct MTT reduction: the MTT solution containing the test item remained yellow. This was taken to indicate that the test item did not reduce MTT.

Any other information on results incl. tables

Quality criteria:

- The relative mean tissue viability for the positive control treated tissues was 3% relative to the negative control treated tissues following the 240-minute exposure period. The positive control acceptance criterion was therefore satisfied.

- The mean OD₅₆₂ for the negative control treated tissues was 0.925. The negative control acceptance criterion was therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:

not classified

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The test item was classified as non-corrosive to the skin. The following classififcation criteria apply: -EU DSD (65/548/EEC): not classified for corrosivity -EU CLP (1272/2008/EC)/UN GHS: not classified for corrosivity-UN Packing Group: non-corrosive.