bis({9-[2-(carboxy-κO)phenyl]-6-(bis(tallow alkyl hydrogenated)amino)-3H-polyheterocycl-3-ylidene}-3-bis(tallow alkyl hydrogenated)iminium) zinc(2+) dichloride

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation / corrosion

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 February 2009 to 20 February 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and EC guidelines and according to GLP regulations.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD 431: In vitro Skin Corrosion: Human Skin Model Test (13 April 2004)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Details on test material:

Substance type: dark purple waxy solid
- Physical state: solid
- Stability under storage conditions: stable
- Storage condition of test material: at room temperature in the dark

Test animals

Species:

other: human-derived epidermal keratinocytes

Details on test animals or test system and environmental conditions:

EpiDerm Skin Model (EPI-200, Lot no.: 11250 kit C). The model 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 foundin vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

Tissues: On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 ml DMEM medium.

DMEM (Dulbecco’s Modified Eagle’s Medium): Supplemented DMEM medium, serum-free supplied by MatTek Corporation.

MTT medium: MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation.

Environmental conditions: All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 73 - 88%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.2 - 37.1°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the humidity (with a maximum of 7%) occurred that were caused by opening and closing of the incubator door, but the time of these deviations did not exceed 1 hour. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Test system

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg
- The solid test substance was crushed and ground in a mortar with pestle to improve the consistency. Twenty-five mg of fine ground test substance was applied after moistening with 25 µl Milli-Q water, to ensure close contact to the tissue. Custom Red #3 was spread to match the size of the tissue.

Duration of treatment / exposure:

3 minutes and 1 hour exposure times

Observation period:

After exposure the skin tissue is thoroughly rinsed to remove the test substance followed by immediate determination of the cytotoxic (corrosive) effect.

Number of animals:

The test was performed on a total of 4 tissues per test substance together with a negative control and positive control. Two tissues were used for a 3-minute exposure (under yellow light) to Custom Red #3 and two for a 1-hour exposure (in the incubator in the dark).

Details on study design:

TEST SITE
- Area of exposure: human skin model
- % coverage: 0.6 cm2
- Type of wrap if used:


REMOVAL OF TEST SUBSTANCE
- Washing (if done): physiological saline
- Time after start of exposure: 3 minutes and 1 hour


SCORING SYSTEM: percentage viability

Results and discussion

In vivo

Results

Irritation parameter:

other: percentage viability

Basis:

other:

Time point:

other: 3 minutes and 1 hour

Score:

ca. 99 - ca. 105

Irritant / corrosive response data:

The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with Custom Red #3 compared to the negative control tissues was 99% and 105% respectively. The mean relative tissue viability for Custom Red #3 was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment.

Any other information on results incl. tables

The absolute mean OD₅₄₀(optical density at 540 nm) of the negative control tissues was within the laboratory historical control data range. The mean relative tissue viability following 3-minute exposure to the positive control was 7%. The maximum inter-tissue variability in viability between two tissues treated identically was less than 9% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 5%. It was therefore concluded that the test system functioned properly.

 

Applicant's summary and conclusion

Interpretation of results:

other: not corrosive

Remarks:

Criteria used for interpretation of results: other: OECD guideline 431

Conclusions:

Custom Red #3 is not corrosive in the in vitro skin corrosion test.