3,6-bis(diethylamino)-9-[2-(methoxycarbonyl)phenyl]xanthylium tetrachlorozincate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 July 2016 to 22 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test"

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3,6-bis(diethylamino)-9-[2-(methoxycarbonyl)phenyl]xanthylium tetrachlorozincate

EC Number:

277-459-0

EC Name:

3,6-bis(diethylamino)-9-[2-(methoxycarbonyl)phenyl]xanthylium tetrachlorozincate

Cas Number:

73398-89-7

Molecular formula:

C29H33N2O3.1/2Cl4Zn

IUPAC Name:

bis{3,6-bis(diethylamino)-9-[2-(methoxycarbonyl)phenyl]xanthenium} tetrachlorozincate(2-)

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Greenish powder
- Storage conditions of test material: At room temperature
- Stable until: 10 June 2021 (expiry date)

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Recommended test system in international guidelines

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200, kit J)
- Tissue batch number(s): Lot no.: 24306
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 organised 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 EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.
- Source: MatTek Corporation, Ashland MA, USA

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37.0 ± 1.0 °C
- Temperature of post-treatment incubation (if applicable): 37 °C

CELL CULTURE
- 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 supplemented DMEM medium (Dulbecco’s Modified Eagle’s Medium, serum-free).
- MTT medium: MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM).
- Environmental conditions: All incubations, with the exception of the test material incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 to 100 %, containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0 °C.

NUMBER OF REPLICATE TISSUES: 2 per exposure time

TEST FOR THE INTERFERENCE OF THE TEST MATERIAL WITH THE MTT ENDPOINT
The test material was checked for possible colour interference before the study was started. To assess the colour interference, at least 25 mg or 50 μL Milli-Q water as a negative control were added to 0.3 mL Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0 °C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue / purple colour change was observed.
Since the test material induced colour interference in aqueous conditions, in addition to the normal procedure, two tissues must be treated with test material for 3 minutes and two tissues for 1-hour. Instead of MTT solution these tissues will be incubated with DMEM medium.

TEST FOR REDUCTION OF MTT BY THE TEST MATERIAL
The test material was checked for possible direct MTT reduction before the study was started. To assess the ability of the test material to reduce MTT, at least 25 mg was added to 1 mL MTT solution (1 mg/mL) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0 °C. A negative control, sterile Milli-Q water was tested concurrently.

APPLICATION/TREATMENT OF THE TEST MATERIAL
The skin tissues were kept in the refrigerator the day they were received. The next day, at least 1 hour before the assay was started the tissues were transferred to 6-well plates containing 0.9 mL DMEM medium per well. The level of the DMEM medium was just beneath the tissue. The plates were incubated for approximately 2 hours at 37.0 ± 1.0 °C. The medium was replaced with fresh DMEM medium just before the test material was applied. The test was performed on a total of 4 tissues per test material together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test material and two for a 1-hour exposure. The skin was moistened with 25 μL Milli-Q water to ensure close contact of the test material to the tissue and 25.2 to 28.5 mg of the solid test material was added into the 6-well plates on top of the skin tissues. The remaining tissues were treated with 50 μL Milli-Q water (negative control) and with 50 μL 8 N KOH (positive control), respectively.
In addition to the normal procedure, two tissues were treated with test material for 3 minutes and two tissues for 1-hour. Instead of MTT solution these tissues were incubated with medium. After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test material, however the purple test material was incorporated into the tissue. Rinsed tissues were kept in 24 well plates on 300 μL DMEM medium until 6 tissues (= one application time) were dosed and rinsed.

CELL VIABILITY MEASUREMENT
The DMEM medium was replaced by 300 μL MTT-medium and tissues were incubated for 3 hours at 37 °C in air containing 5 % CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol overnight at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as percentage of the mean of the negative control tissues. Skin corrosion potential of the test material was classified according to remaining cell viability following exposure of the test material with either of the two exposure times.

INTERPRETATION
- Acceptability of the assay
The in vitro skin corrosion test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range.
b) The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
c) In the range 20 to 100 % viability, the Coefficient of Variation (CV) between tissue replicates should be ≤30 %.
d) The non-specific colour should be ≤30 % relative to the negative control OD.

- Data evaluation and statistical procedures
A test material is considered corrosive in the skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50 %.
b) In addition, a test material considered non-corrosive (viability ≥ 50 %) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test material is decreased below 15 %.
A test material is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50 %.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15 %.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25.2 to 28.5 mg of the solid test material was added into the 6-well plates on top of the skin tissues.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL Milli-Q water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL KOH
- Concentration (if solution): 8 N

Duration of treatment / exposure:

3 minutes of exposure and 1 hour of exposure

Duration of post-treatment incubation (if applicable):

incubated for 3 hours with MTT

Number of replicates:

2 per exposure time

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Remarks on result:

not determinable because of methodological limitations

Other effects / acceptance of results:

The test material was checked for colour interference in aqueous conditions and for possible direct MTT reduction by adding the test material to MTT medium. As a purple colour change was observed in aqueous conditions it was concluded that the test material showed colour interference. In addition to the normal procedure, two tissues were treated with test material for 3 minutes and two tissues for 1-hour but instead of MTT solution these tissues were incubated with medium. The non-specific colour by the test material was 49 and 132 % of the negative control tissues after 3-minutes and 1 hour, respectively.

Skin corrosion is expressed as the remaining cell viability after exposure to the test material. The mean tissue viability of the test material could not be calculated due to the too high non-specific colour.

ACCEPTANCE OF RESULTS
The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The mean relative tissue viability following the 1 hour exposure to the positive control was 8 %. In the range of 20 - 100 % viability the Coefficient of Variation between tissue replicates was <14 %, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the conditions of this study, the non-specific colour was above acceptance criteria therefore the test material is not compatible with the test system and no conclusion can be made on the corrosive potential of the test material.

Executive summary:

The potential of the test material to cause skin corrosion was assessed in an in vitro skin corrosion test using a human skin model in accordance with the standardised guidelines OECD 431 and EU Method B.40 BIS under GLP conditions.

Skin tissue was moistened with 25 μL of Milli-Q water and at least 25 mg of test material was applied directly on top of the skin tissue. The test consists of topical application on the skin tissue for 3-minute and 1-hour exposure periods. After exposure the skin tissue is thoroughly rinsed to remove the test material, followed by immediate determination of the cytotoxic (corrosive) effect. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The test material showed colour interference. In addition to the normal procedure, two tissues were treated with test material for 3 minutes and two tissues for 1-hour, however instead of MTT solution these tissues were incubated with medium. The non-specific colour by the test material was 49 and 132 % of the negative control tissues after 3-minutes and 1 hour, respectively.

The positive control had a mean relative tissue viability of 8 % after the 1 hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. In the range of 20 - 100 % viability the Coefficient of Variation between tissue replicates was < 14 %, indicating that the test system functioned properly.

Under the conditions of this study, the non-specific colour was above acceptance criteria therefore the test material is not compatible with the test system and no conclusion can be made on the corrosive potential of the test material.