Fatty Acids, C16-18 and C18-unsatd., diesters with 2-(3-hydroxypropyl)-6-[(3-hydroxypropyl)amino]-1H-benz[de]isoquinoline-1,3(2H)-dione)

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 February 2018 to 23 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline 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:

Fatty Acids, C16-18 and C18-unsatd., diesters with 2-(3-hydroxypropyl)-6-[(3-hydroxypropyl)amino]-1H-benz[de]isoquinoline-1,3(2H)-dione)

EC Number:

814-602-3

Cas Number:

190454-06-9

Molecular formula:

Variable

IUPAC Name:

Fatty Acids, C16-18 and C18-unsatd., diesters with 2-(3-hydroxypropyl)-6-[(3-hydroxypropyl)amino]-1H-benz[de]isoquinoline-1,3(2H)-dione)

Test material form:

liquid

Details on test material:

- Appearance: Amber liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Recommended in international guidelines

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200)
- Source: MatTek Corporation, Ashland MA, U.S.A.
- Tissue Lot no.: 27958. Kits F and G
- The model consists 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 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.

TEST FOR COLOUR INTERFERENCE BY THE TEST MATERIAL
- The test material was checked for possible colour interference before the study was started. Some non-coloured test materials may change into coloured materials in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the colour interference, 50 μL of the test material 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.

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, 50 μL of the test material or 50 μL Milli-Q water as a negative control were 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. At the end of the exposure time it was checked if a blue / purple colour change or a blue / purple precipitate was observed.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37 °C with MTT

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 per well. The level of the DMEM was just beneath the tissue. The plates were incubated for approximately 1 hour at 37.0 ± 1.0 °C. The medium was replaced with fresh DMEM 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. An excessive amount of the undiluted test material was added into the 6-well plates on top of the skin tissues.
- For the negative and positive controls, 2 tissues were treated with 50 μL Milli-Q water (negative control) and 2 tissues were treated with 50 μL 8N KOH (positive control) for both the 3-minute and 1-hour time point.

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test material. During both the 3 minute and 1 hour treatment, the test material was difficult to remove from the skin. Therefore a small amount of residual test material could be left on the skin. The skin inserts were carefully dried. Rinsed tissues were kept in 24 well plates on 300 μL DMEM until 6 tissues (= one application time) were dosed and rinsed.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- The DMEM 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 over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.

NUMBER OF REPLICATE TISSUES: 2

INTERPRETATION
A test material is considered corrosive in the in vitro skin corrosion test if:
- The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50 %.
- 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:
- The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50 %.
- In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15 %.

STEP 1:
< 50 % viability after 3 minute exposure = Corrosive
≥ 50 % viability after 3 minute exposure AND < 15 % viability after 1 hour exposure = Corrosive
≥ 50 % viability after 3 minute exposure AND ≥ 15 % viability after 1 hour exposure = Non-corrosive
STEP 2 (for substances/mixtures identified as Corrosive in step 1):
< 25 % viability after 3 minute exposure = Optional Sub-category 1A
≥ 25 % viability after 3 minute exposure = A combination of optional Sub-categories 1B and 1C

ANALYSIS
- Calculation of Cell Viability:
Optical Density readings were transferred into Microsoft Excel to allow further calculations to be performed.
The corrected OD (ODc) for each sample or control was calculated by subtracting the value of the blank mean (ODbl) from each reading (ODraw).
ODc = ODraw – ODbl
The OD value representing 100 % cell viability is the average OD of the negative controls (ODlt_u+MTT).
The %Viability for each sample and positive control is calculated as follows:
%Viability = (ODc/mean ODlt_u+MTT) * 100

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: an excessive amount of the viscous liquid test material was applied directly on top of the tissue. The test material was spread to match the size of the tissue.

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration: 8 N

Duration of treatment / exposure:

3 minutes and 1 hour

Duration of post-treatment incubation (if applicable):

3 hours with MTT

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-Minute Application

Value:

107

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-Hour Application

Value:

93

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- The test material was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test material to MTT medium. Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that the test material did not interfere with the MTT endpoint.
- Skin corrosion is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test material compared to the negative control tissues was 107 % and 93 % respectively. Because the mean relative tissue viability for the test material was not below 50 % after 3 minutes treatment and not below 15 % after 1 hour treatment the test material is considered to be not corrosive.
- The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥ 0.8 and upper acceptance limit ≤ 2.8) and the laboratory historical control data range. The mean relative tissue viability following the 1-hour exposure to the positive control was 6.0 %. In the range of 20 – 100 % viability the Coefficient of Variation between tissue replicates was <10 %, indicating that the test system functioned properly.

Any other information on results incl. tables

Table 1: Mean Tissue Viability in the in vitro Skin Corrosion Test with The Test Material

Treatment	3-Minute Application Viability (Percentage of Control)	1-Hour Application Viability (Percentage of Control)
Negative Control	100	100
Test Material	107	93
Positive Control	7.5	6.0

 

Table 2: Coefficient of Variation between Tissue Replicates

Treatment	3 Minutes	1 Hour
Negative Control	9.9	8.6
Test Material	4.1	3.0
Positive Control	14	12

CV (%) = 100 - [(lowest OD570/highest OD570) x 100 %]

Applicant's summary and conclusion

Interpretation of results:

other: Not classified in accordance with EU criteria

Conclusions:

Under the conditions of this study, the test material was not corrosive in the in vitro skin corrosion test.

Executive summary:

The skin corrosion potential of the test material was investigated in accordance with the standardised guidelines OECD431 and EU Method B.40 BIS, under GLP conditions.

The objective of this study was to evaluate the test material for its ability to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of the test material was tested through topical application for 3 minutes and 1 hour. An excessive amount of the test material was applied directly on top of the skin tissue.

The positive control had a mean relative tissue viability of 6.0 % after the 1-hour exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥ 0.8 and upper acceptance limit ≤ 2.8) and the laboratory historical control data range. In the range of 20 – 100 % viability the Coefficient of Variation between tissue replicates was <10 %, indicating that the test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test material compared to the negative control tissues was 107 % and 93 %, respectively. Because the mean relative tissue viability for the test material was not below 50 % after the 3-minute treatment and not below 15 % after the 1-hour treatment the test material is considered to be not corrosive.

Under the conditions of this study, the test material was not corrosive in the in vitro skin corrosion test.