Indene

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 Jul 2018 - 10 Aug 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:

29 July 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:

31 May 2008

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Indene

EC Number:

202-393-6

EC Name:

Indene

Cas Number:

95-13-6

Molecular formula:

C9H8

IUPAC Name:

indene

Test material form:

liquid

Details on test material:

Identification: Indene

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

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin corrosion tests is the EPIDERM test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Test system: EpiDerm Skin Model (EPI-200, Lot no.: 28864 Kit H, Kit I and Kit J).
- 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 organized 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.
-Media used: Supplemented, serum-free DMEM (Dulbecco’s Modified Eagle’s Medium) supplied by MatTek Corporation.

ENVIRONMENTAL CONDITIONS
- Test item incubation was carried out for 3 minutes at room temperature.
- All other incubations were carried out in a controlled environment: humid atmosphere of 80 - 100% (actual range 67 - 84%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.4 - 36.7°C).

TEST FOR INTERFERENCES
- The test substance was checked for possible color interference and reduction of MTT before the study. No interference was found.

APPLICATION OF TEST ITEM
Before the assay was started, the tissues were transferred to 6-well plates containing 0.9 mL DMEM per well. The plates were incubated for approximately 1.5 hours at 37.0 ± 1.0 ºC. The medium was replaced with fresh DMEM just before Indene was applied. The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to Indene and two for a 1-hour exposure. 50 μL of the undiluted test item was added into the 6-well plates on top of the skin tissues. In addition, since the test item reacted with the MTT medium, two freeze-killed tissues were treated with test item and two freeze-killed non treated tissues were used per exposure time for the cytotoxicity evaluation with MTT. 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. After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item. 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.

REMOVAL OF TEST MATERIAL AND CONTROLS
After the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item.

MEASUREMENT OF CELL VIABILITY
- MTT concentrate (5 mg/mL) was diluted 1:5 with supplemented DMEM: MTT-medium
- MTT-medium was added to the wells (300 μL)
- Incubation time: 3 hours at 37°C in air containing 5% CO2.
- Formazan was extracted with 2 mL isopropanol (MatTek Corporation) 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.

ACCEPTABILITY CRITERIA
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 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%.
d) The non-specific MTT reduction should be ≤ 30% relative to the negative control OD.

INTERPRETATION
A test item is considered corrosive in the in vitro 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 item 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 item is decreased below 15%.
A test item 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

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

50 μL

Duration of treatment / exposure:

3 minutes (2 tissues)
1 hour (2 tissues)

Duration of post-treatment incubation (if applicable):

3 hours with MTT-medium

Number of replicates:

- 2 tissues were used for a 3-minute exposure to Indene and two for a 1-hour exposure.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure (mean of two experiments)

Value:

109

Negative controls validity:

valid

Remarks:

100 %

Positive controls validity:

valid

Remarks:

8.4 %

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour exposure (mean of two experiments)

Value:

52

Negative controls validity:

valid

Remarks:

100 %

Positive controls validity:

valid

Remarks:

8.3 %

Other effects / acceptance of results:

- The test item values are corrected for the non-specific MTT reaction (6.94% and 15% of the negative control tissues at the 3 minute and 1 hour treatment, respectively).
- 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 of the positive control was 8.3%.
- In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 21%, indicating that the test system functioned properly.
- Individual results are included below, under "Any other information on results incl. tables".

Any other information on results incl. tables

Table 1

Absorption in the in vitro Skin Corrosion test with Indene

	3 minute application				1 Hour application
	A (OD570)	B (OD570)	Mean (OD570)	SD	A (OD570)	B (OD570)	Mean (OD570)	SD
Negative control	1.456	1.704	1.580	0.175	1.728	1.921	1.825	0.137
Indene	1.585	1.854	1.719	0.190	0.837	1.056	0.946	0.155
Positive control	0.137	0.128	0.133	0.006	0.113	0.191	0.152	0.055

 

Table 2

Mean tissue viability in the in vitro Skin Corrosion Test with indene

	3-minute application Viability (percentage of control)	1-hour application Viability (percentage of control)
Negative control	100	100
Indene	109	52
Positive control	8.4	8.3

 

Table 3: Historical Control Data for in vitro Skin Corrosion Studies

 

	Negative control		Positive control
	3-minute treatment (OD570)	1-hour treatment (OD570)	3-minute treatment (OD570)	1-hour treatment (OD570)
Range	1.258–2.615	1.371–2.371	0.0172–0.56	0.046–0.339
Mean	1.80	1.82	0.19	0.14
SD	0.26	0.22	0.09	0.05
n	111	110	106	103

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting

all data over the period of November 2014 to November 2017.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

An in vitro Skin Corrosion Test was performed with Indene according to OECD/EC guidelines and GLP principles. The results do not indicate that Indene has corrosive properties.

Executive summary:

The human three dimensional epidermal model EpiDerm (EPI-200) was used to assess the skin corrosion hazard potential of Indene, according to the most recent OECD guideline 431 and following GLP principles. Indene was applied undiluted. Results of the negative and positive control tissues were within the laboratory historical control data range, indicating that the system functioned properly.

Indene was found to minimally interact with MTT and the results were corrected for the non-specific reduction. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with Indene compared to the negative control tissues was 109% and 52%, respectively. Indene is therefore concluded to be not corrosive to the skin and it does not need classification and labelling for skin corrosion according to UN GHS.