Indene

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Sept 2018 - 21 Sept 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

No relevant information was available that allowed hazard assessment. Therefore testing was carried out in accordance with the requirements of Annexes VII of the REACH Regulation. As the BCOP assay is an accepted in vitro method and recommended for identifying substances that cause serious eye damage, this test was performed first. The outcome of a BCOP assay did not allow conclusion on classification. Therefore, as a next step, a second in vitro test was performed. The EpiOcular™ Cornea Epithelial Model was chosen as it is an internationally accepted test method for serious eye damage/eye irritation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

25 June 2018

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

Test animals / tissue source

Species:

human

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 μL of the undiluted test item was added into the 6-well plates on top of the tissues


VEHICLE
- No

Duration of treatment / exposure:

30 ± 2 minutes at 37.0 ± 1.0°C

Duration of post- treatment incubation (in vitro):

Post-soak: 12 ± 2 minute immersion incubation at room temperature.
Incubation in 6-well plate containing 1.0 mL of warm Assay Medium: 120 ± 15 minutes at 37°C.
With MTT: 180 ± 10 minutes at 37°C.

Number of animals or in vitro replicates:

Two tissues per test item
Two tissues per control (positive and negative)
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.

Details on study design:

TEST SYSTEM
- EpiOcular™ OCL-200-EIT MatTek Corporation
- Lot: 27446 Kit C
- The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes. It models the cornea epithelium with progressively stratified, but not cornified cells. The tissue is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.

TEST FOR THE INTERFERENCE WITH MTT ENDPOINT
- Test for Color Interference by the Test Item: Yes.
- Test for Reduction of MTT by the Test Item: Yes.

APPLICATION TEST ITEM
- 2 tissues per test item together with a negative control and positive control

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes, with Ca2+Mg2+-free D-PBS at room temperature
- Time after start of exposure: 30 ± 2 minutes at 37.0 ± 1.0°C

SCORING SYSTEM: mean absorption at 570 nm.

TOOL USED TO ASSESS SCORE: TECAN Infinite® M200 Pro Plate Reader.

ACCEPTABILITY CRITERIA
The in vitro eye irritation 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 > 0.8 and < 2.5.
b) The mean relative tissue viability of the positive control should be <50% relative to the negative control.
c) The difference between the % tissue viabilities of the two identically treated replicates should be <20.
d) The non-specific MTT reduction should be ≤ 50% relative to the negative control OD.

INTERPRETATION
The test chemical is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and postexposure incubation is less than or equal (≤) to 60%.

Results and discussion

In vitro

Results

Irritation parameter:

other: mean relative tissue viability (%)

Run / experiment:

mean of two experiments

Value:

31

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

(mean cell viability 20%)

Remarks on result:

other: considered to be potentially irritant or corrosive to the eye

Other effects / acceptance of results:

INTERFERENCE WITH MTT ENDPOINT
- Test item did not induce color interference.
- Test item showed color change in presence of MTT. Therefore it was concluded that the test item interacted with MTT. In addition to the normal procedure, two freeze-killed tissues were treated with test item and one freeze-killed negative control treated tissue were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT (NSMTT) by Indene was -0.5% of the negative control tissues. Since the NSMTT was ≤ 0.0%, there is no need to correct for interference of the test item because it was considered a minor effect.

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY:
- The difference between the percentage of viability of two tissues treated identically was less than 10%, indicating that the test system functioned properly.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes, mean cell viability after 30 ± 2 minutes exposure of 20%.
- The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range.

- Individual results are included under "Any other information on results incl. tables"

Any other information on results incl. tables

Table 1

Mean Absorption in the EpiOcular™ Test with Indene

	A (OD570)	B (OD570)	Mean (OD570)	SD
Negative control	1.731	1.729	1.730	0.001
Indene	0.513	0.576	0.544	0.044
Positive control	0.425	0.256	0.340	0.119

OD = optical density

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background absorption (0.043). Isopropanol was used to measure the background absorption.

 

Table 2

Mean tissue viability in the EpiOcular™ Test with Indene

	Mean tissue viability (percentage of control)	Difference between two tissues (percentage)
Negative control	100	0.11
Indene	31	3.62
Positive control	20	9.76

 

Table 3: Historical Control Data for EpiOcular™ Studies

 

	Negative control (absorption; OD570)	Positive control (absorption; OD570)	Positive control (viability; %)
Range	1.077–1.805	0.029–0.793	2.11–48.25
Mean	1.52	0.42	26.86
SD	0.21	0.23	14.11
n	16	16	16

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 January 2013 to May 2017.

Applicant's summary and conclusion

Interpretation of results:

other:

Remarks:

Potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1)

Conclusions:

An EpiOcular™ test was performed with Indene according to OECD guideline 492 and GLP principles. Indene was found to be potentially irritant or corrosive under the experimental conditions of this test.

Executive summary:

The reconstructed Human EpiOcular™ Model was used to assess the eye hazard potential of Indene, according to the most recent OECD guideline 492 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 test system functioned properly. Indene was found to minimally interact with MTT (non-specific reduction of MTT by Indene of -0.5%), however the interference was considered minimal and was not further taken into account. The relative mean tissue viability obtained after treatment with Indene was 31%. Indene is therefore concluded to be potentially irritant or corrosive to the eye and potentially to require classification and labelling according to UN GHS (Category 2 or Category 1).