Indene

Endpoint summary

Description of key information

In order to evaluate skin and eye irritation and corrosion, a set of in vitro tests was performed for Indene (skin: EpiDerm and EpiSkin test; eye: BCOP and Epiocular). These tests were performed according to the most recent OECD guidelines and following GLP principles. Indene was concluded to be irritant in the in vitro skin irritation test. Indene was found not to be corrosive to the skin. Indene was found to be potentially irritant or corrosive to the eye, but the current data-set does not allow final conclusion on classification.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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

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

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 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.

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 %

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".

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.

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.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 Oct 2018 - 19 Nov 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

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 irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, B.46 In vitro Skin Irritation: Reconstructed Human Epidermis Model Test. Official Journal of the EU No. L142; Amended by EC No. 640/2012 OJ No. L193

Version / remarks:

20 July 2012

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE (living tissue)
- Model used: EPISKIN Small Model™
- Tissue batch number: 18-EKIN-046
- Surface: 0.38 cm^2
- Expiration date: 19 November 2018
- Killed tissues (EPISKIN-Small Model™, 0.38 cm2, Lot no.: 18 EKIN 033 and 18 EKIN 045): Living epidermis was transferred to 12 well plates and incubated with 2 mL Milli-Q for 48 ± 1 hours. After incubation, killed epidermis was stored at ≤ -15°C. Killed tissues were thawed by placing them for 1 hour at room temperature in 12 well plates on 2 mL maintenance medium.

TEST FOR THE INTERFERENCE OF THE TEST ITEM WITH THE MTT ENDPOINT
- The test substance was checked for possible color interference and reduction of MTT before the study. Interference was found.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during exposure to the test item: room temperature.
- Temperature of incubations(°C): 37.0 ± 1.0°C (actual range 36 – 36.7°C)
- Humidity(%): 80 - 100% (actual range 70 - 87%)

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: tissues were washed with phosphate buffered saline (1 washing step)
- Observable damage in the tissue due to washing: no

APPLICATION/TREATMENT OF TEST ITEM
- Number of replicate tissues: 3 for the test substance. Additionally, 3 tissues for the negative and the positive control each.
- In addition, three killed tissues treated with test item and three killed untreated tissues were used for the cytotoxicity evaluation with MTT.
- Volumen test item, positive and negative control: 10 μL.
- The positive control was re-spread after 7 minutes contact time.
- Exposure period: 15 minutes.
- Incubation period after exposure: 42 hours at 37°C.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1 single run

MEASUREMENT OF CELL VIABILITY
- After incubation, tissues were dried and incubated with 2 mL MTT-solution (0.3 mg/mL in PBS).
- Incubation time: 3 hours at 37°C in air containing 5% CO2.
- Formazan was extracted with 500 μL isopropanol (MatTek corporation)
- The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

INTERPRETATION (see Table 1)
- A test item is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- A test item is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

ACCEPTABILITY CRITERIA
a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤ 18.
b) The mean relative tissue viability of the positive control should be ≤ 40% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤ 18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤ 18.
d) The non-specific MTT reduction should be ≤ 30% relative to the negative control OD.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

- The test was performed on a total of 3 tissues per test item together with negative and positive controls.
- Test item: 3 tissues were treated with 10 μL of the undiluted test item, added undiluted on top of the skin tissues.
- Negative control: 3 tissues were treated with 10 μL PBS.
- Positive control: 3 tissues were treated with 10 μL 5% SDS. The positive control was re-spread after 7 minutes contact time.
- In addition. 3 killed tissues treated with test item and three killed untreated tissues were used for the cytotoxicity evaluation with MTT.

Duration of treatment / exposure:

15 ± 0.5 minutes at room temperature.

Duration of post-treatment incubation (if applicable):

42 hours at 37°C + 3 hours with MTT.

Number of replicates:

The test was performed on a total of 3 tissues per test item together with negative and positive controls.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Experiment 1

Value:

5.8

Negative controls validity:

valid

Remarks:

(Mean cell viability: 100%)

Positive controls validity:

valid

Remarks:

(Mean cell viability: 11%)

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- Visible damage on test system: No.
- Direct-MTT reduction: The non-specific reduction of MTT by Indene was 0.46%. Therefore, the net OD of the treated killed tissues was subtracted from the ODs of the test item treated viable tissues.
- Individual results are included below, under "Any other information on results incl. tables".

Table 1

Mean Absorption in the In Vitro Skin Irritation Test with Indene

 

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)	SD
Negative control	1.213	1.214	1.078	1.169	0.078
Indene	0.068	0.068	0.066	0.068	0.001
Positive control	0.102	0.138	0.137	0.126	0.020

Table 2

Mean Tissue Viability in the In Vitro Skin Irritation Test with Indene

 

	Mean tissue viability (percentage of control)	Standar deviation (percentage)
Negative control	100	6.7
Indene	5.8	0.1
Positive control	11	1.7

Table 3: Historical Control Data for In Vitro Skin Irritation Studies

 

	Negative control (absorption; OD570)	Positive control (absorption; OD570)	Positive control (viability; %)
Range	0.422–1.547	0.023–0.449	2.41–46
Mean	0.96	0.12	13.03
SD	0.17	0.09	9.58
n	174	173	173

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 2015 to November 2018.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

An in vitro Skin Irritation Test was performed with Indene according to OECD/EC guidelines and GLP principles. Indene is irritant in the in vitro skin irritation test and should be classified category 2 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

Executive summary:

The human skin model (EPISKIN Small Model ™) was used to assess skin irritation potential of Indene, according to the most recent OECD guidelines 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. The mean cell viability of Indene was 5.8 %, which implies that Indene is irritant in the in vitro skin irritation test and should be classified category 2 according to the Global Harmonized System of Classification and Labeling of Chemicals (GSH) of the United Nations.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

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.

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

Species:

human

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%.

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"

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.

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).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

As the results from in vitro studies did not allow a conclusive decision on the classification, further in vivo testing was considered. However, since Indene is registered in Annex VII, and this Annex does not foresee in vivo testing for serious eye damage/eye irritation, further animal testing is omitted. Although indene was found to be potentially irritant or corrosive to the eye in an in vitro test, the current data does not allow final conclusion on classification for this endpoint.

Justification for classification or non-classification

Indene is classified as skin irritant (cat. 2) according to Regulation 1272/2008 and its amendments. Indene was found to be potentially irritant or corrosive to the eye, but the current data-set does not allow a final conclusion on classification for effects on the eye.