4,4a,5,9b-tetrahydroindeno[1,2-d]-1,3-dioxin

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 -29 Apr 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

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)

Deviations:

yes

Remarks:

no historical data of positive and negative controls

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Test material information

Constituent 1

Reference substance name:

4,4a,5,9b-tetrahydroindeno[1,2-d]-1,3-dioxin

EC Number:

241-997-4

EC Name:

4,4a,5,9b-tetrahydroindeno[1,2-d]-1,3-dioxin

Cas Number:

18096-62-3

Molecular formula:

C11H12O2

IUPAC Name:

2H,4H,4aH,5H,9bH-indeno[1,2-d][1,3]dioxine

Test animals / tissue source

Species:

human

Strain:

other: EpiOcular™

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: approx. 50 mg

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

in duplicates for each treatment and contol group

Details on study design:

- RhCE tissue construct used, including batch number: EpiOcular™ (MatTek Corporation, Bratislava, Slovakia), Lot No.: 23707
- Tissue viability: The quality of the final product was assessed by undertaking an MTT cell viability test. The determined OD (540 - 570 nm) was 1.918 ± 0.095 (acceptance criteria: 1.1 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 0.3% Triton X-100. The ET-50 value was determined to be 15.12 min (acceptance criteria: 12.2 - 37.5 min).
- Contamination: The cells used to produce the EpiOcular tissue were screened for the presence of viruses, bacteria, yeast and other fungi.
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: 6 hours exposure (37 °C), 25 min post-exposure immersion (room temperature), 18 hours post-exposure (37 °C)
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water or isopropanol did not led to a change in colour. Therefore, an additional test with viable tissues without MTT addition was not necessary. Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent did not show blue colour. Therefore, an additional test with freeze-killed tissues was not necessary.
- Description of the method used to quantify MTT formazan: The absorbance at 570 nm of each well was measured with a plate reader (Versamax Molecular Devices, Ismaning, Germany). No reference wavelength measurement was used.
- Decision criteria/Prediction model: If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labeled non-irritant. If the test item-treated tissue viability is ≤ 60% relative to negative control treated tissue viability, the test item is labeled irritant.
- Acceptance criteria: The results are acceptable if 1) the negative control OD is > 0.8 and < 2.5, 2) the mean relative viability of the positive control is below 50% of the negative control viability and 3) the difference of viability between the two relating tissues of a single test substance is < 20% in the same run (for positive and negative control tissues and tissues of test substances).

Results and discussion

In vitro

Results

Irritation parameter:

other: % mean relative absorbance of 2 tissues

Run / experiment:

6 hours exposure

Value:

85.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control OD (1.352 and 1.573) was in the range of > 0.8 and < 2.5.
- Acceptance criteria met for positive control: The mean relative viability of the positive control is below 50% of the negative control viability (13.0%).

The difference of viability between the two relating tissues of a single item is < 20% (values between 0.0% to 15.1%) in the same run (for positive and negative control tissues and tissues of single test items).

Any other information on results incl. tables

Table 2. Results after 6 hours incubation time

Test group	Mean absorbance*		Mean absorbance of 2 tissues*	Rel. absorbance (%)**		Absolute value of the difference of the rel. absorbance (%) Tissue 1 and 2	Rel. absorbance (% of negative control)
	Tissue 1	Tissue 2		Tissue 1	Tissue 2
Negative control	1.352	1.573	1.463	92.4	107.6	15.1	100.0
Positive control	0.189	0.190	0.190	13.0	13.0	0.0	13.0
Test substance	1.332	1.159	1.245	91.1	79.2	11.9	85.1

* Mean of two replicate wells after blank correction

** Relative absorbance (rounded values): 100 × (absorbance test substance/positive control) / (absorbance negative control)

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008

Conclusions:

Under the conditions of the conducted test, the test substance is non irritating towards human-derived epidermal keratinocytes in the EpiOcular™ model.