1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylindeno[5,6-c]pyran

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 May 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

09 October 2017

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylindeno[5,6-c]pyran

EC Number:

214-946-9

EC Name:

1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylindeno[5,6-c]pyran

Cas Number:

1222-05-5

Molecular formula:

C18H26O

IUPAC Name:

4,6,6,7,8,8-hexamethyl-1H,3H,4H,6H,7H,8H-indeno[5,6-c]pyran

Test material form:

liquid: viscous

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Eyes from adult cattle (typically 12 to 60 months old) from a local abattoir
- Storage, temperature and transport conditions of ocular tissue: after slaughter eyes were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL) and transported to the test facility over ice packs on the same day of slaughter.
- Time interval prior to initiating testing: the corneas were prepared immediately on arrival.
- indication of any existing defects or lesions in ocular tissue samples: all eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL

Duration of treatment / exposure:

10 minutes

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
QUALITY CHECK OF THE ISOLATED CORNEAS: The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.
NUMBER OF REPLICATES: 3
NEGATIVE CONTROL USED: Sodium chloride 0.9% w/v
POSITIVE CONTROL USED: Ethanol
APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL of the test item or control items were applied at 32 ± 1 ºC for 10 minutes.
REMOVAL OF TEST SUBSTANCE AND POST-EXPOSURE INCUBATION: After exposure the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed. The holders were incubated, at 32 ± 1 ºC for 120 minutes and a final opacity reading was taken.
The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes. After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 μL of media representing each cornea was dispensed into the appropriate wells of a pre-labeled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.
METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD490) The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea.
- Others (visual observations, histopathology): The condition of the cornea was visually assessed post treatment and post incubation. The corneas were retained after testing for possible conduct of histopathology.
SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
DECISION CRITERIA: In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value). Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score. IVIS≤ 3 UN GHS: No Category. IVIS >3; ≤ 55, UN GHS No prediction can be made. IVIS > 55 UN UN GHS Category 1.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

0.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: The corneas treated with the test item were clear post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

ACCEPTANCE OF RESULTS:
In Vitro Irritancy Score: Test Item: 1.5; Negative Control: 0.4; Positive Control: 47.4
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Applicant's summary and conclusion

Interpretation of results:

other: not irritating

Remarks:

in accordance with EU CLP (EC no 1272/2008 and its amendments)

Conclusions:

The substance is not an eye irritant in the BCOP test (OECD guideline 437).

Executive summary:

The eye irriation/corrosion potential of the test item was tested in an OECD TG 437 test, in accordance with GLP. The undiluted test item was applied to bovine eyes for 10 minutes followed by an incubation period of 120 minutes. Negative (sodium chloride 0.9% w/v) and positive (ethanol) control items were tested concurrently, and all tests were performed in triplicate. The two endpoints decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). The corneas treated with the test item and with the negative control were clear post treatment and post incubation, while the corneas treated with the positive control were cloudy post treatment and post incubation. The positive and negative acceptance criteria were fulfilled. The IVIS observed for the test material was 1.5 (negative 0.4 and positive 47.7), therefore the substance is not an eye irritant in the BCOP test.