9H-Fluoren-9-ol, 9,9'-[1,1'-biphenyl]-4,4'-diylbis-

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-02-26 to 2014-03-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: In Vitro Testing Strategy for Ocular Hazard Classification and Labeling of Antimicrobial Cleaning Products, Appendix B: ICCVAM-Recommended EpiOcular™ (EO) Test Method Protocol, published 2010.

Version / remarks:

published 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

9-[4'-(9-hydroxy-9H-fluoren-9-yl)-[1,1'-biphenyl]-4-yl]-9H-fluoren-9-ol

EC Number:

804-773-2

Cas Number:

427165-44-4

Molecular formula:

C38H26O2

IUPAC Name:

9-[4'-(9-hydroxy-9H-fluoren-9-yl)-[1,1'-biphenyl]-4-yl]-9H-fluoren-9-ol

Constituent 2

Reference substance name:

9,9’-biphenyl-4,4’-diylbis(9H-fluoren-9-ol)

IUPAC Name:

9,9’-biphenyl-4,4’-diylbis(9H-fluoren-9-ol)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Purity: >98.5%
Physical form: White solid powder

Test animals / tissue source

Species:

other: in vitro EpiOcular-EIT Model

Details on test animals or tissues and environmental conditions:

The following EpiOcular™-EIT Model was used:
Lot. No. 19146; OCL-200-EIT, MatTek In Vitro Life Science Laboratories, s.r.o Mlynské Nivy 73, 821 05 Bratislava II, Slovak Republic.

Test system

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

Test item was applied as solid.
50 mg of test item and 50 μL of the negative and positive controls were administered neat by topical application onto the tissue surface, so that the
surface of the tissue was completely covered by the test item.

Duration of treatment / exposure:

6-hour treatment period

Duration of post- treatment incubation (in vitro):

18+/- 0.25 h

Number of animals or in vitro replicates:

n=3 tissues/condition

Details on study design:

Assessment of coloured or staining materials
- To identify the potential of the test item to interfere with the MTT assay, the following checks were performed:
50 mg test item were added to 1 ml sterile deionised water and incubated in the dark at 37°C in a humidified atomosphere of 5% CO2 for 60 minutes. Furthermore 50 mg test item were added to 2 ml isopropanol, incubated for 2 hours. No discoloration of the test item either in water or
isopropanol was noted. Hence, no possible interacting with the MTT measurement had to be considered and no additional test had to be performed.

PRE-TREATMENT:
- Wetted tissues were incubated at standard culture conditions for 30 +/- 2 minutes

TREATMENT:
- 50 mg of test item was applied as solid to the tissue, forming a relatively smooth even layer on the surface
- Three replicate tissues were employed
- Positive control item was metyl acetate
- Negative control was sterile deionised water
- 50 μL of negative and positive controls were used
- Tissues were placed back into the culture medium after dosing and incubated at standard culture conditions for 6 hours ± 15 minutes

POST-TREATMENT INCUBATION:
- Test item and controls were removed by extensively rinsing the tissues with Ca++Mg++free D-PBS
- After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously-warmed assay medium in a pre-labeled 12-well
plate for a 25 ± 2 minute immersion incubation at room temperature
- Each insert was removed from the assay medium, the medium was decanted off the tissues, the inserts were blotted on absorbent material and
transferred to the appropriate wells of the pre-labeled 6-well plate containing 1 mL of warm assay medium
- Tissues were incubated for 18 ± 0.25 hours at standard culture conditions

MTT ASSAY:
- After post-treatment incubation of 18 hours, the MTT assay was performed
- 300 μL of the MTT solution was added to each designated well of a pre-labeled 24-well plate, each insert was removed from the 6-well plate and
gently blotted on absorbent material
- Tissues were placed into the 24-well plate containing 0.3 mL of MTT solution. Three construct tissues were used for each treatment and concurrent control groups
- All tissues were placed into the 24-well plate, the plate was incubated for 180 ± 10 minutes at standard culture conditions
-Each insert was removed from the 24-well plate after 180 ± 10 minutes, bottom of each insert was blotted on absorbent material and transferred
to a pre-labeled 24-well plate containing 2.0 mL of propanol-2
- Plates were sealed with parafilm and stored overnight at 2-8°C in the dark for extraction
- Subsequently plates were placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature
- Extract solution was mixed and 200 μL aliquots were transferred to the appropriate wells of pre-labeled 96-well plates
- Optical density (OD) was measured at a wavelength of 570 nm in a spectrophotometer (Tecan Sunrise Magellan Version 6.47)
- Measurements were made for each of the three tissues in duplicate

Results and discussion

In vitro

Results

Irritation parameter:

other: % tissue viability compared to control

Remarks:

test meets acceptance criteria

Value:

93.3

Negative controls validity:

valid

Remarks:

sterile deionized water (100% viability)

Positive controls validity:

valid

Remarks:

methyl acetate (19.5% viability relative to the negative control)

Remarks on result:

no indication of irritation

Any other information on results incl. tables

Acceptance criteria for test results

The results are acceptable if:

- the negative control OD > 1.0 and < 2.3

- the mean relative viability of the positive control methyl acetate is below 60% of negative control viability

- the difference of viability between the tissues of a single chemical is < 20% in the same run (for positive and negative control

tissues and tissues of single chemicals).

Data evaluation and interpretation

The individual % of control OD570 values were averaged to calculate the mean percent of control. The relative tissue viability was determined against the negative control. If the test item-treated tissue viability is > 60% relative to negative controltreated tissue viability, the test item is predicted to be non-irritant. If the test item-treated tissue viability is ≤ 60% relative to negative controltreated tissue viability, the test article is predicted to be irritant.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be non-cytotoxic and predicted to be non-irritant.

Executive summary:

The purpose of the experiment was to evaluate the potential ocular irritation of the test item by measuring

3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) dye conversion by the EpiOcular™ tissue construct after topical exposure to test item.

The EpiOcular (OCL-200) was used. Three construct tissues were used for each treatment and concurrent control groups. The optical density (OD) was determined by photometrical measurement of formazan production as a result of enzymatic reduction of the vital dye MTT (3-[4,5 - dimethylthiazol-2-yl] - 2,5 - diphenyltetrazolium bromide) and expressed as relative percentage of viability of the negative control-treated constructs.

50 mg of test item and 50 μL of the concurrent negative (sterile deionised water) or positive control (methyl acetate) were administered by topical application onto the construct on cultures for 6 hours, followed by a post-incubation for 18 hours.

The mean viability of the cells exposed to the test item was 93.3% of the mean negative control value. Hence, the OD570 values were above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of > 60%.

Result: The test item was considered to be non-cytotoxic and predicted to be non-irritant.

The mean optical density (OD) of the negative control of 3 tissues was 1.498 and was well within the acceptable range of ≥ 1.0 to ≤ 2.3. The viability of cells treated with the positive reference item, methyl acetate, was 19.5% of the negative controls and below the 60% cut-off value of the negative controls. The standard deviation of all triplicates determined was below the limit of acceptance of 20%. Hence, all acceptance criteria required were fulfilled.