Benzyltriphenylphosphonium, salt with 4,4'-[2,2,2-trifluoro-1-(trifluoromethyl)ethylidene]bis[phenol] (1:1)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 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)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Benzyltriphenylphosphonium, salt with 4,4'- [2,2,2- trifluoro-1- (trifluoromethyl)ethylidene]bis[phenol] (1:1)

Cas Number:

75768-65-9

IUPAC Name:

Benzyltriphenylphosphonium, salt with 4,4'- [2,2,2- trifluoro-1- (trifluoromethyl)ethylidene]bis[phenol] (1:1)

Test material form:

solid

Details on test material:

Purity: 98 %

Specific details on test material used for the study:

Purity: > 98%

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Bovine eyes were obtained from a local abattoir as a by-product from freshly slaughtered animals (J.W. TREUTH & SONS, Inc., Baltimore, MD).
- Number of animals: not reported
- Characteristics of donor animals (e.g. age, sex, weight): not reported
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The eyes were excised and then placed in Hanks' Balanced Salt Solution, containing penicillin/streptomycin (HBSS), and transported to the laboratory on ice packs.
- Time interval prior to initiating testing: Immediately upon receipt of the eyes into the laboratory, preparation of the corneas was initiated.
- indication of any existing defects or lesions in ocular tissue samples: The eyes were grossly examined for damage and those exhibiting defects were discarded.
- Indication of any antibiotics used: eyes were transported in HBSS containing penicillin/streptomycin

Test system

Vehicle:

water

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Concentration (if solution): 20% (w/v)

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

90 minutes

Number of animals or in vitro replicates:

4 replicates for test substance
3 replicates each for negative and positive controls

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- The tissue surrounding the eyeball was carefully pulled away and the cornea was excised such that a 2 to 3 mm rim of sclera was present around the cornea. The isolated corneas were then stored in a petri dish containing HBSS until they were mounted in a corneal holder. The corneas were mounted in the holders with the endothelial side against the O-ring of the posterior chamber. The anterior chamber was then positioned on top of the cornea and the screws were tightened. The chambers in the corneal holder were filled with Minimum Essential Medium without phenol red, containing 1% fetal bovine serum and 2 mM L-glutamine (Complete MEM (without phenol red)). The corneal holders were incubated at 32±1°C for a minimum of 1 hour.

QUALITY CHECK OF THE ISOLATED CORNEAS
-After a minimum of 1 hour of incubation, the corneas were removed from the incubator. The medium was removed from both chambers and replaced with fresh Complete MEM (without phenol red). The initial opacity was determined for each cornea using an Electro Designs OP-KIT opacitometer. The treatment of each cornea was identified with the test article number written in permanent marker on colored tape, affixed to each holder. The medium was then removed from the anterior chamber and replaced with the test article, positive control, or negative control.

NUMBER OF REPLICATES
- 3 positive controls, 3 negative controls, 4 corneas with test substance

NEGATIVE CONTROL USED
-Sterile, deionized water

POSITIVE CONTROL USED
- 20% (w/v) dilution of imidazole in Complete MEM without phenol red.

APPLICATION DOSE AND EXPOSURE TIME
- The solid test article was tested as a 20% (w/v) dilution in sterile, deionized water. An aliquot of 750 μL of the test article, positive control, or negative control was introduced into the anterior chamber while slightly rotating the holder to ensure uniform distribution over the cornea. Three corneas were incubated in the presence of the negative control at 32 ± 1ºC for approximately 4 hours. Three corneas were incubated in the presence of the positive control at 32 ± 1ºC for approximately 4 hours. Four corneas were incubated in the presence of the test article at 32 ± 1ºC for approximately 4 hours.

TREATMENT METHOD: open chamber

REMOVAL OF TEST SUBSTANCE
- After the 4-hour exposure period, the control or test article treatments were removed. The epithelial side of the corneas treated with the controls was washed at least three times with Complete MEM (containing phenol red) to ensure total removal of the controls. The corneas were then given a final rinse with Complete MEM (without phenol red). For the corneas treated with the test article (open chamber technique), the glass window was removed from the anterior chamber and the test article was rinsed from the treated corneas with Complete MEM (with phenol red). Care was taken not to spray the corneas directly. The chamber windows were returned to the chambers when most or all of the test article had been remove d. The rinsing process continued in the same manner as the positive and negative control corneas. The medium in the posterior chamber was aspirated. The posterior and anterior chambers were refilled with fresh Complete MEM (without phenol red) and then a final opacity measurement was performed immediately (with no further post-exposure incubation).
- Post-exposure incubation:After the final opacity measurement was performed, the medium was removed from the anterior chamber of the holder and replaced with 1 mL of a 5 mg/mL fluorescein solution. The corneas were then incubated in a horizontal position (anterior side up) for approximately 90 minutes at 32 ± 1ºC.

PERMEABILITY
- At the end of the 90-minute incubation period, the medium was removed from the posterior chamber and placed into tubes numbered corresponding to chamber number. Aliquots of 360 μL from the numbered tubes were placed into their designated wells on a 96-well plate. The optical density at 490 nm (OD490) was determined using a Molecular Devices Vmax kinetic microplate reader.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The change in opacity for each cornea (including the negative control corneas) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting from each the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of each cornea for that treatment condition.
- Corneal permeability: The mean OD490 value for the blank wells was calculated. The mean blank OD490 value was then subtracted from the raw OD490 value of each well (corrected OD490). The final corrected OD490 values of the test article and the positive control were then calculated by subtracting the average corrected OD490 value of the negative control corneas from the corrected OD490 value of each treated cornea:

Final Corrected OD490 = (raw OD490 – mean blank OD490) – average corrected negative control OD490

The mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated corneas for that treatment condition.

- Other: After the medium was removed fro the permeability determination, each cornea was carefully separated from its corneal holder and transferred to an individual labeled tissue cassette containing a biopsy sponge. THe endothelial surface of each cornea was placed on the sponge to protect it. The cassettes were placed in 10% neutral buffered formalin to fix the corneal tissue for at least 24 hours. The fixed corneas will be stored up to one year.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS): In Vitro Score = Mean Opacity Value + (15 x Mean OD490 Value)

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

22.4

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: Yes

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was determined to have an in vitro score of 22.4, indicating it is inconclusive regards eye irritation. Using the results of the existing in vivo supportive study, which indicates a slight irritation was reversible after 7 days, one may conclude these results are supporting a non eye irritation classification.

Executive summary:

The purpose of this study was to evaluate the potential ocular irritancy of the test substance, as measured by changes in opacity and permeability (to fluorescein) in isolated bovine corneas according to OECD guideline 437. The test substance was administered to the test system as a 20% (w/v) dilution in sterile, deionized water. Corneas (test substance, positive and negative controls) were incubated in the presence of the test substance at 32 ± 1ºC for 4 hours. The positive control met the acceptance criteria and therefore, the assay was considered valid. The test substance was determined to have an in vitro score of 22.4, indicating it is inconclusive regards eye irritation.