(4-carboxybutyl)triphenylphosphonium bromide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 Feb 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 Ocular Corrosives and Severe Irritants)

Version / remarks:

9th October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(4-carboxybutyl)triphenylphosphonium bromide

EC Number:

241-782-5

EC Name:

(4-carboxybutyl)triphenylphosphonium bromide

Cas Number:

17814-85-6

Molecular formula:

C23H24BrO2P

IUPAC Name:

(4-carboxybutyl)triphenylphosphonium bromide

Test material form:

solid: crystalline

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany
- Number of animals: not reported
- Characteristics of donor animals (e.g. age, sex, weight): 15 month
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Freshly isolated bovine eyes of donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were stored in HBSS (Hank’s Balanced Salt Solution) containing 1% (v/v) penicillin/streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin) in the cooled slaughter-house and during transportation on the same morning to the laboratory using a Styrofoam box. The corneae were isolated on the same day after delivery of the eyes.
- Time interval prior to initiating testing: The corneae were directly used in the BCOP test on the same day.
- indication of any existing defects or lesions in ocular tissue samples: No
- Indication of any antibiotics used: see above

Test system

Vehicle:

other: 0.9% (w/v) NaCl in deionised water

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 20% suspension (w/v) in 0.9% (w/v) NaCl

Duration of treatment / exposure:

240 min

Number of animals or in vitro replicates:

3

Details on study design:

COLLECTION OF BOVINE EYES

Freshly isolated bovine eyes of donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were stored in HBSS (Hank’s Balanced Salt Solution) containing 1% (v/v) penicillin/streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin) in the cooled slaughter-house and during transportation on the same morning to the laboratory using a Styrofoam box. The corneae were isolated on the same day after delivery of the eyes.

PREPARATION OF CORNEAE
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. The corneae were directly used in the BCOP test on the same day.
Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, which consists of anterior and posterior compartments, which interface with the epithelial and the endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure that no air bubbles were present within the compartments.
For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath. At the end of the incubation period, the medium was changed before basal opacity measurement (t0).
Only corneae with a value of the basal opacity < 7 were used. Sets of three corneae were used for treatment with the test item and for the negative and positive controls, respectively.

NUMBER OF REPLICATES
3

SOLVENT CONTROL USED

Saline (0.9% (w/v) NaCl in deionised water)

POSITIVE CONTROL USED
10% (w/v) Benzalkonium chloride (Sigma, lot BCBQ4331) in 0.9% (w/v) NaCl (saline) using sonication for 10 minutes.

APPLICATION DOSE AND EXPOSURE TIME

The anterior compartment received the test item suspension or the negative or positive controls at a volume of 0.75 mL each on the surface of the corneae via open chamber method, respectively. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath.
The incubation time lasted 240 minutes.

TREATMENT METHOD: open chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
at least three times or more until phenol red was still discoloured (yellow or purple), or the test item was still visible

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
opacitometer (OP_KiT opacitometer (Electro Design, 63-Riom, France))
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
IVIS GHS / EU CLP
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
> 55 Category 1

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean of 3 replicates

Value:

66.82

Vehicle controls validity:

valid

Remarks:

IVIS: 1.31

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

IVIS: 91.43

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

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

Any other information on results incl. tables

Results after 240 min treatment time

 

 

Test Group	Opacity value (t0)	Opacity value (t240)	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Standard Deviation in vitro score	Proposed in vitro Irritancy Score
				Mean
Vehicle control	0	1	1	0.33	0.07	0.065	2.05	1.31	0.64	No Category
	0	0	0		0.061		0.82
	0	0	0		0.065		0.98
Positive control	2	80	77.67*		0.551	0.486*	84.95	91.43	5.88	Category 1
	1	84	82.67*		0.749	0.684*	92.92
	2	88	85.67*		0.782	0.717*	96.42
Test item	0	68	67.67*		0.075	0.010*	67.81	66.82	4.54	Category 1
	0	62	61.67*		0.078	0.013*	61.86
	0	71	70.67*		0.073	0.008*	70.78

 

*corrected values

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

The test item caused an increase in corneal opacity. The calculated mean in vitro irritancy score was 66.82. According to OECD 437 fulfils the classification criteria for serious eye damage (EU CLP/GHS Category 1).