Ethyltriphenylphosphonium acetate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 March 2018 - 15 March 2018

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:

2019

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ethyltriphenylphosphonium acetate

EC Number:

252-743-7

EC Name:

Ethyltriphenylphosphonium acetate

Cas Number:

35835-94-0

Molecular formula:

C20H20P.C2H3O2

IUPAC Name:

ethyltriphenylphosphanium acetate

Test material form:

solid

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were refrigerated on arrival and used within 24 hours of receipt.

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 min

Duration of post- treatment incubation (in vitro):

240 min

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.
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.
A pre treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated.
Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
Sodium chloride 0.9% w/v

POSITIVE CONTROL USED
Imidazole

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: calibrated opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD492)
- Others (e.g, pertinent visual observations, histopathology): No histopathology was required since a definitive result was achieved.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS):
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)

DECISION CRITERIA:
IVIS UN GHS EU CLP
≤ 3 No Category Not classified for irritation
>3; ≤ 55 No prediction can be made No prediction can be made
> 55 Category 1 Category 1
H318: Causes serious eye damage

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

79.4

Negative controls validity:

valid

Remarks:

IVIS 1.2

Positive controls validity:

valid

Remarks:

IVIS 108.0

Other effects / acceptance of results:

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

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control gave opacity of ≤2.4 and permeability ≤0.072. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The positive control In Vitro Irritancy Score was within the range of 65.1 to 123.3. The positive control acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number	Opacity				Permeability (OD492)		In Vitro Irritancy Score
		Pre-Treatment	Post-Treatment	Post-Treatment-Pre‑Treatment	Corrected Value		Corrected Value
Negative Control	2	4	4	0		0.017
	3	3	4	1		0.013
	6	3	5	2		0.005
				1.0		0.012		1.2
Positive Control	7	4	92	88	87.0	2.130	2.118
	10	4	78	74	73.0	2.260	2.248
	11	3	76	73	72.0	1.785	1.773
					77.3		2.047	108.0
Test Item	12	3	73	70	69.0	0.011	0.000
	13	3	90	87	86.0	0.006	0.000
	14	4	88	84	83.0	0.019	0.007
					79.3		0.002	79.4

 

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

in this study ETPPAAc caused irreversible effects to the eye.

Executive summary:

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short‑term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item ETPPAAc is assessed by quantitative measurements of changes in corneal opacity and permeability.

The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS) and EU Classification, Labelling and Packaging (CLP) of chemicals (Regulation (EC) No 1272/2008), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS and EU CLP Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS/EU CLP Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS/EU CLP No Category.

The test item was applied at a concentration of 20% w/v in sodium chloride 0.9% w/v for 240 minutes. Negative and positive control items were tested concurrently. 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 In Vitro irritancy scores are summarized as follows:

Treatment	In VitroIrritancy Score
Test Item	79.4
Negative Control	1.2
Positive Control	108.0

 

The test item is classified as Category 1. UN GHS or EU CLP Causes serious eye damage.