Phosphoric acid, 2-ethylhexyl ester, ammonium salt

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 March 2017 to 08 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted in accordance with international guideliens and in accordance with GLP. All guidelines validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium mono(2- ethylhexyl)phosphate

Molecular formula:

C8H22NO4P

IUPAC Name:

Ammonium mono(2- ethylhexyl)phosphate

Constituent 2

Reference substance name:

Ammonium bis(2-ethylhexyl) phosphate

EC Number:

225-615-3

EC Name:

Ammonium bis(2-ethylhexyl) phosphate

Cas Number:

4971-47-5

Molecular formula:

C16H38NO4P

IUPAC Name:

Ammonium bis(2- ethylhexyl)phosphate

Constituent 3

Reference substance name:

2-ethylhexyl diphosphate ammonium salts / esters of 2-ethylhexan-1-ol with diphosphoric acid and their corresponding ammonium salts

Molecular formula:

C8H23NO7P2

IUPAC Name:

2-ethylhexyl diphosphate ammonium salts / esters of 2-ethylhexan-1-ol with diphosphoric acid and their corresponding ammonium salts

Test material form:

liquid

Details on test material:

Storage Conditions: Room temperature.

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: local abattoir.
- 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 removed after slaughter, completely immersed in Hanks’ Balanced Salt Solution (containing penicillin at 100 IU/mL and streptomycin at 100 µg/mL) in a suitably sized container and transported on the same day to the testing facility.
- Time interval prior to initiating testing: same day
- indication of any existing defects or lesions in ocular tissue samples: only corneas free of defects were used.
- Indication of any antibiotics used: penicillin at 100 IU/mL and streptomycin at 100 µg/mL solution used to transport eyes to testing facility.

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): 750 µL
- Concentration (if solution): n/a

VEHICLE
- Amount(s) applied (volume or weight with unit): n/a
- Concentration (if solution): n/a
- Lot/batch no. (if required): n/a
- Purity: n/a

Duration of treatment / exposure:

10 mins

Duration of post- treatment incubation (in vitro):

Following exposure and rinsing, corneas were incubated (horizontally) for 2 hours after which, corneal opacity was measured and then the anterior chamber emptied. For the permeability endpoint, 1 mL of sodium fluorescein (4 mg/mL solution) was added into the anterior chamber and the corneas were incubated in the vertical position for 1 hour and 25 minutes at 32±1°C.

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: On arrival at the test facility the eyes were carefully examined for defects including increased opacity, scratches and neovascularisation. Only corneas free from such defects were used.

QUALITY CHECK OF THE ISOLATED CORNEAS: The opacity of each cornea was measured using an opacitometer. Any corneas found to have scratches or increased neovascularization or an opacity of >7 opacity units when examined prior to treatment were discarded.

NUMBER OF REPLICATES: 3 per treatment group.

NEGATIVE CONTROL USED: The negative control substance was 0.9% sodium chloride solution supplied by Baxter Healthcare Ltd., Thetford, UK.

SOLVENT CONTROL USED (if applicable): n/a

POSITIVE CONTROL USED: The positive control substance was dimethylformamide supplied by Sigma-Aldrich Company Ltd., Poole, UK.

APPLICATION DOSE AND EXPOSURE TIME: 10 mins

TREATMENT METHOD: Not reported

POST-INCUBATION PERIOD: No

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After exposure, each cornea was washed with media containing phenol red (as a pH indicator) until this indicator showed no pH effect occurring (and demonstrating that the test article had been removed successfully). Cotton buds were used to aid the removal of the test article. The corneas were then washed once in media without phenol red.

- POST-EXPOSURE INCUBATION: After test item exposure and rinsing, the corneas were incubated (horizontally) for 2 hours after which, corneal opacity was measured and then the anterior chamber emptied. For the permeability endpoint, 1 mL of sodium fluorescein (4 mg/mL solution) was added into the anterior chamber and the corneas were incubated in the vertical position for 1 hour and 25 minutes.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opaciometer.
- Corneal permeability: Passage of sodium fluorescein dye measured with the aid of spectrophotometry (OD 490).
- Others (e.g, pertinent visual observations, histopathology): (please specify) n/a

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used. As per OECD 437.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean value

Value:

33.93

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control yields opacity and permeability values that are less than the established upper limits for these endpoints for bovine corneas as treated at the testing facility.
- Acceptance criteria met for positive control: The positive control yields an IVIS within 2 standard deviations of the historical control mean.
- Range of historical values if different from the ones specified in the test guideline: n/a

Any other information on results incl. tables

Table 1       Corneal Opacity

Test Substance	Cornea Number	Initial Opacity	Post Incubation Opacity	Change in Opacity	Mean Change in Opacity	Corrected Opacity	Mean Corrected Opacity
Test article	7	-2	9	11	N/A	11.0	12.7
	8	-2	11	13		13.0
	15	-2	12	14		14.0
Negative	1	-2	-2	0	0.00	0.0	0.0
	2	-2	-2	0		0.0
	6	-2	-2	0		0.0
Positive	3	-3	66	69	N/A	69.0	73.3
	16	-2	72	74		74.0
	17	-2	75	77		77.0

 

Table 2       Corneal Permeability

Test Substance	Cornea Number	Mean Blank OD490	OD490	Corrected OD490	Mean Corrected OD490	Final Corrected OD490	Mean Group Corrected OD490
Test article	7		1.435	1.435	N/A	1.426	1.417
	8		1.444	1.444		1.435
	15		1.400	1.400		1.391
Negative	1	0.000	0.009	0.009	0.009	0.000	0.000
	2		0.005	0.005		-0.004
	6		0.014	0.014		0.004
Positive	3		0.274	0.274	N/A	0.265	0.421
	16		0.308	0.308		0.299
	17		0.708	0.708		0.698

 

Table 3       Calculated IVIS

Test Chemical	Mean Opacity	Mean Permeability	IVIS (Mean Opacity + (15 x Mean Permeability))
Test article	12.7	1.417	33.93
Negative control	0.00	0.000	0.000
Positive control	73.3	0.421	79.64

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

It was concluded that under the condition of this study, the test item, produced an IVIS score of 33.93 and therefore no prediction could be made in respect of its potential to cause eye irritation.

Executive summary:

OECD 437 (2017) - The Bovine Corneal Opacity and Permeabilty (BCOP) test was conducted using reaction mass of the test item in accordance with 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" (2013). Undiluted test item was applied evenly to the surface of three corneas before being washed off with media solution after 10 minute test item contact time. A negative and positive control group, each containing 3 corneas, were also prepared. Measurements for corneal opacity were made after 2 h incubation in the horizontal position with fresh media. Measurements for corneal permeability were made following 1 h and 25 min incubation in the vertical position with sodium fluorescein. Corneal opacity and corneal permeability media solutions were analysed by a spectrophotometer at 490 nanometers (OD₄₉₀). The mean corrected opacity reading and permeability readings for the test item were 12.7 and 1.417, resulting in an In Vitro Irritation Score (IVIS) of 33.93. As a result, no prediction could be made in respect to the test items potential to cause eye irritation.