Phosphoric acid, hexadecyl esters, potassium salts with cetyl alcohol and isostearyl isostearate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 07, 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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:

Phosphoric acid, hexadecyl esters, potassium salts with cetyl alcohol and isostearyl isostearate

EC Number:

947-751-9

Molecular formula:

C16H34O4P1K1 (representative: mono- C16 PSE, K+) C16H34O1 (representative alcohol: C16 alcohol) C36H72O2 (isostearyl isostearate)

IUPAC Name:

Phosphoric acid, hexadecyl esters, potassium salts with cetyl alcohol and isostearyl isostearate

Test material form:

solid

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Eyes from adult cattle were obtained from local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee and placed in Hanks’s Balanced Salt Solution (HBSS), supplemented with Penicillin/Streptomycin, and transported to the laboratory on ice packs. The eyes were refrigerated on arrival and used within 24 of receipt.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Neat test substance. For the purpose of this study the test substance was pulverised and chopped to a suitable consistency before use.

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

First incubation, with the test substance for 240 min. Subsequently a post treatment opacity reading was taken and each cornea was visually observed.
Second incubation: 1 mL of sodium fluorescein solution (5 mg/mL) at 32 ± 1 ºC for 90 minutes.

Number of animals or in vitro replicates:

Three corneas were allocated to the negative, positive control and the test substance.

Details on study design:

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 cornea were immersed (epithelial side uppermost) 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 minimum essential medium (MEM) and plugged. The holders were incubated at 32 ± 1 °C for at least 60 minutes. At the end of incubation period each cornea was examined fr defects. Only corneas free of damage were used.

Selection of Corneas and Opacity Reading
The medium from both chambers of each holder was replaced with fresh complete MEM. 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 also numerically allocated to the negative control substance and three corneas to the positive control test substance.

Treatment of Corneas
The MEM was removed from the anterior chamber of the BCOP holder and the neat test substance or control substances were applied to the cornea. The holders were gently tilted back and forth to ensure a uniform application of the substance over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 °C for 240 minutes. At the end of exposure period the test substance and control substances were removed from the anterior chamber and the cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM. The anterior and posterior chambers were refilled with fresh complete MEM. A post-treatment opacity reading was taken and each cornea were visually observed.

Application of Sodium Fluorescein
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Permeability Determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 μL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured. If values greater than 1.500 OD492 were obtined a 1 in 5 dilution of the medium in complete MEM was performed and the measurement repeated. The modified value was multiplied by 5 to reflect dilution.

Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10 % neutral buffered formalin.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

240 min

Value:

1

Negative controls validity:

valid

Remarks:

3.3

Positive controls validity:

valid

Remarks:

99.6

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Criteria:
- A test substance that induces an In Vitro Irritancy Score >55.1 is defined as an ocular corrosive or severe irritant.
Result: the test substance was considered not to be an occular corrosive or severe irritant.

- 20 % w/v Imidazole was used for positive control purposes. The test is acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean for this testing facility. In this case should be within: 55.8 - 126.1.
Result: passed

Applicant's summary and conclusion

Interpretation of results:

other: not classified based on EU CLP criteria

Conclusions:

Under the study conditions, the test substance was determined to be non-corrosive or non-irritating to the eye.

Executive summary:

An in vitro study was conducted to determine the corneal damage potential by the test substance, 'mono- and di- C16 PSE, K+ and C16-OH and isostearyl isostearate' (100%), using the Bovine Corneal Opacity and Permeability (BCOP) method according to OECD Guideline 437, in compliance with GLP. One valid experiment was performed with three replicates for negative control, positive control and the test substance. The undiluted test substance was applied in a way that as much as possible of the corneas surface was covered. Subsequently corneas were incubated for 240 minutes at 32 ± 1°C in BCOP corneal holder. At the end of the exposure period the test substance and control substances were removed from the anterior chamber and the cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM. The anterior and posterior chambers were refilled with fresh complete MEM. A post treatment opacity reading was taken and each cornea was visually observed. Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium was replaced with 1 mL of sodium fluorescein solution (5 mg/mL) and corneas were incubated again at 32 ± 1°C for 90 minutes. After incubation 360 μL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured. The two endpoints opacity and permeability were combined in an empirically derived formula to generate an in vitro irritancy score (IVIS). The obtained in vitro irritancy scores for the test substance, negative control and positive control were 1 (which is well below the threshold for no classification for eye), 3.3, and 99.6, respectively. The study was considered to have met all the validity criteria. Under the study conditions, the test substance was determined to be non-corrosive or non-irritating to the eye (Harlan, 2012).