Condensation products of fatty acids, tall oil with 2-amino-2-ethylpropanediol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 June 2017-15 January 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:

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:

Condensation products of fatty acids, tall oil with 2-amino-2-ethylpropanediol

EC Number:

946-010-7

Molecular formula:

None

IUPAC Name:

Condensation products of fatty acids, tall oil with 2-amino-2-ethylpropanediol

Test material form:

liquid: viscous

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

Bovine eyes were obtained from a local abattoir as a by-product from freshly slaughtered animals (J.W. TREUTH & SONS, Inc., Baltimore, MD). The eyes were excised and then placed in Hanks' Balanced Salt Solution, containing Penicillin/Streptomycin (HBSS), and transported to the laboratory on ice packs. Immediately upon receipt of the eyes into the laboratory, preparation of the corneas was initiated.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Details on study design:

Preparation of Corneas
The eyes were grossly examined for damage and those exhibiting defects were discarded. 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. Starting with the posterior chamber, the two chambers were then filled with Minimum Essential Medium (EMEM) without phenol red, containing 1% fetal bovine serum and 2 mM L-glutamine (Complete MEM (without phenol red)). Each corneal holder was uniquely identified with a number written in permanent marker, on both the anterior and posterior chambers. The corneal holders were incubated at 32 ± 1ºC for a minimum of 1 hour.

Test Substance Preparation
Alkaterge E, was administered to the test system without dilution.

Test Substance pH Determination
The pH of the test substance was determined using pH paper (EMD Millipore Corporation; Billerica, MA). Initially, each test substance was added to 0-14 pH paper with 1.0 pH unit increments to approximate a narrow pH range. Next, the test substances were added to 0-6 and 5-10, or 7.5-14.0 pH paper with 0.5 pH unit increments, to obtain more accurate pH values.

Bovine Corneal Opacity and Permeability Assay
The liquid test substances Alkaterge E was tested on 17 July 2017. 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 OPKIT opacitometer. Any cornea with an initial opacity greater than 7 was not used in the assay. The treatment of each cornea was identified with the test substance 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 substance, positive control, or negative control.

Alkaterge E was tested neat. An aliquot of 750 μL of the test substance, positive control, or negative control was introduced into the anterior chamber while slightly rotating the holder to ensure uniform distribution over the cornea. Each treated cornea was completely covered with the test substance.
Three corneas were incubated in the presence of the negative or positive control at 32 ± 1ºC for 10 minutes. Four corneas were incubated in the presence of each test substance at 32 ± 1ºC for 10 minutes. After the 10-minute exposure times, the control or test substance treatments were removed. The epithelial side of the corneas was washed at least three times with Complete MEM (containing phenol red) to ensure total removal of the control or test substances. The corneas were then given a final rinse with Complete MEM (without phenol red). The anterior chambers were refilled with fresh Complete MEM (without phenol red) and an opacity measurement was performed. The corneas were returned to the incubator for approximately 2 hours after which a final measure of opacity was obtained. After the final opacity measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was filled with fresh Complete MEM (without phenol red) and 1 mL of a 4 mg/mL fluorescein solution was added to the anterior chamber. The corneas were then incubated in a horizontal position (anterior side up) for approximately 90 minutes at 32 ± 1ºC. 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. If the OD490 value of a control or test substance sample was 1.500 or above, a 1:5 dilution of the sample was prepared in Complete MEM (without phenol red) (to bring the OD490 value within the linear range of the platereader). A 360 μL sample of each 1:5 dilution was transferred to its specified well on the 96-well plate. The plate was read again and the final reading was saved to a designated print file.

Fixation of Corneas
After the medium was removed for the permeability determination, each cornea was carefully separated from its corneal holder and transferred to an individual prelabeled 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.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

Alkaterge E

Value:

2.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

Ethanol (positive control)

Value:

46.3

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

positive indication of irritation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance is not irritating to isolated bovne eyes.

Executive summary:

The Bovine Corneal Opacity and Permeability Assay with Optional Histology was used assess the potential ocular corrosivity or severe irritancy of Alkaterge E to isolated bovine corneas. The ocular irritancy potential of the test substances were evaluated using the protocol that is consistent with the OECD Test Guideline 437 “Bovine Corneal Opacity and Permeability Test Method for Identifying Chemicals Inducing Serious Eye Damage and Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage”. A test substance was predicted as a GHS Category I if the In Vitro Score was > 55. A test substance was predicted to not require classification or labelling for eye irritation or serious eye damage (GHS No Category), if the In Vitro score was ≤ 3.0. According to the current prediction model, Alkaterge E, was predicted to not require classification or labelling for eye irritation or serious eye damage (GHS No Category).