2-amino-2-methylpropane-1,3-diol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July-November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

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 (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-amino-2-methylpropane-1,3-diol

EC Number:

204-100-7

EC Name:

2-amino-2-methylpropane-1,3-diol

Cas Number:

115-69-5

Molecular formula:

C4H11NO2

IUPAC Name:

2-amino-2-methylpropane-1,3-diol

Details on test material:

- Name of test material (as cited in study report): Aminomethyl propanediol, AMPD

Specific details on test material used for the study:

AMPD ULTRA PC (ANGUS Chemical Company)
- CAS name: 2-amino-2-methyl-1,3-propanediol
- CAS number: 115-69-5
- appearance: solid, white crystals
- molecular weight: 105.1 g/mol
- batch #VK014801I1
- purity: 99.5%
- sum of impurities: 0.48%

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.

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 system

Vehicle:

water

Controls:

yes, concurrent positive control

Amount / concentration applied:

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 OP-KIT 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 article 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 article, positive control, or negative control. The solid test article, AMPD, was tested as a 20% (w/v) dilution in sterile, deionized water. An aliquot of 750 µL of the test article, positive control, or negative control was introduced into the anterior chamber while slightly rotating the holder to ensure uniform distribution over the cornea.

Duration of treatment / exposure:

Three corneas were incubated in the presence of the negative or positive control at 32 ± 1ºC for approximately 4 hours. Five corneas were incubated in the presence of the test article at 32 ± 1ºC for approximately 4 hours. After the 4-hour exposure period, the control or test article treatments were removed. The epithelial side of each cornea was washed at least three times with Complete MEM (containing phenol red) to ensure total removal of the control or test articles. The corneas were then given a final rinse with Complete MEM (without phenol red). The anterior and the posterior chambers were refilled with fresh Complete MEM (without phenol red) and a final opacity measurement was performed immediately (with no further post-exposure incubation).

Duration of post- treatment incubation (in vitro):

After the final opacity measurement was performed, the medium was removed from the anterior chamber of the holder and replaced with 1 mL of a 5 mg/mL fluorescein solution. 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 sample was 1.500 or above, a 1:5 dilution of the sample was prepared in Complete MEM (without phenol red). 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.

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. The fixed corneas will be stored up to one year.

Results and discussion

In vitro

Results

Irritation parameter:

cornea opacity score

Value:

63.9

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The BCOP assay was accepted as the positive control (imidazole) caused an in vitro score that fell within two standard deviations of the historical mean.

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

The test substance is considered to be corrosive in the in vitro assay with isolated bovine eyes.

Executive summary:

The purpose of this study was to evaluate the potential ocular irritancy of AMPD as measured by changes in opacity and permeability (to fluorescein) in isolated bovine corneas. Four to five corneas were treated with the test article. Based on changes in corneal opacity and permeability (relative to the control corneas), an in vitro score was determined for the test article. After a 4 hour exposure, the in vitro score for AMPD was 63.9. Therefore, the test substance is considered to be corrosive in the in vitro assay with isolated bovine eyes.