Carbamic acid, (2-hydroxypropyl)-, compd. with 1-amino-2-propanol (1:1)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Principles of method if other than guideline:

The HCE model is currently involved in the eye irritation validation conducted by COLIPA following ECVAM guidelines. Furthermore, it is routinely used by the major Cosmetic and Pharmaceutical companies, and has already been prevalidated in 2004 (van Goethem et. al., Tox in Vitro 20, 2006, 1-17). This model is recognized as the model of choice and scientifically relevant as documented by several publications (Cotovio et. al., Tox. in Vitro, 24, 2010, 523-537).

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(2-hydroxypropyl)carbamic acid; 1-aminopropan-2-ol

EC Number:

605-539-0

Cas Number:

169115-74-6

Molecular formula:

C7 H18 N2 O4

IUPAC Name:

(2-hydroxypropyl)carbamic acid; 1-aminopropan-2-ol

Details on test material:

- Composition of test material, percentage of components: approximately 50% active compound in ethylene glycol

Test animals / tissue source

Species:

other: Assessment of ocular irritation potential of the test substance by determination of cytotoxic effects on a human corneal epithelium (HCE) cell model (exposure 60 min./rt followed by 16 hours incubation at 37 °C, subsequently MTT).

Details on test animals or tissues and environmental conditions:

The experiment was carried out on a Human Corneal Epithelial (HCE) Model, which is standardized and commercially available (SkinEthic, France). Inserts were of 0.5 cm² size. When cultivated at the air-liquid interface in a chemically defined medium, the immortalized human cornea epithelial cells reconstruct a corneal epithelial tissue (mucosa), without a stratum corneum, ultra-structurally (tissue morphology and thickness) similar to the corneal mucosa of the human eye.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: as negative control: Phosphate Buffered Saline (PBS, 30 µL)

Amount / concentration applied:

30 µl per insert

Duration of treatment / exposure:

60 min at room temperature

Observation period (in vivo):

post-exposure incubation: 16 hours (37°C, 5% CO2, maximum humidity)

Details on study design:

The irritation potential of the test item is assessed by determination of its cytotoxic effect on an reconstructed human ocular epithelia. The test principle is based on the MTT assay reflecting the cell viability after exposure of the cornea equivalent to topically applied test item.
Tests were performed in triplets. The test item was applied at a 100% concentration, i.e. 30 µl per insert, for 60 min at room temperature. After the exposure period the inserts were washed carefully with PBS. After a post-exposure incubation of 16 hours in the incubator (37 +/- 2 °C, 5 % CO2, maximum humidity) MTT reduction was performed. Cell viability was measured by the amount of MTT reduction, i.e. an OD value following exposure to the negative or positive control substances or the test item.

Positive control: 1H-1,2,4-Triazole-3-thiol (30 mg, plus 30 µl PBS for moistening)

Results and discussion

Any other information on results incl. tables

A test substance is predicted to be an ocular irritant if the mean relative tissue viability (%) exposed to the test substance is ≤ 50 %.

Sample No.	Test item	%Viability
1 -3	negative control PBS	100.00
4 -6	positive control 1H-1,2,4 -Triazole-3 -thiol	22.63
16 -18	test substance	0.42

The test item was detected as irritant under the conditions of this test model.

Applicant's summary and conclusion

Interpretation of results:

other: irritant property in vitro

Executive summary:

An in vitro study for assessing ocular irritation of chemicals using a human 3D-corneal epithelial (HCE) cell model was conducted with carbamic acid, (2-hydroxypropyl)-, compound with 1-amino-2-propanol (1:1) in ethylene glycol. The cell model, routinely used by the major Cosmetic and Pharmaceutical companies, has been prevalidated in 2004 (van Goethem et. al., Tox in Vitro 20, 2006, 1-17). The pure test item (30 µl per insert) was applied topically to the reconstructed HCE tissue. After an exposure period of 60 minutes followed by a 16 hours post-treatment incubation period the cell viability was measured as about 0.4 % by the MTT conversion assay. A test item is predicted to be an ocular irritant if the mean relative cell viability is ≤ 50 %

after exposure. Therefore it is concluded that the test item it is an ocular irritant under the conditions of this test method.