Methylidynetri-p-phenylene triisocyanate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key 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:

2013

Report date:

2013

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:

Methylidynetri-p-phenylene triisocyanate

EC Number:

219-351-8

EC Name:

Methylidynetri-p-phenylene triisocyanate

Cas Number:

2422-91-5

Molecular formula:

C22H13N3O3

IUPAC Name:

1-[bis(4-isocyanatophenyl)methyl]-4-isocyanatobenzene

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).

Strain:

other: not applicable

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:

other: ethyl acetate

Controls:

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

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)

Number of animals or in vitro replicates:

not applicable

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

It is not advisable to test isocyanates in aqueous vehicles. Therefore, ethyl acetate had been chosen to moisture the test item before application to the cornea epithelium. Ethyl acetate is classified as an eye irritant which was confirmed by the results of this assay. The results of this assay show that the test substance Triphenylmethane-4,4’,4’’-triisocyanate also has an irritant effect, which however increases the irritant property of ethyl acetate.

Therefore the test item was detected as irritant to the cornea epithelium under the conditions of this test (s. Table 1)

Table1: Summary of results

Compound	Cell Viability [%]	Evaluation
negative control PBS	100.00	non-irritant
positive control 1H-1,2,4 -Triazole-3 -thiol	24.41	irritant
test substance	2.76	irritant

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 Triphenylmethane-4,4',4''-triisocyanate. 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 mg 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 3 % 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 Triphenylmethane-4,4´,4´´-triisocyanate is an ocular irritant under the conditions of this test method.