Hexamethylene diisocyanate, oligomers, reaction products with 3-methoxypropylamine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Ferbuary 15, 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:

2017

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:

Hexamethylene diisocyanate, oligomers, reaction products with 3-methoxypropylamine

EC Number:

500-740-9

EC Name:

Hexamethylene diisocyanate, oligomers, reaction products with 3-methoxypropylamine

Cas Number:

162492-07-1

Molecular formula:

C38H69N9O9

IUPAC Name:

Hexamethylene diisocyanate, oligomers, reaction products with 3-methoxypropylamine

Specific details on test material used for the study:

Batch No.: 1591ZG-101; CAS No.: 162492-07-1; EINECS No.: 500-740-9; Purity: >98 %; Appearance: homogeneous, white powder

Test animals / tissue source

Species:

cattle

Strain:

other: Bos primigenius Taurus

Details on test animals or tissues and environmental conditions:

Specification:
Species Bos primigenius Taurus (fresh bovine corneas)

Origin:
Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH. On the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hank’s balanced salt solution (supplemented with 0.01% streptomycin and 0.01% penicillin). Then, the corneas were dissected and incubated in medium at 32 ± 1 °C in an incubation chamber for 1 hour.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Neat test substance tested in three replicates:
Replicate 1: 643.3 mg;
Replicate 2: 616.8 mg;
Replicate 3: 667.6 mg;

Duration of post- treatment incubation (in vitro):

4 h at 32 ± 1 °C

Details on study design:

Preparations:
After having carefully cleaned and sterilised the cornea holders, they were kept in the incubation chamber at 32 ± 1 °C. On the day of the assay, the MEM without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1% fetal calf serum (= complete MEM) and stored in a water bath at 32 ± 1 °C. The same was performed with the MEM with phenol red, but without addition of sodium bicarbonate. After the arrival of the corneas, they were examined and only corneas which were free from damages were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C.

Method description:
After the initial incubation, the medium was changed and the baseline opacity for each cornea was recorded. None of the corneas showed tissue damage, therefore, all corneas were used. For each treatment group (negative control solution, test substance and positive control solution), three replicates were used. After removal of the pre-incubation medium (cMEM without phenol red), 750 μL negative control solution, test substance or positive control solution were applied to each replicate.

Open Chamber Method:
The “open chamber-method” is used for solid substances. In order to apply the test substance, the nut was unscrewed to remove the glass disc. The test substance could be applied directly on the cornea now. The following amounts of the test substance were tested neat and applied directly on the cornea using a weighing funnel: 643.3 mg, 616.8 mg, 3 667.6 mg. The test substance was applied on the epithelium in such a manner that as much as possible of the cornea was covered with test substance. Exposure time on the corneas was 4 hours at 32 ± 1 °C. After thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, both chambers were filled with cMEM without phenol red, and the final opacity value of each cornea was recorded. The cMEM without phenol red was then removed from the front chamber and 1 mL sodium fluorescein solution (concentration: 5 mg/mL) was added to the front chamber. The chambers were then closed again and incubated for 90 minutes at 32 ± 1 °C. After incubation, the content of the posterior chamber was thoroughly mixed. Then, the permeability of the cornea was measured as optical density of the liquid at 492 nm.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

-0.39

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

According to the guideline, the test is considered valid if the positive control causes an IVIS that falls within the two standard deviations of the current historical mean value. The negative control has to show an IVIS ≤ 3.
IVIS of negative control HBSS-solution: 2.13 (criterion: ≤ 3)
IVIS of positive control 20% imidazole solution: 113.47 (criterion: 73.75 – 163.55)
Values for negative and positive controls were within the range of historical data of the test facility. Therefore, the test system was acceptable.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the study conditions, the test substance showed no effects on the cornea of the bovine eye. The calculated IVIS was established at -0.39.

Executive summary:

A study was conducted to determine the corneal damage potential of the test substance according to OECD Guideline 437, in compliance with GLP. In this in vitro method, damage by the substance is assessed by quantitative measurements of changes in corneal opacity and permeability. Bovine corneas were collected from slaughtered cattle which were between 12 and 60 months old. One valid experiment was performed with three replicates for negative control, positive control and the test substance. The neat test substance was brought onto the cornea of bovine eyes which had been incubated with cMEM without phenol red at 32 ± 1°C for 1 h and whose opacity had been measured. The test substance was incubated on the cornea for 4 hours at 32 ± 1°C. After removal of the test substance, opacity and permeability values were measured. HBSS-solution was used as negative control. Imidazole solution was used as positive control. The negative control showed no irritating effect on the cornea and the calculated IVIS was 2.13. The positive control induced serious eye damage on the cornea and fell within the standard deviations of the current historical mean values. The calculated IVIS was 113.47. Under the study conditions, the test substance showed no effects on the cornea of the bovine eye. The calculated IVIS was established at -0.39 (Andres 2017).