Reaction products of N-methyldiethanolamine and Boric Acid (1:1.5)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 March, 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

Test guideline 1

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)

Version / remarks:

2017

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of N-methyldiethanolamine and Boric Acid (1:1.5)

EC Number:

947-350-9

Molecular formula:

N.A. (UVCB)

IUPAC Name:

Reaction products of N-methyldiethanolamine and Boric Acid (1:1.5)

Test material form:

liquid: viscous

Test animals / tissue source

Species:

cattle

Strain:

other: Bos primigenius Taurus

Details on test animals or tissues and environmental conditions:

- Species: Bos primigenius Taurus (fresh bovine corneas)
- Origin: fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH (Enzstr. 2-4, 75217 Birkenfeld, DE) on the day of the test.
- Age of test animals: between 12 and 60 months old
- Transportation of samples: eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled container within 1 hour 5 minutes

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 µL

Duration of treatment / exposure:

Incubation time: 10 min

Duration of post- treatment incubation (in vitro):

2 hours

Number of animals or in vitro replicates:

3

Details on study design:

PREPARATION
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 item and positive control), three replicates were used. After removal of the pre-incubation medium (cMEM without phenol red), negative control solution, test item and positive control were applied to each replicate. According to the characteristics of the test item, the following treatment procedure was performed:
- Closed Chamber Method
The respective substance (negative control solution, test item or positive control) was applied by pipetting 750 µL of the appropriate liquid through the refill hole in the holder on the cornea. The test item was given on the epithelium in such a manner that as much as possible of the cornea was covered with the test item.
Exposure time of the test item on the corneas was 10 minutes at 32 ± 1 °C. After thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, the anterior chamber was filled with cMEM without phenol red, and the corneas were stored for additional 2 hours at 32 ± 1 °C (post-incubation).
After post-incubation time, the cMEM without phenol red was renewed in both chambers. Then, the final opacity value of each cornea was recorded.
- Permeability Test
After the recording of the final opacity value, the cMEM without phenol red was removed from the front chamber, and 1 mL sodium fluorescein solution was added to the front chamber for the detection of permeability of the corneas.
For the closed chamber method, a sodium fluorescein solution with a concentration of 4 mg/mL was used.
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, its optical density at 492 nm was measured with the microtiter plate photometer.
 
EVALUATION
- Calculation of Opacity
The change of opacity value of each treated cornea with test item, positive control and negative control was calculated by subtracting the initial basal opacity from the post treatment opacity reading for each cornea.
The average change in opacity of the negative control cornea was calculated and this value was subtracted from the change in opacity of each treated cornea with test item and positive control to obtain a corrected opacity.
Opacity= (I0/I -b)/a
where:
a = 0.0251 and b = 0.9894 being Opacitometer-specific empirically determined variables
I0 = the empirically determined illuminance through a cornea holder with windows and
Medium, here: Io= 1070.84
I = the measured illuminance (unit: LUX)
Calculation of Permeability
The corrected OD492 value of each cornea treated with test item and positive control was calculated by subtracting the mean negative control cornea value from the original permeability value for each cornea.
The mean OD492 value for each treatment group (test item, positive control and negative control) was determined by averaging the final corrected OD492 values of the treated corneas for one treatment group.

- Calculation of IVIS (In Vitro Irritancy Score)
The IVIS of each replicate of the negative control was calculated from the following equation:
IVIS = opacity difference + (15 x corrected OD492 value)
The IVIS of each replicate of the positive control and of the test item were calculated from the following equation:
IVIS = (opacity difference – mean opacity difference of the negative control) + [15 x (OD492 – mean OD492 of the negative control)]

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Remarks:

IVIS

Run / experiment:

mean

Value:

0.31

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Interpretation of results:

other: not classified according to the CLP Regulation (EC) No. 1272/2008

Conclusions:

The test item showed no effects on the cornea of the bovine eye. The calculated IVIS score is 0.31.

Executive summary:

In order to assess the corneal damaging potential of the test item, an in vitro study was performed by quantitative measurement of the change in bovine corneal opacity and permeability (Bovine Corneal Opacity and Permeability (BCOP) test), according to the OECD Guideline 437 (2017) and the EU Method B.47 (2017). Bovine eyes of cattle aged 12 to 60 months were incubated in cMEM without phenol red at 32 °C ± 1 °C for 1 hour and opacity was measured. The test item was then applied the bovine cornea and incubated for 10 minutes at 32 °C ± 1 °C. 2 hours post-incubation, the test item was removed from corneas, and opacity and permeability values were measured. A negative control (Hank’s Balanced Salt Solution (HBSS)) and a positive control (Dimethylformamide (DMF); undiluted) were tested simultaneously with the test item. IVIS (In Vitro Irritancy Scores) were calculated.

The negative control showed no irritating effect on the cornea and the calculated IVIS (In Vitro Irritancy Score) is 1.50. The positive control induced serious eye damage on the cornea and was within two standard deviations of the current historical mean. The calculated IVIS score was 80.32. The test item showed no effects on the cornea of the bovine eye. The calculated IVIS of the test item was found to be 0.31. As the IVIS was ≤ 3, the test item requires no classification for eye irritation or serious eye damage according to the CLP Regulation (EC) No. 1272/2008.