D-Glucitol, 1-deoxy-1-(methylamino)-, N-[C18-C18 (unsaturated) acyl] derivs.

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-10-31 to 213-12-03

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

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals, number 437 “Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants” (adopted: 26 July 2013)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und dLebensmittelsicherheit, München, Germany)

Test material

Test material information

Constituent 1

Reference substance name:

(6E)-N-[(2R,3S,4S,5S)-6-hydroxy-2,3,4,5-tetramethylhexyl]-N-methyloctadec-6-enamide; N-[(2R,3S,4S,5S)-6-hydroxy-2,3,4,5-tetramethylhexyl]-N-methyloctadecanamide

EC Number:

940-373-5

Cas Number:

1591782-99-8

Molecular formula:

C25H49NO6 (C18 unsatd.) C25H51NO6 (C18 satd.)

IUPAC Name:

(6E)-N-[(2R,3S,4S,5S)-6-hydroxy-2,3,4,5-tetramethylhexyl]-N-methyloctadec-6-enamide; N-[(2R,3S,4S,5S)-6-hydroxy-2,3,4,5-tetramethylhexyl]-N-methyloctadecanamide

Test material form:

other: Waxy solid

Test system

Vehicle:

physiological saline

Amount / concentration applied:

The test item was diluted with physiological saline 0.9% NaCl to gain a 20% concentration.

Duration of treatment / exposure:

750 microL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method).
After 4 hours ± 5 minutes incubation at 32 +/- 1 °C either the test substance or the control substance was removed and the epithelium washed
at least three times with MEM (containing phenol red).

Details on study design:

Test System

Preparation of the Corneas:
The assay uses isolated corneas obtained as a by-product from an abattoir from freshly slaughtered animals (from Attenberger Fleisch GmbH & Co. KG).
On the test day, fresh eyes were collected from the slautherhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.
The eyes were carefully examined for defects and any defective eyes were discarded.
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (MC2, Clermont, France) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 +/- 1 °C in a water bath.

Calibration of the Opacitometer:
The opacitometer had been switched on 15 min before the calibration procedure was started. Empty cornea holders were placed into the opacitometer and the readout was adjusted to zero using the “BAL”-turning knob. For calibration the polyester foil no. 1 was introduced into the test chamber and the readout was adjusted to 75 using the “CAL”-turning knob. To test the linearity of the measurement, two additional calibration foils, polyester foil no. 2 and polyester foil no. 3, were measured. For these, the opacitometer was supposed to display 150 and 225, respectively (± 3%). If this had not been the case, the calibration procedure would have had to be repeated. The calibration procedure was performed before each test and was documented in the raw data.

Treatment of the Corneas:
After the equilibration period, the medium was removed from both chambers and replaced with fresh Complete RPMI. An initial opacity measurement was performed on each of the corneas using an opacitometer (MC2, Clermont, France). Three corneas with opacity readings approximately equivalent to the median opacity of all corneas were selected as negative-control corneas. The opacity of each cornea was read against an air-filled chamber and recorded. Corneas that have an initial opacity reading above 7 units were not dosed. The medium was removed from the anterior chamber and replaced with the test item or control.
750 microL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method).
After 4 hours ± 5 minutes incubation at 32 +/- 1 °C either the test substance or the control substance was removed and the epithelium washed
at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an opacity measurement was performed.
After the opacity measurement the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 5 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes
at 32 +/- 1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined,
using a spectrophotometer.

Test Groups:
3 corneas for the test item
3 corneas as negative controls treated with physiological saline 0.9% NaCl
3 corneas as positive control treated with imidazole 20% in physiological saline 0.9% NaCl
The BCOP assay is considered to be valid if the in vitro score obtained with the positive control falls within the two standard deviations of the current historical mean.

Evaluation of Results:
The change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading. These values were
corrected by subtracting from each the average change in opacity observed for the negative-control corneas. The mean opacity value for each
treatment was calculated by averaging the corrected opacity values of each cornea for a given treatment.
The mean OD490 for the blank wells were calculated. The mean blank OD490 was subtracted from the OD490 of each well (corrected OD490).
Any dilutions that were made to bring the OD490 values into the linear range of the spectrophotometer (OD490 should be less than 1.500), were
taken into account by multiplying the OD490 value of the dilution by the dilution factor. The final-corrected OD490 of the test article and the positive
control were calculated by subtracting the average corrected OD490 of the negative control corneas from the corrected OD490 value of each
treated cornea:
Final-corrected OD490 = (OD490 – mean blank OD490) – average-corrected negative control OD490
The mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated corneas
for that treatment condition.
The following formula was used to determine the in vitro score:
In vitro score = mean opacity value + (15 x mean OD490 value)

Results and discussion

In vivo

Results

Irritation parameter:

other: in vitro irritation score (IVIS)

Basis:

mean

Time point:

other: 240 minutes

Score:

3.47

Reversibility:

other: not measured in test system

Irritant / corrosive response data:

The following mean in vitro irritation score was calculated: 3.47

The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.

Applicant's summary and conclusion

Interpretation of results:

other: mild irritant

Remarks:

Criteria used for interpretation of results: other: OECD

Conclusions:

Based on the results of this in vitro study and under the given conditions the test item C18/C18 unsatd.-Glucamide is considered to be a mild irritant to the eye. However, since the IVIS score 3.47 is slightly above the regulatory non-classification threshold, no C&L decision is based on these study results.

Executive summary:

The eye irritancy potential of the undiluted test substance was investigated in a GLP compliant bovine corneal opacity and permeability assay according to OECD TG 437 (BCOP). Evaluation of effects on the opacity and the permeability of the bovine corneas were performed on three corneas per test group. After 1 hour equilibration the corneas were exposed to a 20% (w/v) suspension in physiological saline for 240 minutes. After treatment the test item was rinsed off the corneas and the opacity was measured. Based on the results of this study a mean in vitro score of 3.47 was obtained. The test substance therefore is considered to be a mild eye irritant. However, as the IVIS score is slightly above the regulatory treshold for non-classification, no C&L decision is based on this outcome. The in vitro score obtained with the positive control was within the current historical mean and therefore this assay is considered to be valid.