Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 August 2011 to 27 September 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
yes
Remarks:
The study integrity was not adversely affected by the deviation.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
N-tert-butylglycyl chloride hydrochloride (1:1)
EC Number:
618-771-2
Cas Number:
915725-52-9
Molecular formula:
C6H13Cl2NO
IUPAC Name:
N-tert-butylglycyl chloride hydrochloride (1:1)
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Purity Not indicated by the sponsor; treated as 100% pure Test substance storage In refrigerator (2-8°C) in the dark Stability under storage conditions Stable Expiry date 21 July 2012

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Test System: Bovine eyes were used as soon as possible after slaughter on the same day.

Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing (1-6). As a consequence a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test.

Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.

Transport: Eyes were collected and transported in physiological saline in a suitable container

All eyes were carefully examined for defects by holding the eyes submersed in physiological saline. Those exhibiting unacceptable defects, such as opacity, scratches, pigmentation and neovascularization were discarded.

The isolated corneas were stored at 32 +/- 1oC in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Invitrogen Corporation, Breda, The Netherlands) containing 1% (v/v) L-glutamine (Invitrogen Corporation) and 1% (v/v) Foetal Bovine Serum (Invitrogen Corporation)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont, France) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 +/- 1oC. The corneas were incubated for the minimum of 1 hour at 32 +/- 1oC.

Test system

Vehicle:
water
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
The test substance was applied as a 16.7% and a 20% (w/w) solution (750 µl) directly on top of the corneas in the first and second experiment, respectively.
Duration of treatment / exposure:
240 ± 10 minutes
Number of animals or in vitro replicates:
3
Details on study design:
Preparation of corneas
All eyes were carefully examined for defects by holding the eyes submersed in physiological saline. Those exhibiting unacceptable defects, such as opacity, scratches, pigmentation and neovascularization were discarded.

The isolated corneas were stored at 32 +/- 1oC in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Invitrogen Corporation, Breda, The Netherlands) containing 1% (v/v) L-glutamine (Invitrogen Corporation) and 1% (v/v) Foetal Bovine Serum (Invitrogen Corporation)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont, France) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 +/- 1oC. The corneas were incubated for the minimum of 1 hour at 32 +/- 1oC.

Cornea selection and Opacity reading
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (OP-KIT, MC2, Clermont, France). The opacity of each cornea was read against an air filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

Treatment of corneas and opacity measurements
In the initial experiment, the medium from the anterior compartment was removed and 750 l of the negative control, 20% (w/v) Imidazole solution (positive control) or a 16.7% (w/w) test substance solution were introduced onto the epithelium of the cornea. In the second experiment, the medium from the anterior compartment was removed and 750 ul of the negative control, 20% (w/v) Imidazole solution (positive control) or a 20% (w/w) test substance solution were introduced onto the epithelium of the cornea. The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 +/- 10 minutes at 32 +/- 1C. After the incubation the solutions were removed and the epithelium was washed at least three times with MEM with phenol red (Eagle’s Minimum Essential Medium, Invitrogen Corporation). Possible pH effects of the test substance on the corneas were recorded. The anterior and the posterior compartment were refilled with fresh cMEM and an opacity determination was performed without any further incubation. After the completion of the incubation period each cornea were inspected visually for dissimilar opacity patterns and the opacity determination was performed.

Opacity measurement
The opacitometer determined the difference in the light transmission between each control or treated cornea and an air filled chamber. The numerical opacity value (arbitrary unit) was displayed and recorded. The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each positive control or test substance treated cornea was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each positive control or test substance treated cornea. The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

Application of sodium fluorescein
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Merck) was evaluated.

The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of 5 mg Na-fluorescein/ml cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 +/- 5 minutes at 32 +/- 1oC.

Permeability determinations
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 ul of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.

The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution was performed, the OD490 of each reading was corrected for the mean negative control OD490 before the dilution factor was applied to the readings.

Electronic data capture
Observations/measurements in the study were recorded electronically using the following programme: Magellan Tracker 7.0 (TECAN, Austria) for optical density measurement.

Interpretation
In vitro irritancy score
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:

In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

Additionally the opacity and permeability values were evaluated independently to determine whether the test substance induced irritation through only one of the two endpoints.

A test substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.


The assay is considered acceptable if:

- The positive control gives an in vitro irritancy score that falls within two standard deviations of the laboratory historical mean value. - The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
16.7% (w/w) solution of N-t-butylglycine acid chloride HCl
Value:
ca. 37
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
20% (w/w) solution
Value:
ca. 24
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Although the opacity of one cornea of the negative control was less than the lower limit of the laboratory historical range and consequently the IVIS was less than the lower limit of the laboratory historical range, the acceptability criteria were considered valid.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The mean in vitro irritancy score was 37 after 240 minutes of treatment with a 16.7% (w/w) solution of N-t-butylglycine acid chloride HCl and 24 with a 20% (w/w) solution of N-t-butylglycine acid chloride HCl. Since the mean in vitro irritancy score for N-t-butylglycine acid chloride HCl was below 55.1 after 240 minutes treatment in both experiments N-t-butylglycine acid chloride HCl is considered to be not severe irritant or corrosive.
Executive summary:

Screening for the eye irritancy potential of N-t-butylglycine acid chloride HCl using the Bovine Corneal Opacity and Permeability test (BCOP test).

This report describes the ocular irritation properties of N-t-butylglycine acid chloride HCl on an isolated bovine cornea. The possible ocular irritancy of N-t-butylglycine acid chloride HCl was tested through topical application for 240 ± 10 minutes.  

The study procedures described in this report were based on the most recent OECD and EC guideline.

Batch # 11560006 of N-t-butylglycine acid chloride HCl was a beige powder. The test substance was applied as a 16.7% and a 20% (w/w) solution (750 µl) directly on top of the corneas in the first and second experiment, respectively.  

The negative control responses of the opacity and permeability values were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy scores of the positive control (20% (w/v) Imidazole) were 94 and 105, in the first and second experiment, respectively and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.  

The mean in vitro irritancy score was 37 after 240 minutes of treatment with a 16.7% (w/w) solution of N-t-butylglycine acid chloride HCl and 24 after 240 minutes of treatment with a 20% (w/w) solution of N-t-butylglycine acid chloride HCl. Since the mean in vitro irritancy score for N-t-butylglycine acid chloride HCl was below 55.1 after 240 minutes treatment in both experiments N-t-butylglycine acid chloride HCl is considered to be not severe irritant or corrosive.