Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 20 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
adopted October 09, 2017
Deviations:
yes
Remarks:
One of the negative control eye was excluded from analysis because opacity value was above historical data. This does not affect the study outcome.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by the sponsor, batch No. 160405
- Expiration date of the lot/batch: 31 January 2019
- Purity test date: only purity (>98%) was given, no certificate was added.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature desiccated
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: soluble in physiological saline
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: A 20% w/v solution was performed from TMAO anhydrous in physiologicalsaline
- Preliminary purification step (if any): not applicabme
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: prepared in solution

FORM AS APPLIED IN THE TEST (if different from that of starting material) in solution

Test animals / tissue source

Species:
cattle
Strain:
other: bovine. not more details
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Vitelco, -'s Hertogenbosch, The Netherlands
- Number of animals: 9 eyes were used
- Characteristics of donor animals (e.g. age, sex, weight): young cattle
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Time interval prior to initiating testing: not detailed
- indication of any existing defects or lesions in ocular tissue samples: no lesions
- Indication of any antibiotics used: not specified

Test system

Vehicle:
physiological saline
Controls:
yes
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):750 µL
- Concentration (if solution): 20% (w/v) in physiological saline
Duration of treatment / exposure:
240 ± 10 minutes
Duration of post- treatment incubation (in vitro):
90 ± 5 minutes
Number of animals or in vitro replicates:
triplicates were used per condition
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded. The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Fetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) 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 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.


QUALITY CHECK OF THE ISOLATED CORNEAS
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 (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM 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.

NUMBER OF REPLICATES
Triplicates were used

NEGATIVE CONTROL USED
Physiological saline

SOLVENT CONTROL USED (if applicable)
not applicable

POSITIVE CONTROL USED
Imidazole 20% (w/v) in physiological saline

APPLICATION DOSE AND EXPOSURE TIME
The medium from the anterior compartment was removed and 750µl of either the negative control, positive control (20% (w/v) Imidazole solution) or 20% (w/v) solution of the test item was 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.

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: yes. 90 minutes (for application of sodium fluorescein)

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions were removed and the epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium Life Technologies).
- POST-EXPOSURE INCUBATION: No

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
The opacity value (measured with the device OP-KIT) was calculated according to: Opacity = ((Io/I) - 0.9894)/0.0251
With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea. 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 treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control 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.

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
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)


DECISION CRITERIA: ≤ 3 No Category

> 3; ≤ 55 No prediction can be made
>55 Category 1

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
Negative control
Value:
3.6
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
Positive control
Value:
164
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
TMAO anhydrous
Value:
-0.7
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
Negative control
Value:
2.7
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
Positive control
Value:
131
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
TMAO anhydrous
Value:
-0.8
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein leakage
Run / experiment:
Negative control
Value:
0.055
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein leakage
Run / experiment:
Positive control
Value:
2.161
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein leakage
Run / experiment:
TMAO anhydrous
Value:
0.007
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: the positive control condition showed proficiency of the test system to measure effect

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The individual in vitro irritancy scores for the negative controls ranged from 1.8 to 5.3. One of the negative control eyes was excluded from the analysis since the opacity was above the historical data base which resulted in an IVIS outside the historical data base. Since the mean of the two remaining eyes completely met the criteria and the test item results were not influenced by this result, this does not affect the study outcome
- Acceptance criteria met for positive control: yes

Any other information on results incl. tables

Table1          
Summary of Opacity, Permeability and In Vitro Scores

Treatment

Mean

Opacity

Mean

Permeability

Mean In vitro Irritation Score1, 2

Negative control

2.7

0.055

3.6

Positive control

131

2.161

164

TMAO anhydrous

-0.8

0.007

-0.7

1     Calculated using the negative control mean opacity and mean permeability values for the positive control and test item.

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


 

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, under the experimental condition of the study, IVIS score of the test item TMAO was below 3, no classification is required for eye irritation or serious eye damage according to CLP criteria.
Executive summary:

The objective of this GLP compliant study was to evaluate the eye hazard potential of TMAO anhydrous as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test according to OECD TG 437 method).

This studydescribes the potency of chemicals to induce serious eye damage using isolated bovine corneas.  The eye damage of TMAO anhydrous was tested through topical application for approximately 240 minutes.  

Batch 160405 of TMAO anhydrous was a white powder.  The test item was applied as a 20% (w/v) solution (750 µL) directly on top of the corneas.  

The negative control responses for opacity and permeability 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 score of the positive control (20% (w/v) Imidazole) was 164 and within two standard deviations of the current historical positive control mean.  It was therefore concluded that the test conditions were adequate and that the test system functioned properly.  

TMAO anhydrous did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 2.2 after 4 hours of treatment.  

In conclusion, since TMAO anhydrous induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.