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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 December 2016 - 17 January 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:
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:
26 July 2013
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
solid: crystalline
Details on test material:
Appearance: white crystalline powder
Storage conditions: at room temperature protected from light
Specific details on test material used for the study:
Stability at higher temperatures: stable, maximum temperature: 40 °C, maximum duration: 2 days
pH: 4-5 (10% solution)

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 750 μl of a 20% (w/v) solution of test substance prepared in physiological saline per cornea

NEGATIVE CONTROL
- Amount applied: 750 µl of physiological saline per cornea

POSITIVE CONTROL
Amount applied: 750 µl 20% (w/v) Imidazole solution prepared in physiological saline
Duration of treatment / exposure:
240 ± 10 minutes
Number of animals or in vitro replicates:
Test item: 3
Negative control: 3
Positive control: 3
Details on study design:
TEST SITE
- Isolated bovine cornea, obtained from the slaughterhouse, where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

REMOVAL OF TEST SUBSTANCE
- Washing: yes, three times with MEM with phenol red (Eagle’s Minimum Essential Medium, Life Technologies)
- Time after start of exposure: 240 minutes

SCORING SYSTEM:
- 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).

TOOL USED TO ASSESS SCORE:
- opacitymeter (OP-KIT) and microplate reader (TECAN Infinite® M200 Pro Plate Reader).

DATA EVALUATION:
A test substance that induces an IVIS ≤ 3 is not classified for eye irritancy (UN GHS: no category);
A test substance that induces an IVIS > 55 is defined as a corrosive or severe irritan (UN GHS: catgegory 1);
For a test substance that induces an IVIS >3 and ≥ 55, no prediction on irritant potency can be made.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Remarks:
Mean of 3 replicates
Run / experiment:
1st experiment
Value:
3.3
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Remarks:
(2.4)
Positive controls valid:
yes
Remarks:
(159.7)
Remarks on result:
other: Individual scores were spread over 2 categories
Irritation parameter:
in vitro irritation score
Remarks:
Mean of 3 replicates
Run / experiment:
2nd experiment
Value:
3.4
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Remarks:
(0.2)
Positive controls valid:
yes
Remarks:
(129.9)
Remarks on result:
other: Individual scores were spread over 2 categories
Other effects / acceptance of results:
- In the first experiment the individual IVIS for the negative controls were 2.0-2.8. The 3 cornea were slightly translucent but since all opacity and permeability values were within the historical data control range this had no effect on the study. Individual IVIS of the positive control were 143-184, the corneas were turbid after 240 minutes of treatment. The corneas treated with test item showed opacity scores of 1.6-6.4 and permeability values of -0.030 - 0.061. The corneas were clear after 240 minutes of treatment. A pH effect of the test item was observed on the rinsing medium. Therefore the corneas were rinsed until no colour change of the medium was observed. Hence, the IVIS ranged from 1.3-5.9 after 240 minutes of treatment. Because the individual IVIS were spread over 2 categories, the test was inconclusive and a second experiment was performed.
- In the second experiment the individual IVIS for the negative controls were -0.6-0.9 and for the positive control 106-175. The corneas treated with the positive control were turbid after 240 minutes of treatment. The corneas treated with test item showed opacity values of 1.0-5.0 and permeability values of 0.000-0.013. The corneas were slightly translucent after the 240 minutes of treatment. In addition, two corneas showed a spot. a pH effect of the test item was observed. Hence, the IVIS ranged from 1.2 to 5.2 after 240 minutes if treatment. Since individual IVIS were spread over 2 categories, the test was inconslusive.
- Proper functioning of the test system in both experiments was shown by the results from the positive and negative controls, which were within the historical data range. Therefore, the acceptability criteria were met and the test was considered to be valid.
- Since both experiments provided inconclusive results, no conclusion can be drawn for eye irritation or serious eye damage.

Any other information on results incl. tables

Table 1 First experiment: Summary of opacity, permeability and in vitro scores

Treatment

Mean

Opacity

Mean

Permeability

Mean In vitro Irritation Score1, 2

Negative control

1.8

0.037

2.4

Positive control

135.9

1.588

159.7

Bicin

3.2

0.001

3.3

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

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

Table 2 Second experiment: Summary of opacity, permeability and in vitro scores

Treatment

Mean

Opacity

Mean

Permeability

Mean In vitro Irritation Score1, 2

Negative control

0.1

0.004

0.2

Positive control

109.3

1.379

129.9

Bicin

3.2

0.009

3.4

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

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

Applicant's summary and conclusion

Interpretation of results:
other: Results were spread in two categories and were therefore inconclusive.
Conclusions:
Based on the outcome of a Bovine Corneal Opacity and Permeability test (BCOP) performed according to OECD guideline and GLP principles, no conclusion can be drawn for eye irritation of the substance. The mean IVIS in two experiments were 3.3 and 3.4, but individual scores were spread over two categories.