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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:
2017-04-03 to 2017-05-29
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:
adopted 26 July, 2013
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):
On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing 1% Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.

- Time interval prior to initiating testing:
At least 1 hour

- indication of any existing defects or lesions in ocular tissue samples:
Before the corneae were mounted in corneal holders 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.

- Indication of any antibiotics used:
1% Penicilin/Streptomycin

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Concentration (if solution): 20% solution in physiological saline

VEHICLE

- Concentration (if solution): 0.9% NaCl physiological saline
Duration of treatment / exposure:
4 h at 32°C
Duration of post- treatment incubation (in vitro):
90 min at 32°C
Number of animals or in vitro replicates:
each 3 corneae were used for the test item, the positive control and the negative control
Details on study design:
SELECTION AND PREPARATION OF CORNEAE
Isolated corneae were obtained as a by-product from animals freshly slaughtered at an abattoir. Fresh eyes were collected from the slaughterhouse and transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.
The tissue surrounding the eyeball was carefully pulled away and the cornea will be excised leaving a 2 to 3 mm rim of sclera. The isolated corneae will be stored in a petri dish containing HBSS. Before mounting the corneae in corneal holders with the endothelial side against the O-ring ofthe posterior chamber, they were visually examined for defects and any defective cornea were discarded.

QUALITY CHECK OF THE ISOLATED CORNEAS
The eyes were visually checked for any defects of the cornea.

NUMBER OF REPLICATES
3

SOLVENT CONTROL USED (if applicable)
yes, pyhsiological saline

POSITIVE CONTROL USED
20% imidazole

APPLICATION DOSE AND EXPOSURE TIME
20% solution of the test item was applied and the corneae incubated for 4 h at 32°C

TREATMENT METHOD:
The eyes were incubated applying the closed chamber method.

POST-INCUBATION PERIOD:
No.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
The eyes were washed at least three times

- POST-EXPOSURE INCUBATION:
Yes. 90 min at 32°C.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
Corneal opacity was measured using an opacitometer (BASF-OP3.0, Duratec)

- Corneal permeability:
passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490)

- Others (e.g, pertinent visual observations, histopathology):
Beside illuminance measurement the corneae were observed visually and pertinent observations were recorded

SCORING SYSTEM:
In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
The following IVIS cut-off values for identifying test substances as inducing serious eye damage (UN GHS Category 1) and test substances not requiring classification for eye irritation or serious eye damage (UN GHS No Category) were used:
IVIS UN GHS
≤ 3 No Category
>3; ≤ 55 No prediction can be made
> 55 Category 1

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
mean IVIS of three experiments
Value:
7.28
Vehicle controls validity:
valid
Remarks:
IVIS 0.59
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
IVIS 121.72
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system:
No

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The BCOP assay is considered to be valid if the in vitro irritation score obtained with the positive control falls within the two standard deviations of the current historical mean.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control responses should result in opacity and permeability values that are less than the established upper limits (Mean VaIue + 2 x Standard Deviation = 2.89) for background bovine corneae treated with the respective negative control. Mean in vitro score = 0.59.

- Acceptance criteria met for positive control:
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. Mean in vitro score = 121.72.


Historical mean IVIS of the positive control:
Mean value (MV): 125.72
Standard deviation (SD): 18.54
MV-2 x SD: 88.65
MV+2 x SD: 162.80
Number of Replicates providing Historical Mean: 21

Historical mean IVIS of the negative control:
Mean value (MV): 1.27
Standard deviation (SD): 0.81
MV-2 x SD: -0.34
MV+2 x SD: 2.89
Number of Replicates providing Historical Mean: 21

Any other information on results incl. tables

Table 1: Opacity

Cornea No.

Test Item

Initial

Opacity

Final

Opacity

Change of Opacity Value

Corrected

Opacity Value

1

2

3

MV

Negative Control

2.20

2.01

2.08

2.10

3.09

2.05

2.12

2.42

0.89

0.04

0.04

0.32

 

4

5

6

MV

Positive

Control

3.20

3.16

3.05

3.14

94.43

96.42

92.88

94.58

91.23

93.25

89.84

91.44

90.91

92.93

89.52

91.12

7

8

9

MV

Test Item

0.98

2.85

0.88

1 .57

7.10

8.37

10.45

8.64

6.12

5.52

9.57

7.07

5.80

5.19

9.25

6.75

MV: mean value

Table 2: Permeability

Cornea No.

Test Item

OD490

Corrected

OD490 Value

1

2

3

MV

Negative Control

0.009

0.009

0.036

0.018

 

4

5

6

MV

Positive

Control

1 .435

2.415

2.325

2.058

1.417

2.397

2.307

2.040

7

8

9

MV

Test Item

0.022

0.037

0.101

0.053

0.004

0.019

0.083

0.035

MV: mean value

Table 3: In Vitro Irritation Score

Cornea No.

Test Item

Corrected Opacity

Corrected

OD490 Value

 

1

2

3

MV

Negative Control

0.89

0.04

0.04

0.32

0.009

0.009

0.036

0.018

0.59

4

5

6

MV

Pos itive Control

90.91

92.93

89.52

91.12

1 .417

2.397

2.307

2.040

121.72

7

8

9

MV

Test Item

5.80

5.19

9.25

6.75

0.004

0.019

0.083

0.035

7.28

MV: mean value

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
In the present study the treatment of bovine corneae with m-Xylylenebismaleimide for 4h at 32°C resulted in a mean IVIS of 7.28. There were signs of opacity in of the treated corneae after 4 h exposure and the corneae showed a yellowish colouration. However, in accordance with OECD Testguideline 437 (adopted July 26, 2013) the following cut-off values for classification according to GHS criteria the IVIS must be < 3 for no classification and > 55 to idenify a substance as inducing serious eye damage (UN GHS Category 1). All values between the afore mentioned values do not provide conclusive results, thus, no prediction can be made for the test item.
Due to the lack of further information, m-Xylylenebismaleimide is classified in Category 2 'causes serious eye irritation' according to Regulation EC No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS) for precautionary reasons.