Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-03-28
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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
2017-10-09
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2015-06-05

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- State of aggregation: solid, white powder
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, in a tightly closed container

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: abattoir A. Moksel AG, Buchloe, Germany
- Characteristics of donor animals (e.g. age, sex, weight): age of the cattle was between 18 and 23 months
- Storage, temperature and transport conditions of ocular tissue: fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories
- Time interval prior to initiating testing: immediately after arrival of the eyes, cornea preparation was initiated and was used for BCOP testing on the same day.

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): about 750 µL of the test item
The test item was treated as a neat surfactant. The test item was suspended in 0.9 % NaCl to give a 10% w/v concentration.
The test item was mixed with physiological saline 0.9 % NaCl to give a 10 % w/v concentration using ultrasonique technique. The sonicated suspension was incubated at 32 °C for 1 hour. Prior to application, the mixture was re-suspended by vortexing and administered directly.
Duration of treatment / exposure:
10 minutes
Observation period (in vivo):
not applicable
Duration of post- treatment incubation (in vitro):
2 hours
Number of animals or in vitro replicates:
Number of bovine corneae per dose:
Test item: triplicates
Negative control: triplicates
Positive control: triplicates
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
- eyes were examined for defects and any defective eyes were discarded. Eyes with scratches or any kind of opacity were not used.
- tissue surrounding the eyeball was pulled away and the cornea was excised.
- isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) 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 pre-warmed RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI).
- corneas were incubated for one hour at 32 ± 1 °C for equilibration in an air incubator.

QUALITY CHECK OF THE ISOLATED CORNEAS
- after the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI.
- an initial measurement was performed on each of the corneas using the opacitometer.
- three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas.
- the illuminance of each cornea was read and recorded.
- only corneas that had an initial illuminance reading I > I0/1.1651 lux (an equivalent to the opacity threshold of 7 as listed in OECD 437) were used for the assay.

APPLICATION DOSE AND EXPOSURE TIME
- medium was removed from the anterior chamber and replaced with the test item or control.
- 750 µL of the test substance mixture and control substances were introduced into the anterior chamber (closed-chamber method).
- after 10 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed.

REMOVAL OF TEST SUBSTANCE/CONTROL SUBSTANCES
- epithelium was 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).

METHODS FOR MEASURED ENDPOINTS:
- anterior chamber was refilled with complete RPMI and an illuminance measurement was performed after 2 hours incubation at 32 ± 1 °C.
- corneas were visually examined for tissue peeling, residual test chemical and non-uniform opacity patterns and observation were recorded.
- after the illuminance measurement was performed, the medium was removed from both chambers of the holder.
- posterior chamber was refilled with fresh complete RPMI.
- 1 mL of a 4 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 (Jenway 6405 UV/VIS).

Evaluation of the opacity:
- the following formula was used to calculate the opacity, whereas the values a and b are equipment-specific variables empirically determined by the manufacturer:
Opacity = ((I0/I) - b)/ a
with a = 0.025 and b = 0.9894
- value I0 is the illuminance through a holder without cornea, but with windows and liquid. This value is determined by taking the mean for a set of cornea holders and is reevaluated periodically. This I0 value was than calculated to the respective data of the opacitometer and the data according to guideline (opacity < 7). So the initial illuminance could be calculated and corneas below this value were discarded.
- change in opacity for each cornea (test item, positive and negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values of test item treated cornea or positive control were corrected by subtracting from each the average change in opacity observed for the negative-control corneas to obtain a corrected opacity. The mean corrected opacity value for each treatment was calculated by averaging the corrected opacity values of each cornea for a given treatment.

Evaluation of the permeability:
- mean OD490 for the blank cuvettes was calculated.
- mean blank OD490 was subtracted from the OD490 of each cuvette (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.
- 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
- mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated corneas for that treatment condition.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula was used to determine the in vitro irritation score (IVIS):
IVIS = mean opacity value + (15 x mean permeability OD490 value)
To determine the IVIS of the positive control and the test item, the corrected opacity and OD490 values were used.
For the 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) please refer to table 1 in the field "Any other information onmaterial and methods incl. tables" below.

ACCEPTABILITY CRITERIA
- 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.
- the negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Remarks:
(mean)
Value:
10.05
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
All 3 corneas treated with Magnesium 2-ethylhexanoate showed moderate opacity of the tissue.
Relative to the negative control, the test item caused an increase of corneal opacity and permeability in all 3 corneas.

Acceptance of results:
- Acceptance criteria met for negative control: the negative control responses resulted in opacity and permeability values that are less than the respective established upper limits for background opacity and permeability.
- Acceptance criteria met for positive control: the IVIS of the positive control falls within two standard deviations of the current historical mean

Please also refer for results to the field "Any other information on results incl. tables" below

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

Negative
Control

2.20

2.46

0.26

 

2

2.73

3.94

1.21

 

3

2.27

2.96

0.68

 

MV

2.40

3.12

0.72

 

4

Positive
Control

3.54

26.20

22.66

21.94

5

3.98

26.01

22.03

21.31

6

3.70

26.01

22.31

21.60

MV

3.74

26.08

22.34

21.62

7

Test Item

2.88

6.10

3.22

2.50

8

1.11

5.06

3.94

3.22

9

2.92

7.42

4.51

3.79

MV

2.30

6.19

3.89

3.17

MV = mean value

Table 2: Permeability

Cornea
No.

Test Item

OD490

Corrected
OD490 Value

1

Negative
Control

0.011

 

2

0.004

 

3

0.008

 

MV

0.008

 

4

Positive
Control

1.407

1.399

5

1.354

1.346

6

1.355

1.347

MV

1.372

1.364

7

Test Item

0.568

0.560

8

0.336

0.328

9

0.495

0.487

MV

0.466

0.459

MV = mean value

Table 3: In vitro irritation score

Cornea
No.

Test Item

Corrected
Opacity

Value

Corrected
OD490

Value

IVIS

1

Negative
Control

0.26

0.011

 

2

1.21

0.004

 

3

0.68

0.008

 

MV

0.72

0.008

0.83

4

Positive
Control

21.94

1.399

 

5

21.31

1.346

 

6

21.60

1.347

 

MV

21.62

1.364

42.08

7

Test Item

2.50

0.560

 

8

3.22

0.328

 

9

3.79

0.487

 

MV

3.17

0.459

10.05

MV = mean value

Table 4: Historical mean in vitro irritation score of the positive control from February 2015 until March 2018

 

IVIS
Positive Control - Ethanol 100 %

Mean Value (MV)

48.69

Standard Deviation (SD)

9.75

MV- 2xSD

29.18

MV+2xSD

68.19

Number of Replicates providing Historical Mean: 49

Positive controls are updated after every single experiment or at least every 3 months.

Table 5: Historical data on opacity and permeability of the positive control (ethanol 100 %) from August 2017 until March 2018

Number of Replicates Providing Historical Mean

Cornea No.

Opacity

Permeability

IVIS

Change of
Opacity Value

Corrected
Opacity Value

OD490 Value

Corrected
OD490 Value

2017

1

4

37.495

37.899

1.013

1.004

53.36

 

5

27.335

27.739

1.147

1.138

 

6

30.218

30.622

2.120

2.111

 

2

4

24.492

23.921

1.442

1.436

49.70

 

5

31.754

31.182

1.108

1.102

 

6

30.259

29.688

1.755

1.749

3

4

40.460

39.546

1.270

1.266

49.84

5

28.925

28.010

0.949

0.945

6

29.064

28.149

1.381

1.377

 

4

4

22.710

22.292

1.182

1.178

52.16

 

5

35.986

35.567

1.780

1.776

 

6

24.036

23.618

2.050

2.046

2018

6

4

25.61

24.97

2.025

2.014

63.38

 

5

28.89

28.25

2.920

2.909

 

6

27.82

27.18

2.405

2.394

 

7

4

22.66

21.94

1.407

1.399

42.08

 

5

22.03

21.31

1.354

1.346

 

6

22.31

21.60

1.355

1.347

Mean Value (MV)

28.448

27.972

1.592

1.586

51.75

Standard

Deviation (SD)

5.375

5.473

0.533

0.532

6.92

MV- 2xSD

17.699

17.025

0.526

0.522

37.91

MV+2xSD

39.197

38.918

2.658

2.649

65.59

Table 6: Historical mean in vitro irritation score of the negative control from February 2015 until March 2018

 

IVIS Negative Control -

NaCl 0.9 %

Mean Value (MV)

0.80

Standard Deviation (SD)

0.65

MV- 2xSD

-0.49

MV+2xSD

2.10

Number of Replicates providing Historical

Mean: 49

Negative controls are updated after every single experiment or at least every 3 months.

Table 7: Historical data on opacity and permeability of the negative control (NaCl 0.9 %) from August 2017 until March 2018

Number of Replicates Providing Historical Mean

Cornae No.

Opacity

Permeability

IVIS

Change of
Opacity Value

OD490 Value

2017

1

1

-0.57

0.009

-0.27

2

-0.11

0.013

3

-0.53

0.004

2

1

-0.25

0.004

0.66

2

0.80

0.005

3

1.17

0.008

3

1

0.11

0.004

0.48

2

0.68

0.005

3

0.47

0.003

2018

4

1

0.19

0.008

0.18

2

-0.12

0.005

3

0.00

0.018

 

1

0.11

0.005

 

5

2

0.74

0.022

0.81

 

3

1.06

0.007

 

6

1

0.26

0.011

0.83

2

1.21

0.004

3

0.68

0.008

Mean Value

(MV)

0.15

0.01

0.37

Standard Deviation (SD)

0.54

0.00

0.43

MV- 2xSD

-0.92

0.00

-0.49

MV+2xSD

1.22

0.02

1.23

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
According to the bovine corneal opacity and permeability assay, since the IVIS was between 3 and 55, no prediction can be made regarding the test item magnesium 2-ethylhexanoate.