Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-08-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
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:
2013-07-26
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2015-06-05

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: flakes
Details on test material:
- State of aggregation: solid flakes
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: dry; < 30 °C

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: cattle was between 19 and 34 months old.
- 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
Remarks:
0.9 % NaCl
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μL of the test substance mixture
- Concentration (if solution): 20 % w/v concentration
The test item was mixed with the vehicle to give a concentration of 20 % w/v using ultrasonic 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:
4 hours ± 5 minutes
Observation period (in vivo):
not applicable
Duration of post- treatment incubation (in vitro):
not required
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 or the control substance was introduced into the anterior chamber (closed-chamber method).
- after 4 hours ± 5 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.
- each cornea was observed visually and pertinent observations were recorded.
- 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 5 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 annually. This I0 value is 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 was calculated by subtracting the initial opacity reading from the final opacity reading. These values were corrected by subtracting from each the average change in opacity observed for the negative-control corneas. The mean 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)
For the IVIS cut-off values for identifying test substances as inducing serious eye damage and test substances not including eye irritation or serious eye damage 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:
58.66
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
All 3 corneas treated with m-toluic acid showed slight opacity of the tissue.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.
- 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.

The evaluation of acceptability criteria was performed by using the following historical data:
- for evaluation of the validity of the positive control, the historical mean IVIS score as obtained from 2015 until 2017 was used (please refer to the tables in the field "Any other information on results incl. tables").
- for the evaluation of the validity of the negative control, the historical upper limits of the opacity and permeability values as obtained in 2017 were used (please refer to the tables in the field "Any other information on results incl. tables" below)

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

1.98

2.96

0.98

 

2

2.09

6.01

3.92

 

3

2.16

3.78

1.62

 

MV

2.08

4.25

2.17

 

4

 

Positive Control

3.50

112.05

108.5

106.38

5

3.54

83.03

79.49

77.32

6

3.74

87.36

83.62

81.45

MV

3.59

94.15

90.55

88.38

7

 

Test Item

2.31

49.11

46.80

44.63

8

0.97

59.42

58.45

56.28

9

1.69

73.82

72.13

69.96

MV

1.66

60.78

59.13

56.95

MV = mean value

Table 2: Permeability

Cornea No.

Test Item

OD490

Corrected OD490 Value

1

Negative Control

0.019

 

2

0.022

 

3

0.028

 

MV

0.023

 

4

 

Positive Control

1.473

1.450

5

2.465

2.442

6

3.065

3.042

MV

2.334

2.311

7

 

Test Item

0.091

0.068

8

0.092

0.069

9

0.228

0.205

MV

0.137

0.114

MV = mean value

Table 3: In vitro irritation score

Cornea No.

Test Item

Corrected Opacity

Corrected OD490 Value

IVIS

1

Negative Control

0.98

0.019

 

2

3.92

0.022

 

3

1.62

0.028

 

MV

2.17

0.023

2.52

4

 

Positive Control

106.38

0.624

 

5

77.32

1.182

 

6

81.45

2.222

 

MV

88.38

1.343

123.05

7

 

Test Item

44.63

0.068

 

8

56.28

0.069

 

9

69.96

0.205

 

MV

56.95

0.114

58.66

MV = mean value

Table 4: Historical mean in vitro irritation score of the positive control from February 2015 until August 2017

 

IVIS Positive Control – Imidazole 20%

Mean Value (MV)

125.12

Standard Deviation (SD)

17.75

MV-2xSD

89.63

MV+2xSD

160.62

Number of Replicates providing Historical Mean: 28

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 (Imidazole 20 %) from August 2017 until October 2017

Incubation; 240 min

Number of Replicates providing Historical Mean

 

 

Cornea No.

Opacity

Permeability

 

 

IVIS

 

Change of Opacity Value

 

Corrected Opacity Value

 

OD490 Value

 

Corrected OD490 Value

1

4

122.785

121.861

0.662

0.624

 

 

5

117.173

116.249

1.220

1.182

133.42

 

6

102.655

101.731

2.260

2.222

 

2

4

108.553

106.381

1.473

1.450

 

 

5

79.491

77.319

2.465

2.442

123.05

 

6

83.618

81.446

3.065

3.042

 

3

4

55.644

56.308

2.200

2.209

 

 

5

71.511

72.175

1.348

1.337

92.64

 

6

65.148

65.812

2.040

2.029

 

Mean Value (MV)

89.620

88.809

1.859

1.837

116.370

Standard Deviation (SD)

23.998

23.370

0.740

0.745

21.195

MV-2xSD

41.624

42.069

0.379

0.347

73.980

MV+2xSD

137.615

135.549

3.340

3.328

158.760

Table 6: Historical mean in vitro irritation score of the negative control from February 2015 until August 2017

 

IVIS Negative Control – NaCl 0.9 %

Mean Value (MV)

1.23

Standard Deviation (SD)

0.77

MV-2xSD

-0.31

MV+2xSD

2.78

Number of Replicates providing Historical Mean: 28

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 October 2017

Number of Replicates providing Historical Mean

 

 

Cornea No.

 

Opacity

 

Permeability

 

 

IVIS

 

Change of Opacity Value

 

OD490 Value

1

1

0.234

0.008

 

1.49

 

2

1.738

0.008

 

3

0.800

0.098

2

1

0.978

0.019

 

2.52

 

2

3.920

0.022

 

3

1.617

0.028

3

1

-0.149

0.009

 

-0.50

 

2

-0.415

0.015

 

3

-1.427

0.009

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

Mean Value (MV)

0.520

0.017

0.780

Standard Deviation (SD)

1.296

0.023

1.254

MV-2xSD

-2.072

-0.029

-1.727

MV+2xSD

3.112

0.064

3.287

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
According to the bovine corneal opacity and permeability assay, since the IVIS was > 55 (IVIS score: 58.66), the test item m-toluic acid can be considered as requiring classification for eye irritation or serious eye damage.
According to the Regulation (EC) No 1272/2008 and subsequent adaptions, the substance is classified for serious eye damage (Category 1; H318).