Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 August 2017 - 29 August 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
Test material form:
solid: particulate/powder
Details on test material:
Physical appearance: white powder
Storage conditions: at room temperature

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: young cattle, obtained from the slaughterhouse
- Storage, temperature and transport conditions of ocular tissue: eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Subsequently, eyes were collected and transported in physiological saline in a suitable container under cooled conditions. The isolated corneas were stored in a petri dish with cMEM containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum. The corneas were incubated for the minimum of 1 hour at 32 ± 1 °C.
- The eyes were checked for unacceptable defects and those exhibiting defects were discarded.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 321.6 to 387.5 mg (complete coverage of the cornea)

NEGATIVE CONTROL:
- Amount applied: 750 µL

POSITIVE CONTROL:
- Amount applied: 750 µL
Duration of treatment / exposure:
240 +/- 10 minutes
Duration of post- treatment incubation (in vitro):
90 +/- 5 minutes in sodium fluorescein
Number of animals or in vitro replicates:
3
Details on study design:
TREATMENT METHOD:
Two individual experiments were performed. The medium from the anterior compartment was removed and 750 µL of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. The test item was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (321.6 to 387.5 mg).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.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red.
- POST-EXPOSURE INCUBATION: 90 ±5 minutes 32 ± 1 °C in sodium-fluorescein for permeability determinations.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacity meter (OP-KIT)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microtiter plate reader (TECAN Infinite® M200 Pro Plate Reader, OD490). OD490 values of less than 1.500 were used in the permeability calculation.
- Other: possible pH effects of the test substance on the corneas were recorded.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

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

ACCEPTABILITY CRITERIA:
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Remarks:
mean of 3 replicates
Run / experiment:
Repeated experiment
Value:
65
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
Mean IVIS: 121
Other effects / acceptance of results:
Opacity scores, permeability scores and in vitro irritancy scores are summarized in table 2, 3 and 4 in the section 'any other information on results'.
Since the IVIS were spread over 2 categories (78, 59 and 16), the test was inconclusive and a repeat experiment was performed.

OTHER EFFECTS:
- Visible damage on test system: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, in the first experiment, the individual in vitro irritancy scores for the negative controls ranged from -0.6 to 2.8. In the second test, the individual in vitro irritancy scores for the negative controls ranged from 0.1 to 1.4. These results were within the historical data range.
- Acceptance criteria met for positive control: yes, in the first experiment the individual positive control in vitro irritancy scores ranged from 91 to 147 and the corneas treated with the positive control were turbid after the 240 minutes of treatment. In the second experiment, the individual positive control in vitro irritancy scores ranged from 111 to 134 and the corneas treated with the positive control were turbid after the 240 minutes of treatment.

Any other information on results incl. tables

Table 2 Opacity scores

First experiment:

Treatment

Opacity

before treatment

Opacity

after treatment

Final Opacity1

Negative control corrected Final Opacity2

Mean Final Opacity

 

Negative control

3.2

3.0

-0.2

 

0.7

3.3

6.1

2.8

3.8

3.2

-0.6

 

Positive control

2.7

125.1

122.4

122

104

4.9

123.8

119.0

118

2.2

76.1

73.9

73

 

Test item

3.0

4.4

1.5

0.8

6.7

1.3

12.4

11.1

10

3.5

13.2

9.7

9.0

Second experiment

Treatment

Opacity

before treatment

Opacity

after treatment

Final Opacity1

Negative control corrected Final Opacity2

Mean Final Opacity

 

Negative control

2.2

2.0

-0.2

 

0.4

2.2

3.5

1.3

2.7

2.9

0.2

 

Positive control

3.1

90.0

86.9

86

100

2.9

116.5

113.6

113

4.3

105.9

101.5

101

 

Test item

2.4

5.0

2.6

2.2

0.1

3.1

3.0

-0.2

-0.6

4.2

3.2

-1.0

-1.4

1  Final Opacity = Opacity after treatment – Opacity before treatment.

2  Negative control corrected Final Opacity = Final opacity – Mean final opacity negative control

3    Calculations are made without rounding off.

Table 3 Permeability scores (corrected)

First experiment

Treatment

Dilution factor

Negative control corrected OD490 11

Negative control corrected OD490 21

Negative control corrected OD490 31

Negative control corrected OD490

Average

Negative control corrected final

OD490

Average OD

 

Positive control

6

0.277

0.278

0.279

0.278

1.667

1.390

1

1.308

1.293

1.323

1.308

1.308

1

1.206

1.183

1.195

1.195

1.195

 

Test item

1

0.863

0.850

0.864

0.859

5.153

2.964

1

0.552

0.544

0.533

0.543

3.257

1

0.489

0.477

0.476

0.481

0.481

Second experiment

Treatment

Dilution factor

Negative control corrected OD490 11

Negative control corrected OD490 21

Negative control corrected OD490 31

Negative control corrected OD490

Average

Negative control corrected final

OD490

Average OD

 

Positive control

6

0.270

0.271

0.276

0.272

1.634

1.391

1

1.403

1.406

1.402

1.404

1.404

1

1.129

1.138

1.140

1.136

1.136

 

Test item

6

0.758

0.761

0.765

0.761

4.568

5.275

6

0.881

0.883

0.890

0.885

5.308

6

0.995

0.984

0.995

0.991

5.948

1  OD490 values corrected for the mean final negative control permeability (0.017).

2    Calculations are made without rounding off.

Table 5 In vitro irritancy score

First experiment

Treatment

Final Opacity2

Final OD4902

In vitroIrritancy Score1

 

Negative control

-0.2

0.002

-0.2

2.8

-0.003

2.8

-0.6

-0.002

-0.6

 

Positive control

122

1.667

147

118

1.308

138

73

1.195

91

 

Test item

0.8

5.153

78

10

3.257

59

9.0

0.481

16

Second experiment

Treatment

Final Opacity2

Final OD4902

In vitroIrritancy Score1

 

Negative control

-0.2

0.015

0.1

1.3

0.009

1.4

0.2

0.027

0.6

 

Positive control

86

1.634

111

113

1.404

134

101

1.136

118

 

Test item

2.2

4.568

71

-0.6

5.308

79

-1.4

5.948

88

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

2  Positive control and test item are corrected for the negative control.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Remarks:
Category 1 according to Regulation (EC) No. 1272/2008
Conclusions:
Based on the outcome of a Bovine Corneal Opacity and Permeability test (BCOP) performed according to OECD guideline 437 and GLP principles, it is concluded that HYDROPIP-NR is classified Category 1 under GHS (IVIS ranging from 71 to 88 after 240 minutes of treatment).
Executive summary:

The eye hazard potential of HYDROPIP-NR was assessed in a Bovine Corneal Opacity and Permeability test (BCOP test), performed according to OECD guideline 437 and GLP principles. Three corneas from young cattle were exposed to 321.6 to 387.5 mg (complete coverage) of the test item, next to a negative control group of physiological saline (n=3) and a positive control of 20% (w/v) imidazole (n=3). Duration of treatment was approximately 240 minutes. Results of the negative and positive controls were within the historical data. Therefore, it was shown that the negative control did not induce irritancy on the corneas, the test conditions were adequate and the test systems functioned properly. Since the IVIS of the test item were spread over 2 categories in the first experiment (78, 59 and 16), the test was inconclusive and a repeat experiment was performed. The results of the repeated experiment showed a mean irritancy score of 65. Based on this result (IVIS > 55), the test item is classified as categry 1 for eye irritation according to GHS criteria and Regulation (EC) No. 1272/2008.