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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:
13-April-2010 to 14-April-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD guideline 437 “Bovine corneal opacity and permeability (BCOP) test method for identifying ocular corrosives and severe irritants”
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): Ammonium thiocyanate
- Substance type: White crystals
- Physical state: Solid
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark in desiccator

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
Bovine eyes were used as soon as possible after slaughter on the same day. Bovine eyes from young cattle were obtained from the slaughterhouse
(Vitelco, 's Hertogenbosch, The Netherlands), 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.

Preparation of corneas
All eyes were carefully examined for defects by holding the eyes submersed in physiological saline. Those exhibiting unacceptable defects, such as opacity, scratches, pigmentation and neovascularization were discarded.
The isolated corneas were stored at 32 +/- 1°C in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Invitrogen Corporation, Breda, The Netherlands) containing 1% (v/v) L-glutamine (Invitrogen Corporation) and 1% (v/v) Foetal Bovine Serum (Invitrogen Corporation)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont, France) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32+/- 1°C. The corneas were incubated for the minimum of 1 hour at 32 +/- 1°C.

Cornea selection and Opacity reading
with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (OP-KIT, MC2, Clermont, France). The opacity of each cornea was read against an air filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 3 were not used. Three corneas were selected at random for each treatment group.

Test system

Vehicle:
physiological saline
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µl per cornea
- Concentration (if solution): 20% (w/w)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µl of physiological saline per cornea

POSITIVE CONTROL
Amount(s) applied (volume or weight with unit): 750 µl per cornea
Concentration (if solution): 20% (w/w) Imidazole (According to the OECD guideline (437) a positive control should result in an appropriate response. Whether this is 20% (w/v) or 20% (w/w) has no influence on the study integrity. Since the response was within the historical control data range, this deviation has no effect on the study integrity.)
Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
Three corneas were selected at random for each treatment group.
Details on study design:
TEST SITE
- Isolated bovine cornea

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: 240 minutes

SCORING SYSTEM:
- After exposure the cornea is thoroughly rinsed to remove the test substance followed by immediate opacity measurement and permeability evaluation of the cornea.
- 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 and microplate reader

DATA EVALUATION:
A test substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant

Results and discussion

In vivo

Results
Irritation parameter:
other: In vitro irritancy score (IVIS)
Basis:
mean
Time point:
other: 240 minutes
Score:
121
Reversibility:
not reversible

Any other information on results incl. tables

Table 1 summarizes the opacity, permeability and in vitro irritancy scores of Sodium thiocyanate and the controls. The opacity, permeability and in vitro scores of the individual corneas are shown in Tables 2-5.

 

The individual in vitro irritancy scores for the negative controls ranged from -0.9 to 0.0. The individual positive control in vitro irritancy scores ranged from 102 to 110 (Table 5).

 

The corneas treated with the positive control were turbid after the 240 minutes of treatment.

 

The corneas treated with Ammonium thiocyanate showed opacity values ranging from 60 to 83 and permeability values ranging from 3.036 to 3.690. The corneas were turbid after the 240 minutes of treatment with Ammonium thiocyanate. Hence, the in vitro irritancy scores ranged from 106 to 132 after 240 minutes of treatment with Ammonium thiocyanate.

 

 

Table 1 Summary of opacity, permeability and in vitro scores

 

Treatment

Mean
Opacity

Mean
Permeability

Mean In vitro
Irritation Score1,2

Negative control

0

0.000

0

Positive control

76

1.998

106

Ammonium thiocyanate

71

3.328

121

1 Calculated using the negative control mean opacity and mean permeability values.

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

 

 

INDIVIDUAL OPACITY, PERMEABILITY AND IN VITRO SCORES

 

Table 2 Opacity score

 

Eye

Opacity
before
treatment

Opacity
after
treatment

Final
Opacity1

Negative control
corrected Final
Opacity

Mean
Opacity

Negative control

1

0

0

0

-1

0

2

0

1

1

0

3

0

1

1

0

Positive control

4

1

82

81

80

76

5

0

74

74

73

6

0

76

76

75

Ammonium thiocyanate

10

0

84

84

83

71

11

1

61

61

60

12

0

72

72

71

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

 

 

Table 3 Permeability score individual values (uncorrected)

 

Eye

Dilution
factor

OD490
1

OD490
2

OD490
3

Average
OD

Final
OD

Mean final
negative
control

Negative control

1

1

0.018

0.020

0.019

0.019

0.019

0.014

2

1

0.008

0.010

0.007

0.008

0.008

3

1

0.013

0.012

0.016

0.014

0.014

Positive control

 

4

6

0.310

0.305

0.304

0.306

1.836

 

5

6

0.422

0.422

0.418

0.421

2.526

 

6

6

0.314

0.312

0.317

0.314

1.884

 

Ammonium thiocyanate

 

10

6

0.565

0.561

0.545

0.557

3.342

 

11

6

0.524

0.519

0.516

0.520

3.120

 

12

6

0.628

0.633

0.626

0.629

3.774

 

 

 

Table 4 Permeability score individual values (corrected)

 

Eye

Dilution
factor

OD490
1(1)

OD490
2(1)

OD490
3(1)

Average
OD

Final
OD

Mean final
negative
control

Negative control

1

1

0.004

0.006

0.005

0.005

0.005

0.000

2

1

-0.006

-0.004

-0.007

-0.006

-0.006

3

1

-0.001

-0.002

0.002

0.000

0.000

Positive control

4

6

0.296

0.291

0.290

0.292

1752

1.998

5

6

0.408

0.408

0.404

0.407

2442

6

6

0.300

0.298

0.303

0.300

1800

Ammonium thiocyanate

10

6

0.551

0.547

0.531

0.543

3.258

3.328

11

6

0.510

0.505

0.502

0.506

3.036

12

6

0.614

0.619

0.612

0.615

3.690

1 OD490 values corrected for the mean final negative control permeability (0.014).

 

 

Table 5 In Vitro irritancy score

 

Eye

Negative control
corrected Final
Opacity

Negative control
corrected Final
OD490

In vitro Irritancy
Score1

Negative control

1

-1

0.005

-0.9

2

0

-0.006

-0.1

3

0

0.000

0.0

Positive control

4

80

1.752

106

5

73

2.442

110

6

75

1.800

102

Ammonium thiocyanate

10

83

3.258

132

11

60

3.036

106

12

71

3.690

126

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

 

 

Table 6 Historical control data for the BCOP studies

 

Negative control

Positive control

Opacity

Permeability

In vitro Irritancy
Score

In vitro Irritancy
Score

Range

0 - 1

-0.007 - 0.009

-0.1 - 1.1

96 - 146

Mean

0.24

0

0.24

120

SD

0.44

0

0.45

14

n

21

21

21

21

SD = Standard deviation

n = Number of observations

 

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of July 2008 to October 2009.

Applicant's summary and conclusion

Interpretation of results:
corrosive
Remarks:
Migrated information and severe irritant Criteria used for interpretation of results: OECD GHS
Conclusions:
The mean IVIS was 121 after 240 minutes of treatment with Ammonium thiocyanate.
Executive summary:

Screening for the eye irritancy potential of Ammonium thiocyanate using the Bovine Corneal Opacity and Permeability test (BCOP test).

This report describes the ocular irritation properties of Ammonium thiocyanate on an isolated bovine cornea. The possible ocular irritancy of Ammonium thiocyanate was tested through topical application for 240 ± 10 minutes. The study procedures described in this report were based on the most recent OECD 437 guideline.

Ammonium thiocyanate consisted of white crystals with a purity of 99.8% (dried material). The test substance was applied as a 20% (w/w) solution (750 μl) directly on top of the corneas.

The negative control responses of the opacity and permeability values were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% w/w Imidazole) was 106 and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Ammonium thiocyanate induced severe ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 121 after 240 minutes of treatment.

Since Ammonium thiocyanate induced an IVIS ≥ 55.1, it is concluded that Ammonium thiocyanate is a corrosive or severe irritant in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.