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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:
weight of evidence
Study period:
May - Aug 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
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:
09 Oct 2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt Rheinland-Pfalz

Test material

Constituent 1
Chemical structure
Reference substance name:
Dibenzyldimethylammonium chloride
EC Number:
202-903-7
EC Name:
Dibenzyldimethylammonium chloride
Cas Number:
100-94-7
Molecular formula:
C16H20N.Cl
IUPAC Name:
dibenzyldimethylazanium chloride
Test material form:
solid: crystalline
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: 17 Jan 2020
- Physical state / color: Solid / white
- pH value: Ca. 5 (undiluted test substance moistened with deionized water, determined in the lab prior to the start of the GLP study); Ca. 5 (20 % aqueous preparation; determined in the lab prior to start of the GLP study)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, under N2
- Solubility and stability of the test substance in the solvent/vehicle: After stirring with a magnetic stirrer, the test substance was soluble in the vehicle.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance preparation was produced on a weight per volume (w/v) basis shortly before application. After stirring with a magnetic stirrer, the test substance was soluble in the vehicle.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : 20 % (w/v) solution in deionized water

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Schlachthof Alzey, Emil Färber GmbH & Co. KG, Robert-Bosch-Straße 23, 55232 Alzey, Germany
- Characteristics of donor animals (e.g. age, sex, weight): min. 12 months and max. 60 months old animals
- indication of any existing defects or lesions in ocular tissue samples: Corneas free of defects (opacity, scratches, pigmentation etc.)

Test system

Vehicle:
water
Remarks:
deionized
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 20 % (w/v) in deionized water

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µL
Duration of treatment / exposure:
4 h
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS : Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders that consist of anterior and posterior chambers. Both chambers were filled to excess with pre-warmed Eagle’s MEM (without phenol red) and then equilibrated in a vertical position at about 32°C for at least 1 hour.

QUALITY CHECK OF THE ISOLATED CORNEAS : After the equilibration period, the medium in both chambers was replaced by fresh pre-warmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that showed macroscopic tissue damage or an opacity value < 561 opacity units were discarded. The remaining corneas were then distributed into negative control, positive control and treatment groups.

NUMBER OF REPLICATES : 3

NEGATIVE CONTROL USED : deionized water

POSITIVE CONTROL USED : 20 % solution (w/v) of imidazole in deionized water

APPLICATION DOSE AND EXPOSURE TIME : 750 µL, 4 h

TREATMENT METHOD: [closed chamber / open chamber]

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: one

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Before measurement, each cornea was visually observed and observations were recorded. Final corneal opacity readings were taken for each cornea with an opacitometer.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: see table 1

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
1st experiment
Value:
65
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
2nd experiment
Value:
45.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
3rd experiment
Value:
82.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation

Any other information on results incl. tables

Table 2: In Vitro Irritancy score (IVIS) of the test substance, the NC and the PC

Test substance identification

Cornea-No.

Opacity per cornea

Permeability per cornea

IVIS

Per cornea

Per group

Mean

SD

Test substance

7

8

9

63.9

42.8

73.5

0.078

0.171

0.584

65.0

45.3

82.3

64.2

18.5

NC

1

2

3

2.8

9.8

8.5

0.004

0.005

0.008

2.9

9.9

8.6

7.1

3.7

PC

4

5

6

91.0

96.7

88.8

1.606

2.043

2.389

115.1

127.3

124.6

122.4

6.4

Table 3: Opacity score of the test substance, the NC and the PC

Test substance identification

Cornea-No.

Initial opacity

Final opacity

Opacity Change

Corrected Opacity Change

Mean

SD

Test substance

7

8

9

6.5

5.2

6.5

77.4

55.0

87.1

70.9

49.8

80.5

63.9

42.8

73.5

60.0

15.7

NC

1

2

3

6.9

6.0

6.4

9.7

15.8

14.9

2.8

9.8

8.5

NA

NA

NA

7.0

3.7

PC

4

5

6

4.9

6.6

5.6

103.0

110.3

101.4

98.1

103.7

95.8

91.0

96.7

88.8

92.2

4.1

Table 4: Permeability score of the test substance, the NC and the PC

Test substance identification

Cornea-No.

Mean OD490

Dilution Factor

Mean Corrected OD490

Mean

SD

Test substance

7

8

9

0.084

0.176

0.590

1

1

1

0.078

0.171

0.584

0.278

0.270

NC

1

2

3

0.004

0.006

0.008

1

1

1

NA

NA

NA

0.006

0.002

PC

4

5

6

0.322

0.410

0.479

5

5

5

1.606

2.043

2.389

2.013

0.393

Table 5: Historic control data of the BCOP Test

Historic Range of NC (protocol for solids)

Historic period

Jan 2017 – Mar 2019 (No. of tests performed: 12)

Opacity

Mean

SD

Mean + 2 SD

Mean – 2 SD

9.2

3.7

16.5

1.9

Permeability (OD490)

Mean

SD

Mean + 2 SD

Mean – 2 SD

0.003

0.002

0.006

0.000

In Vitro Irritation Score (IVIS)

Mean

SD

Mean + 2 SD

Mean – 2 SD

9.2

3.7

16.6

1.9

Historic Range of PC (20 % Imidazole)

Historic period

Jan 2017 – Mar 2019 (No. of tests performed: 9)

Opacity

Mean

SD

Mean + 2 SD

Mean – 2 SD

93.6

9.9

113.5

73.7

Permeability (OD490)

Mean

SD

Mean + 2 SD

Mean – 2 SD

2.234

0.578

3.389

1.078

In Vitro Irritation Score (IVIS)

Mean

SD

Mean + 2 SD

Mean – 2 SD

127.1

13.6

154.2

100.0

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
In the BCOP Test the mean IVIS (64.2) of the corneas treated with the test substance indicated a serious eye irritation potential (BCOP cut-off threshold 55) of the test substance.
However, some variability between the results of the individual corneas treated with the test substance was noted and the mean IVIS lies minimal below the borderline range (IVIS 45.0 to 65.0) for evaluation.
Nevertheless, the result of the BCOP has to be considered to indivate ocular corrosion or severe irritation of the test substance, because the IVIS values of two corneas and the mean value of all corneas lie well above the BCOP cut-off threshold of 55 and the mean IVIS lies minimal below the upper value of the borderline range.
Executive summary:

The potential of the test substance to cause severe ocular irritation or serious damage to the eyes was assessed by a single topical application of 750 μL 20% test-substance preparation to the epithelial surface of isolated bovine corneas.

Three corneas were treated with the test-substance preparation for an exposure period of 4 hours.

Besides the test substance, a negative control (NC; deionized water) and a positive control (PC; 20% imidazole in deionized water) were applied to three corneas each.

Corneal opacity was measured quantitatively as the amount of light transmitted through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance.

The mean IVIS (64.2) of the corneas treated with the 20% test substance preparation indicated serious eye damage (BCOP cut-off threshold 55) of the test substance.

The results of the test substance, the negative control (deionized water) and the positive control (20% imidazole) fulfilled the acceptance criteria and demonstrate the validity of the assay.