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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:
Feb - Mar 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:
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. certificate)
Remarks:
Landesamt für Umwelt Rheinland-Pfalz

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: L2018-137
- Expiration date of the Batch: 01 Feb 2021
- Purity: 99.6 % (tolerance ± 1.0 %)
- pH value: ca. 4 (undiluted test substance moistened with deionized water, determined in the lab prior to start of the GLP study); ca. 3.5 (20 % aqueous preparation, determined in the lab prior to start of the GLP study)
- Physical state / color: Solid / yellowish

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

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 by stirring with a high-speed homogenizer (Ultra-Turrax) and a magnetic stirrer. The homogeneity of the test substance preparation during application was provided by stirring with a magnetic stirrer.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : 20 % (w/v) suspension 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): minimum 12 months and maximum 60 months of age
- indication of any existing defects or lesions in ocular tissue samples: corneas free of defects (opacity, scratches, pigmentation etc.) were used

Test system

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

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µL
Duration of treatment / exposure:
4 hours
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.

NUMBER OF REPLICATES : 3

NEGATIVE CONTROL USED : deionized water

POSITIVE CONTROL USED : Imidazole 20 % (w/v) in deionized water

APPLICATION DOSE AND EXPOSURE TIME : 750 µL for 4 hours

TREATMENT METHOD: 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 UV/VIS spectrophotometry (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
- IVIS ≤ 3: no UN GHS Category
- IVIS > 3; ≤ 55: No prediction can be made
- IVIS > 55: UN GHS Category 1

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
1st test run
Value:
39.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
2nd test run
Value:
36.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Table 1: 1st test run: 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

19

20

21

30.2

41.5

46.9

0.003

0.003

0.000

30.2

41.5

46.9

39.5

8.5

NC

13

14

15

4.3

12.0

24.2

0.000

0.001

0.003

4.3

12.0

24.3

13.5

10.1

PC

16

17

18

51.5

256.0

61.6

3.670

1.982

2.385

106.6

285.8

97.4

163.2

106.2

Table 2: 2nd test run: 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

37.9

37.7

32.5

0.004

0.005

0.000

38.0

37.7

32.5

36.1

3.1

NC

1

2

3

2.3

6.7

2.9

0.002

0.004

0.003

2.3

6.7

2.9

4.0

2.4

PC

4

5

6

96.0

96.5

104.3

1.360

2.012

1.569

116.4

126.7

127.8

123.6

6.3

Table 3: Historic control data of the BCOP Test: Negative control (protocol for solids), Jan 2017 - Jul 2018 (No. of tests performed: 8)

 

Mean

SD

Mean + 2 SD

Mean – 2 SD

Opacity

9.6

3.7

17.0

2.1

Permeability [OD570]

0.003

0.001

0.005

0.001

Table 4: Historic control data of the BCOP Test: Positive control (20 % Imidazole), Jan 2017 - Jul 2018 (No. of tests performed: 8)

 

Mean

SD

Mean + 2 SD

Mean – 2 SD

Opacity

92.9

10.4

113.7

72.1

Permeability [OD570]

2.307

0.571

3.449

1.165

In Vitro Irritation Score (IVIS)

127.5

14.4

156.4

98.7

Applicant's summary and conclusion

Interpretation of results:
other: not identified as corrosive or severe irritant
Conclusions:
Based on the results of the BCOP and by applying the evaluation criteria, it was concluded that the test substance was not identified as corrosive or severe irritant under the test conditions chosen. No further assumptions can be made based on this in vitro study alone.
Executive summary:

The potential of the test substance to cause 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. In addition to 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 quantitatively measured as the amount of light transmitted through the

cornea. Permeability was quantitatively measured 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 following results were obtained in the BCOP Test:

Two test runs were performed. The results obtained in the 1st test run did not indicate a severe eye irritation potential of the

test substance. However, high variability between the individual corneas treated with the negative control (NC) and positive control (PC) were noted. Therefore, the study was repeated to verify the result and to meet the acceptance criteria of the test.

The obtained results of the test substance in the 2nd test run of the BCOP test verified the findings of the 1st experiment. All acceptance criteria of the test were fulfilled.

Based on the results of the BCOP and by applying the evaluation criteria, it was concluded that the test substance was not identified as corrosive or severe irritant under the test conditions chosen. No further assumptions can be made based on this in vitro study alone.