Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability 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: particulate/powder
Details on test material:
1 MATERIALS AND METHODS
1.2 Test Item
Designation in Test Facility: 17031402G
Date of Receipt: 14. Mar. 2017
Condition at Receipt Room temperature, in proper conditions
1.2.1 Specification.
Name Blendazol Red Blendwell
Batch no. E 328
Appearance Dark Red Powder
Composition 2-Naphtalenesulfonic acid, 7-amino-4-hydroxy-3,8-bis[[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]azo]-, triso dium salt
Purity 95 % by HPLC
Homogeneity homogeneous
Expiry date 17. Feb. 2019
Storage Room Temperature: (20 ± 5°C); Keep away from humidity
CAS No. 607724-47-0
EINECS-No. 612-028-6
Chemical Class not stated
Stability H2O: 96h; EtOH: unknown; acetone: unknown CH3CN: unknown; DMSO: unknown
Solubility H2O: to be determined*; EtOH: unknown; acetone: unknown CH3CN: unknown; DMSO: unknown


* Will be determined in another GLP-study at LAUS GmbH and will be stated in the final report, this
This information is not provided by the sponsor but determined at LAUS GmbH.


Test animals / tissue source

Species:
cattle
Strain:
other: Bovine cornea collected from slaughtered cattle
Details on test animals or tissues and environmental conditions:
- Source: Bos primigenius Taurus (fresh bovine corneas)

Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hank’s balanced salt solu-tion (supplemented with 0.01% streptomycin and 0.01% penicillin). Then, the corneas were dissected and incubated in medium at 32 ± 1 °C in an incubation chamber for 1 h.

- indication of any existing defects or lesions in ocular tissue samples: no existing defects
-- Source:the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany
- Number of animals: not specified
- Characteristics of donor animals (e.g. age, sex, weight): The cattle were between 12 and 60 months old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The eyes were transported to the test facility in Hank’s balanced salt solution (supple-mented with 0.01% streptomycin and 0.01% penicillin).

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
Amounts of Test Item
Tissue Amount
1 423.8 mg
2 444.1 mg
3 464.4 mg
Duration of treatment / exposure:
4 hours at 32 ± 1 °C
Duration of post- treatment incubation (in vitro):
90 min
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS:
the corneas were examined and only corneas which were free from damages were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C

QUALITY CHECK OF THE ISOLATED CORNEAS:
the corneas were examined and only corneas which were free from damages were used

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: HBSS

SOLVENT CONTROL USED (if applicable)

POSITIVE CONTROL USED: Imidazole, 20% solution in HBSS

APPLICATION DOSE AND EXPOSURE TIME: 4h
Tissue Amount
1 423.8 mg
2 444.1 mg
3 464.4 mg

TREATMENT METHOD: open chamber
The “open chamber-method” is used for solid substances.
In order to apply the test item, the nut was unscrewed to remove the glass disc. The test item could be applied directly on the cornea now

POST-INCUBATION PERIOD: yes.
duration : 1h

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:2
- POST-EXPOSURE INCUBATION:90 min

METHODS FOR MEASURED ENDPOINTS:
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS): 10.31

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used. yes
The test item was tested pure.
Under the conditions of this test, the test item Blendazol Red Blendwell showed effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is 10.31.
According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category.

The negative control (HBSS) and the positive control (20% imidazole solution) have met the validity criteria

Results and discussion

In vitro

Results
Irritation parameter:
cornea opacity score
Value:
ca. 10.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes 1.18 ≤ 3
- Acceptance criteria met for positive control: 104.68 witch falls between 72.85 – 165.65
9.3 Validity
According to the guideline, the test is considered as valid if the positive control causes an IVIS that falls within two standard deviations of the current historical mean.
The negative control has to show an IVIS ≤ 3.
The validity criteria and findings are given in the following table:
Table 9.3 a Validity
Parameter Criterion Found Assessment
IVIS of negative control
HBSS-solution ≤ 3 1.18 ok
IVIS of positive control
20% imidazole solution 72.85 – 165.65 104.68 ok

Values for negative and positive controls were within the range of historical data of the test facility (see historical data). Therefore, the test system was acceptable.

- Range of historical values if different from the ones specified in the test guideline:

COMPARISON WITH HISTORICAL DATA
In the following table, the means of the negative control and positive control of all experi-ments which were performed with this positive control up to 06. Apr. 2017 are stated and compared with the values which were found in this study.
Table 14 a Historical Data
Negative Control Positive Control
Mean IVIS 1.95 119.25
Standard Deviation IVIS 1.01 23.20
Range of IVIS (validity) ≤ 3 72.85 – 165.65
Study 17031402G850 1.18 104.68
Mean Opacity 1.70 82.66
Standard Deviation
Opacity 1.00 18.02
Range of Opacity -1.86 – 4.08 42.92 – 133.11
Study 17031402G850 1.10 77.40
Mean Permeability 0.02 2.48
Standard Deviation Per-meability 0.02 1.00
Range of Permeability -0.01 – 0.10 0.75 – 5.89
Study 17031402G850 0.01 1.82

Any other information on results incl. tables

1.1     IVIS Values

The calculated IVIS for each replicate and the corresponding means are presented in the following table:

Table9.2‑a      IVIS

Test Group

IVIS

Mean IVIS

Relative Standard Deviation IVIS

Negative Control
HBSS-solution

2.37

1.18

94.07%

1.02

0.16

Test Item
Blendazol Red Blendwell

13.26

10.31

26.30%

9.72

7.94

Positive Control
20% imidazole

solution

99.47

104.68

23.84%

82.75

131.83

Note: the high relative standard deviation of the IVIS of the negative control is due to mathematical reasons, as the respective means are very small.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category.
In the negative control, no signs of eye irritation were observed.
The positive control induced serious eye damage, which would be classified as GHS cat-egory 1.
The test item Blendazol Red Blendwell showed effects on the cornea of the bovine eye.
The calculated IVIS (in vitro irritancy score) is 10.31.

The experiment is considered as sufficient for the classification of the test item, because all three replicates of the test item lead to the same assessment for the test item
Executive summary:

The eye irritation of the test Blendazol Red was determined using the Bovine Corneal Opacity and Permeability (BCOP) Test Method according to the OECD Guideline 437 and EU Method B.47

Bovine corneas were used. They were collected from slaughtered cattle which were between 12 and 60 months old.

The test item Blendazol Red Blendwell was brought onto the cornea of a bovine eye which had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured.

The test item was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured.

HBSS-solution was used as negative control. The negative control showed no irritating effect on the cornea and the calculated IVIS (in vitro irritancy score) is 1.18.

20% imidazole solution was used as positive control. The positive control induced serious eye damage on the cornea and falls within two standard deviations of the current historical mean. The calculated IVIS (in vitro irritancy score) is 104.68.

 

Under the conditions of this study, the test item Blendazol Red Blendwell showed effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is 10.31.

According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category for eye damage.