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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:
29.11. - 30.11.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
Adopted 14th Feb 2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrazol-5-ylamine
EC Number:
224-581-7
EC Name:
Tetrazol-5-ylamine
Cas Number:
4418-61-5
Molecular formula:
CH3N5
IUPAC Name:
1H-tetrazol-5-amine
impurity 1
Reference substance name:
Unknown impurities
Molecular formula:
Not available
IUPAC Name:
Unknown impurities
Test material form:
solid: crystalline
Details on test material:
white crystals

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Breeding service CHOVSERVIS a.s., division TORO® Hlavečník, Hradec Králové, Czech Republic

- Characteristics of donor animals (e.g. age, sex, weight): The eyes were enucleated as soon as possible after death. No detergent was used. Only healthy animals (12 to 30 months old) considered suitable for entry into the human food chain were used as a source of corneas for use in the BCOP test.

- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):
The risk of contamination was minimized (e.g., by keeping the container containing the eyes on ice, by adding antibiotics to the HBSS used to store the eyes during transport (e.g., penicillin at 100 IU/mL and streptomycin at 100 μg/mL).

- Time interval prior to initiating testing: The time interval between collection of the eyes and use of corneas in the BCOP was minimized (typically collected and used on the same day). All eyes used in the assay were from the same group of eyes collected on a specific day.

- indication of any existing defects or lesions in ocular tissue samples: Only corneas from eyes free of defects including scratched, and neovascularisation were used. The isolated corneas, after achieve normal metabolic activity (inductive incubation at 32 ± 1°C for one hour), were examined again. The corneas that show macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or a baseline opacity >7 opacity units were discarded.

Test system

Vehicle:
Hank's balanced salt solution
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
2g of the test substance was suspended in 10 mL of 0.9% sodium chloride solution.


VEHICLE
Hank`s Balanced Salts Solution (HBSS)
- Lot/batch no. (if required): SLBL4865, Sigma-Aldrich
Duration of treatment / exposure:
4 hrs
Duration of post- treatment incubation (in vitro):
1.5 hr
Number of animals or in vitro replicates:
The results were based on the selection criteria for the eyes, as well as the positive and negative control responses.
Number of corneas per group:
Exposed group (test item) - 3 corneas (No. 16, 18, 20)
Positive control group (20% Imidazole) – 3 corneas (No. 4, 5, 6)
Negative control group (0.9% NaCl) – 3 corneas (No. 1, 2, 3)
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
Selection criteria for eyes used in BCOP: Only corneas from eyes free of defects including scratched, and neovascularisation were used. The isolated corneas, after achieve normal metabolic activity (inductive incubation at 32 ± 1°C for one hour), were examined again. The corneas that show macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or a baseline opacity >7 opacity units were discarded.

Preparation: Corneas free of defects were dissected with a 2 to 3 mm rim of sclera remaining to assist in subsequent handling, with care taken to avoid damage to the corneal epithelium an endothelium.
Isolated corneas were mounted in specially designed corneal holders that consisted of anterior and posterior compartments, which interfaced with the epithelial and endothelial sides of the cornea, respectively.
Both chambers were filled to excess with pre-warmed Eagle's Minimum Essential Medium (EMEM). The device was then equilibrated at 32 ± 1°C for at least one hour in water bath to allow the corneas to equilibrate with the medium and to achieve normal metabolic activity, to the extent possible. Following the equilibration period, fresh pre-warmed EMEM was added to both chambers and a baseline opacity readings were taken for each cornea. Any corneas that showed macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or a baseline opacity >7 opacity units were discarded.
Each test group (test item, concurrent negative and positive controls) consisted of the three eyes. The three corneas with opacity values close to the median value for all corneas were selected as negative control corneas. The remaining corneas were then distributed into treatment and positive control groups.

QUALITY CHECK OF THE ISOLATED CORNEAS
From 24 eyes the 5 eyes were eliminated after inductive incubation, because the baseline opacity values were >7. Nine corneas were used for the study (the corneas No. 1, 2, 3, 4, 5, 6, 16,18 and 20), 4 eyes was superfluous and 6 corneas were used for testing of other substances.

NUMBER OF REPLICATES
Number of corneas per group:
Exposed group (test item) - 3 corneas (No. 16, 18, 20)
Positive control group (20% Imidazole) – 3 corneas (No. 4, 5, 6)
Negative control group (0.9% NaCl) – 3 corneas (No. 1, 2, 3)

NEGATIVE CONTROL USED
0.9% NaCl

SOLVENT CONTROL USED (if applicable)
0.9% NaCl

POSITIVE CONTROL USED
20% Imidazole

APPLICATION DOSE AND EXPOSURE TIME
2g of the test substance was suspended in 10 mL of 0.9% sodium chloride solution.
Closed-chamber method was used, because the test item was applicable by micropipette. The test item (750 µL) to cover the epithelial side of the cornea is introduced into the anterior chamber through the dosing holes on the top surface of the chamber, and the holes were subsequently sealed with the chamber plugs during the exposure.

Exposure time: 4 hrs

TREATMENT METHOD: closed-chamber method

POST-INCUBATION PERIOD: 1.5 hr

REMOVAL OF TEST SUBSTANCE, POST-EXPOSURE INCUBATION
After the exposure period, the negative control substance, the positive control substance and the test item were removed from the anterior chamber with EMEM (containing phenol red - the effectiveness of rinsing acidic or alkaline materials). The corneas were given a final rinse with EMEM (without phenol red). The EMEM (without phenol red) was used as a final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement. The anterior chamber was then refilled with fresh EMEM without phenol red. The opacity and permeability of each cornea were recorded.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: measured quantitatively with the aid of an opacitometer (Opacitometer, MC2 - Le spécialiste du laboratoire – France) resulting in opacity values measured on a continuous scale
- Corneal permeability: The amount of sodium fluorescein that crosses into the posterior chamber was quantitatively measured with the aid of UV/VIS spectrophotometry (Spectrophotometer GENESYSTM 10 UV/VIS Scanning). The values of absorbance measured at 490 nm were recorded as optical density (OD490) values. This term was used because the measuring is performed with visible light spectrophotometer using a standard 1 cm path length.

SCORING SYSTEM:
In Vitro Irritancy Score (IVIS)
IVIS = mean opacity value + (15 x mean permeability OD490 value)

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

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
8.6
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS: Study acceptance criteria were fulfilled.

- Acceptance criteria met for negative control: The value of opacity for negative control (0.9% NaCl) obtained during the study was 0.73 and value of permeability was 0.008. The values obtained during this study not exceeded upper limits, so the study is considered acceptable.

- Acceptance criteria met for positive control: The value of IVIS for positive control (20% imidazole) obtained during the study was 72.71. This value is within the acceptance limit (one standard deviation of the current historical mean), so the study is considered acceptable.

Any other information on results incl. tables

The In Vitro Irritancy Score (IVIS) was computed according the following formula:

IVIS = mean opacity value + (15 x mean permeability OD490 value)

Group

IVIS calculation

Result

NC (0.9% NaCl)

0.73 + 15 x 0.008

0.85

PC (20% Imidazole in 0.9% NaCl)

45.69 + 15 x 1.801

72.71

Test item (5-aminotetrazole)

8.58 + 15 x 0.001

8.60

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
The In Vitro Irritancy Score (IVIS) for 5-aminotetrazole was 8.60. The corneas treated by the test item were without macroscopic damage.
This value of IVIS is > 3 and simultaneously ≤ 55 therefore the classification according to UN GHS criteria for eye irritation or serious eye damage is: no prediction can be made.
Executive summary:

The test item, 5-aminotetrazole, was tested for the evaluation the potential ocular corrosivity or severe irritancy as measured by its ability to induce opacity and increased permeability in an isolated bovine cornea.

The test was performed according to the Method B.47 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, Adopted 14th February 2017

The test was performed using nine isolated bovine corneas. The testing was performed on three groups of corneas: test item treatment group, positive control group and negative control group. Three corneas per group were used.

The test item was tested as suspension prepared from test item at 20% concentration in a 0.9% sodium chloride solution.

The closed-chamber method was used, because the test item was applicable by micropipette. The opacity and permeability of each cornea were measured. The In Vitro Irritancy Score (IVIS) was calculated from the values of opacity and permeability.

The In Vitro Irritancy Score (IVIS) for 5-aminotetrazole was 8.60. The corneas treated by the test item were without macroscopic damage.

This value of IVIS is > 3 and simultaneously ≤ 55 therefore the classification according to UN GHS criteria for eye irritation or serious eye damage is: no prediction can be made.