Tetrazol-5-ylamine

Endpoint summary

Description of key information

The sequential testing strategy was used as it is recommended in supplement to TG 405 (2012). SAR systems are not applicable to this type of test item. Validated and accepted in vitro tests for eye corrosion/irritation have been conducted prior to decide performing the in vivo test. These were In vitro Skin Irritation Test (OECD TG 439 ) BCOP test (OECD TG No. 437) and In vitro Eye Irritation Test (OECD TG No.492). The results did not allow the classification of test item with regard to its potential to cause eye irritation or serious eye damage. Therefore it was decided to perform in vivo eye irritation study.

SKIN

- Study No. 413/17/4AC: 5-aminotetrazole - In vitro Skin Corrosion Test (EpiDermTM Model); VUOS CETA Report No.: 17-693 (acc. OECD TG No. 431)

Result: non-corrosive in the EpiDermTM model

- Study No. 413/17/4AI: 5-aminotetrazole - In vitro Skin Irritation Test (EpiDermTM Model); VUOS-CETA Report No. 17-780, 2017 (acc. OECD TG No. 439; EU Method B.46)

Result: no category with regard to skin irritation

EYE

- Study No. 413/17/5BCOP: 5-aminotetrazole - Bovine Corneal Opacity and Permeability Test; VUOS-CETA Report No. 17-760, 2018 (acc. OECD TG No. 437; EU Method B.47) Result: no prediction can be made

- Study No. 413/17/5AI: 5 -aminotetrazole - In vitro Eye Irritation Test (EpiOcularTM Model); VUOS-CETA Report No. 18 -12, 2018 (acc. OECD TG No.492)

Result: substance potentially requiring classification and labelling, further testing with other test methods required

Under the experimental design average viability of treated tissues by the test substance 5-aminotetrazole was 2.3 % of negative control average value i.e. viability was ≤ 60 %. The effect of the test substance was positive in EpiOcularTM model (tissues were damaged). According to the classification criteria, the test substance, 5-aminotetrazole, is identified as substance potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1). Further testing with other test methods will be required.

- Study No. 413/17/5: 5-aminotetrazole - Acute Eye Irritation/Corrosion; VUOS-CETA Report No. 18 -187, 2018 (acc. OECD TG No. 405, EU Method B.5)

Result: irritating for eye

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24.10. – 02.11.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

Adopted: July 29th, 2016

Deviations:

yes

Remarks:

The medium during MTT direct reduction test changed colour from red to yellow by low pH of the test substance. This deviation had no impact on the outcome of study.

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

a reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek)

Source species:

human

Cell type:

other: normal human-derived epidermal keratinocytes

Cell source:

other: Keranocyte strain: 00267

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The reconstructed human epidermal model EpiDerm™ (EPI-200, MatTek, Bratislava, SR)
- Tissue batch number(s): Lot No. 25851, kit B

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37±1°C
- Temperature of post-treatment incubation (if applicable): 37±1°C

REMOVAL OF TEST MATERIAL AND CONTROLS
tissues were thoroughly rinsed and blotted to remove the test substance (controls)
Detailed procedure is described in internal SOP M/46/3.
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration:
tissues were transferred to 24-well plates containing MTT medium (1 mg·mL-1)
- Incubation time: 3 hr
- Spectrophotometer: Libra S22. Isopropyl alcohol serves as a blank.
- Wavelength: 570±30 nm

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
1. Direct MTT reduction - functional check in tubes: the test substance does not reduce MTT directly.
2. Colour interference: colour of the test substance did not interfere with evaluation
3. MTT test

DECISION CRITERIA
According to the OECD TG 431 as well as to the EU Method B.40, the test substance is considered to be corrosive to skin:
i) if the viability after 3 minutes exposure is less than 50 %, or
ii) if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15 %.
The test substance is considered to be non-corrosive to skin:
i) if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is greater than or equal to 15 %

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

test substance C2: 25 mg of the test substance was placed directly atop to the previously moistened tissue (50 μL of H2O) and it was spread to match size of the tissue.
NC: 50 μL H2O tested with every exposure time
PC: 50 μL 8N KOH tested with every exposure time

Duration of treatment / exposure:

3 and 60 minutes

Duration of post-treatment incubation (if applicable):

180 minutes (in MTT medium)

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

99.7

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct MTT reduction - functional check in tubes:
The test substance did not change colour from red to blue, so other steps were not employed.

- Colour interference
The test substance did not change colour, so other steps were not employed.

ACCEPTANCE OF RESULTS: All assay acceptance criteria have been met.
Negative control: The assay meets the acceptance criterion - OD570 of the NC tissues was 1.790 (3 min) and 1.747 (60 min) which is ≥ 0.8 and ≤ 2.8.
Positive control: Viability of tissues treated with 8N KOH after 60 minutes treatment was 4.6 % which is ≤ 15%.
Coefficient of variance: CV values in all triplets of tissues were ≤ 0.3

MTT test:

OD₅₇₀values obtained at the MTT test, their averages, standard deviations (%), coefficients of variance and relative viabilities

Treatment 3 min	OD570			mean	SD	CV	%NC
water (NC)	1.873	1.766	1.731	1.790	0.060	0.034	100.0
413/17 (C2)	1.790	1.772	1.809	1.790	0.015	0.008	100.0
8N KOH (PC)	0.156	0.126	0.120	0.134	0.016	0.118	7.5
Treatment 60 min	OD570			mean	SD	CV	%NC
water (NC)	1.898	1.647	1.697	1.747	0.108	0.062	100.0
413/17 (C2)	1.734	1.743	1.751	1.743	0.007	0.004	99.7
8N KOH (PC)	0.066	0.083	0.091	0.080	0.010	0.130	4.6

Interpretation of results:

GHS criteria not met

Conclusions:

Under the above-described experimental design, the test substance 5-aminotetrazole was non-corrosive in In vitro Skin Corrosion Test on EpiDermTM tissues.

Executive summary:

The test substance 5-aminotetrazole was assayed for the in vitro skin corrosion in human epidermal model EpiDermTM. The test was performed according to OECD Test Guideline 431, In Vitro Skin Corrosion: Human Skin Model Test, Adopted: 29th July 2016.

The test substance is white powder. There was no colour change after incubation with isopropanol and water.

Direct reduction test in test tubes was performed simultaneously. MTT medium changed colour from red to yellow. It was caused by low pH of the test substance. Hovewer the direct reduction of the MTT medium by the test substance was not observed.

In the MTT test, the test substance (25 mg) was placed atop the previously moistened tissue. Length of exposition was 3 and 60 minutes. Nine tissues were used for the experiment in each time, three per test substance (C2), three for the positive control (PC) and three for the negative control (NC).

After rinsing, tissues were incubated with MTT for three hours and then extracted with isopropyl alcohol overnight at refrigerator without shaking and than for two hours at room temperature with shaking. OD570 of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

Under the above-described experimental design, the average viability of tissues treated with the test substance 5-aminotetrazole was 100.0 % of the negative control average value after 3 minutes treatment and 99.7 % after 60 minutes treatment.

According to test guidelines, the test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is greater than or equal to 15 %.

In the experiment arrangement described above, the test substance 5-aminotetrazole was non-corrosive in the EpiDermTM model.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21.11.2017 - 24.11.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

Adopted: 28th July, 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

Commission Regulation (EC) No. 761/2009, 23rd July 2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MatTek Protocol for: INVITRO EpiDermTM SKIN IRRITATION TEST For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm, Model EPI-200-SIT,

Version / remarks:

Rev. 26/3/2012,1-37

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: tissue for research puposes from accredited institutions

Source strain:

other: Keratinocyte strain 00267

Vehicle:

physiological saline

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
Tissues: the reconstructed human epidermal model EpiDermTM (EPI-200 ver. 2.0, MatTek, Bratislava, Slovakia); Lot No. 25860 kit A

TEMPERATURE USED FOR TEST SYSTEM
culture conditions 37±1°C, 5±1 % CO2, moistened tissue

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: thoroughly rinsed with PBS

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg·mL-1
- Incubation time: 180±5 mins
- Spectrophotometer: Libra S22 at 570 nm. Isopropyl alcohol serves as a blank.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
Based on Certificate of Analysis the model passed all parametres for viability, barrier function, sterility.

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
The test substance was fully rinsed off the tissues. No changes in tissue appearance were observed during the experiment.
The optical density of the extraction solvent alone should be sufficiently small, i.e. OD< 0.1. OD570 of isopropyl alcohol against empty cuvette was measured before measuring of tissue extract. OD570 of isopropyl alcohol was – 0.031.
MTT test: a single testing, composed of three replicate tissues, was run.

PREDICTION MODEL / DECISION CRITERIA
OECD Test Guideline No. 439 (1), par. 36:
- In case the test chemical is found to be non-corrosive (e.g., based on TG 430, 431 or 435), and shows tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%, the test chemical is considered to be irritant to skin in accordance with UN GHS (3) Category 2.
- The test chemical may be considered as non-irritant to skin in accordance with UN GHS No Category if the tissue viability after exposure and post-treatment incubation is more than (>) 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg of the test substance was placed directly on tissue moistened with 25 µL of PBS and spread on the tissue surface.
NC: PBS (phosphate buffered saline), prepared in laboratory 29/09/17 exp. 29/03/18 (washing of tissues) and MatTek DPBS lot. No. 022217ZSB exp. 22/02/2018 (wetting of tissues, negative control)
PC: 5 % SDS (sodium dodecyl sulphate), MatTek, Lot No. 031617MGKA, exp. 16/03/2018

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

100.7

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

All study acceptance criteria were fulfilled.
The mean OD570 of the NC tissue was 1.893 which meets the acceptance criteria of ≥ 0.8 and ≤ 2.8.
The mean viability of the PC tissues expressed as % of the negative control tissues was 2.4 % which meets the acceptance criterion of ≤ 20 %.
The SD calculated from individual % tissue viabilities of the 3 identically treated replicates was 0.2 % for the positive control, 3.0 % for negative control and 8.1 % for the test substance what is < 18 % in all cases.

MTT test

OD₅₇₀values obtained at the MTT test, their averages, standard deviations (%) and relative viabilities

Treatment	OD570			Avg	SD	%NC
PBS (NC)	1.968	1.834	1.876	1.893	0.056
viability (%NC)	104.0	96.6	99.1	100.0	3.0	100.0
413/17 (C1)	1.772	1.824	2.122	1.906	0.154
viability (%NC)	93.6	96.4	112.1	100.7	8.1	100.7
5% SDS (PC)	0.049	0.041	0.047	0.046	0.003
viability (%NC)	2.589	2.166	2.483	2.4	0.2	2.4

PBS - phosphate buffered saline

SDS - sodium dodecyl sulphate

Interpretation of results:

GHS criteria not met

Conclusions:

Under the above-described experimental design, the average viability of tissues treated by the test substance, 5-aminotetrazole, was 100.7 % of negative control average value i.e. viability was > 50 %. The effect of the test substance was negative in EpiDermTM model.
According to the classification criteria given in this report, the test substance is considered to have no category in accordance with UN GHS and is therefore considered a non-irritant.

Executive summary:

The test substance, 5-aminotetrazole, was assayed for in vitro skin irritation in the human epidermal model EpiDermTM. The test was performed according to the OECD Test Guideline No. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (2015) and Protocol for: In Vitro EpiDerm^TMSkin Irritation Test Test for use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT.

After pre-incubation of tissues, 25 mg of the test substance was placed directly on tissue and spread on the entire tissue surface. The length of exposure was 60 minutes. Three tissues were used for the test substance and for positive and negative controls.

After removal of the test substance, tissues were post-incubated for approximately 42 hours. Three hours incubation with MTT and two hours extraction period with shaking followed. Optical density (OD₅₇₀) of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

No complementary experiments for correcting of colour interference and direct reduction were done, because they were performed as a part of the Study No. 413/17/4AC: 5 aminotetrazole - In vitro Skin Corrosion Test (EpiDermTM Model); VUOS CETA Report No.: 17-693. These experiments confirmed no direct reduction by the test substance and no color interference with the endpoint.

Under the above-described experimental design, average viability of treated tissues was 100.7 %, i.e. viability was > 50 %.

The effect of the test substance was negative in EpiDerm^TM model (tissues were not damaged).

According to the classification criteria given in report, the test substance, 5-aminotetrazole, is considered to have no category with regard to skin irritation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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

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)

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.

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

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.

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

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.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12.12.2017 - 14.12.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

Adopted: 28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Protocol: EpiOcularTM EIT for the prediction of acute ocular irritation of chemicals

Version / remarks:

Version 9, June 29, 2015, MatTek corp.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

other: keratinocyte strain 4F1188

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability
Cell damage (cytotoxicity), playing an important, if not the primary, mechanistic role in determining the overall serious eye damage/eye irritation response of a chemical regardless of the physicochemical processes underlying tissue damage, is followed in this test.

This test uses an in vitro procedure allowing the identification of chemicals not requiring classification and labelling for eye irritation or serious eye damage in accordance with UN GHS. This test is not able to distiguih between serious eye damage and eye irritation.


- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live

The RhCE tissues are reconstructed from primary human cells, which have been cultured for several days to form a stratified, highly differentiated squamous epithelium, morphologically similar to that found in the human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.

The reconstructed human cornea-like epithelial model EpiOcular™ comes from MatTek, Bratislava, SK, supplied with Certificate of Analysis. Lot No. of tissues used for this test: 27017 kit B.

On the day of receipt, EpiOcularTM tissues were conditioned to release transport stress related compounds and debris by incubation in assay medium delivered by MatTek for test performance for 62 minutes at standard culture conditions and, after media replacement, overnight (following 18 hours 28 minutes) also standard at culture conditions.

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test substance (50 mg of substance/surface ratio 39.7 uL/cm2) is placed directly atop to the tissue moistened with 20 µL of PBS. The test substance was spread over entire tissue surface.
A single testing, composed of two replicate tissues, was run.

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

25±2 mins immersion incubation (post-soak)
18 hours at standard culture conditions (post-treatment incubation)

Number of animals or in vitro replicates:

2

Details on study design:

- Details of the test procedure used
Direct MTT reduction - functional check in tubes => The test was performed as a part of another study: Study No. 413/17/4AC: 5-aminotetrazole - In vitro Skin Corrosion (EpiDermTM Model), VUOS-CETA Report No. 17-693, 2017. No direct-reducing properties were observed.
Colour interference
The test was performed as a part of another study: Study No. 413/17/4AC: 5-aminotetrazole - In vitro Skin Corrosion (EpiDermTM Model), VUOS-CETA Report No. 17-693, 2017. No change of colour was observed.
MTT test
A single testing, composed of 2 replicate tissues, is run (plus 3 for the positive control (PC) and 3 for negative control (NC)).

- RhCE tissue construct used, including batch number
The reconstructed human cornea-like epithelial model EpiOcular™ comes from MatTek, Bratislava, SK.
Lot No. of tissues used for this test: 27017 kit B

- Doses of test chemical and control substances used
The test substance (50 mg of substance/surface ratio 39.7 mg/cm2) is placed directly atop to the tissue moistened with 20 µL of PBS.
PC: Methyl Acetate 99%, MatTek, Lot No. 032817ISA
NC: water for injection Ardeapharma, Lot. No. 1608120439 exp.08/2018

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)
60 mins at standard culture conditions (37±1°C, 5±1 % CO2, humidified incubator). Tissues were wetted with 20 μl of PBS spread across entire tissue surface. After 30 minutes incubation tissues were topically exposed to the test chemical (50 mg per tissue) for 30 minutes
25 ± 2 minutes immersion incubation (post-soak) at room temperature
18 hours at standard culture conditions (37±1°C, 5±1 % CO2, humidified incubator)

- Description of any modifications to the test procedure:
Assay acceptance criterion was not fulfilled for absorbancies of extracts from positive control tissues. As all the tissues had viabilities under 50% of negative control viability we consider that this deviation has not impact on study results.

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer)
OD570 is measured on a plate reader Biotek Epoch. Isopropyl alcohol serves as a blank. No external filter is used.

- Description of the method used to quantify MTT formazan
Optical density (OD570) of isopropyl alcohol extracts was measured on a plate reader. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.
Then the mean relative tissue viability of two individual tissues exposed to the test substance is calculated – this value is, after correction, used for the comparison with limit value.
Tests for colour interference and direct reduction did not demonstrate influence of colour or reductive properties of the test item on study results. Thus, no steps for correction of results were performed.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model
The percentage tissue viability cut-off value distinguishing classified from non-classified test chemicals is 60%. Results should thus be interpreted as follows:
- The test chemical is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%. In this case no further testing in other test methods is required.
- The test chemical is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post exposure incubation is less than or equal (≤) to 60%.

- Positive and negative control means and acceptance ranges based on historical data
1) The negative control OD > 0.8 and < 2.5,
2) The mean relative viability of the positive control is below 50% of control viability
3) The difference of viability between the two relating tissues of a single chemical is < 20% in the same run. This applies also to the killed controls and the colorant controls which are calculated as percent values related to the viability of the relating negative control.

Irritation parameter:

other: average viablility

Run / experiment:

1

Value:

2.3

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: i.e. viability was ≤ 60 %

Other effects / acceptance of results:

The average negative control (neat extract) OD570 was 1.638 what is > 0.8 and < 2.5. This criterion was fulfilled.
The mean relative viability of the positive control was 20.0 % what is below 50% of negative control viability. This criterion was fulfilled.
The difference of viability between the three relating tissues of the negative control was 11.7 %. The difference of viability between the two relating tissues of the test substance was 12.4 % what is < 20%. These criteria were fulfilled. The difference of viability between the three positive control tissues was 21.0 % what is > 18%. This criterion was not fulfilled (for comment see Any other information ...above).


No problems occurred at treatment but part of the test substance remained not wetted after the treatment period. Bottom layer directly adjoin to tissue was wetted.

Table 1: OD₅₇₀values obtained at the MTT test, their averages, standard deviations (%) and relative viabilities

The data presented are corrected by subtraction of OD570 isopropyl alcohol itself (blank, 0.040).

Code	Treatment	OD570			mean	SD	Viability %
		Tissue 1	Tissue 2	Tissue 3		%SD
NC	water	1.488	1.516	1.909	1.638	0.175	100.0
	% NC	90.9	92.5	116.6	100.0	11.7
C3	413/17	0.034	0.043	NT	0.038	0.005	2.3
	% NC	2.0	2.6	NT	2.3	12.4
PC	99% MA	0.321	0.247	0.414	0.327	0.069	20.0
	% NC	19.6	15.1	25.3	20.0	21.0

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the above-described experimental design average viability of treated tissues by the test substance 5-aminotetrazole was 2.3 % of negative control average value i.e. viability was ≤ 60 %. The effect of the test substance was positive in EpiOcularTM model (tissues were damaged).
According to the classification criteria, the test substance, 5-aminotetrazole, is identified as substance potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1). Further testing with other test methods will be required.

Executive summary:

The test substance, 5 -aminotetrazole, was assayed for the in vitro eye irritation in human cornea-like model EpiOcular^TM. The test was performed according to the OECD Test Guideline No. 492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage. Details of the procedure are given in Protocol: EpiOcular^TMEye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals (MatTek 06/29/2015).

After pre-incubation and wetting of tissues, 50 mg of the test substance was placed directly atop to the tissue and it was spread on the entire tissue surface. Length of exposition was 6 hours at 37±1°C in humidified CO2 incubator (5±1% CO2). Two tissues were used for the test substance and three for every control.

After removal of the test substance, tissues were post-soaked in medium for approximately 25 minutes and post-incubated for about 18 hours at culture conditions. Three hours incubation with MTT and 2-3 hours extraction period with shaking followed then. Optical density (OD₅₇₀) of isopropyl alcohol extracts was measured on a plate reader. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

Tests for colour interference and direct reduction were performed as a part of another study (Study No. 413/17/4AC: 5-aminotetrazole - In vitro Skin Corrosion (EpiDermTM Model), VUOS-CETA Report No. 17-693, 2017). Neither direct reducting properties nor colour interference with endpoint were found.

Under the above-described experimental designaverage viability of treated tissues was 2.3% i.e. viability was ≤60 %.

The effect of the test substancewaspositiveinEpiOcular^TMmodel (tissues were damaged).

According to the classification criteria, the test substance, 5-aminotetrazole, is identified as substance potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1). Further testing with other test methods will be required.

Reference 3

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02.04. – 30.04.2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Version / remarks:

Commission Regulation (EU) 2017/735,
Published in O.J.L. 112, 2017

Deviations:

yes

Remarks:

See Any other information (a temperature deviation without impact on study)

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

Adopted October 2, 2012

Deviations:

yes

Remarks:

See Any other information (a temperature deviation without impact on study)

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: breeding farm VELAZ s.r.o., Lysolaje, Czech Republic, RČH CZ 21760118
- Weight at study initiation: 2.90 – 3.20 kg
- Housing: conventional animal room – individually in metallic cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days, no signs of disease were observed

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3°C, permanently monitored
- Humidity (%): 30 – 70%, permanently monitored
- Photoperiod (hrs dark / hrs light): 12 h/12 h

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

A dose of 0.1g of the test substance was applied to the test site.

Duration of treatment / exposure:

The test substance was placed into the conjunctival sac of one eye of animals after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second in order to prevent loss of the substance.
The other eye, which was untreated, serves as a control.
The substance was solid and has not been removed from the eye of the test animal by physiological mechanisms at the first observation time point of 1 hour after treatment. The eye was rinsed with physiological saline solution.

Observation period (in vivo):

examined at 1, 24, 48 and 72 hours after application

Number of animals or in vitro replicates:

2 animals

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): rinsed with physiological saline solution
- Time after start of exposure: 1h

SCORING SYSTEM:
To the ocular reactions observed at each time interval the grades were assigned according to the grading system given in Method B.5 – Acute Eye Irritation/Corrosion, Commission Regulation (EU) 2017/735, Published in O.J.L. 112, 2017

TOOL USED TO ASSESS SCORE: After recording the observations at 72 hours, the eyes of rabbit were examined with the hand slit-lamp.

Irritation parameter:

cornea opacity score

Basis:

animal: 13

Time point:

24 h

Score:

1

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal: 13

Time point:

48 h

Score:

2

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal: 13

Time point:

72 h

Score:

2

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal: 13

Time point:

other: 4th day - 5th day

Score:

2

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal: 13

Time point:

other: 6th day -14thday

Score:

1

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal: 13

Time point:

other: 15th day - 16th day

Score:

0

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal: 14

Time point:

48 h

Score:

3

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal: 14

Time point:

24 h

Score:

3

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal: 14

Time point:

other: 4th day - 6th day

Score:

2

Max. score:

4

Irritation parameter:

iris score

Basis:

animal: 13

Time point:

24 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: next 24 h

Irritation parameter:

conjunctivae score

Basis:

animal: 13

Time point:

24/48/72 h

Score:

1

Max. score:

3

Irritation parameter:

chemosis score

Basis:

animal: 13

Time point:

24/48/72 h

Score:

1

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal: 14

Time point:

72 h

Score:

2

Max. score:

4

Irritation parameter:

iris score

Basis:

animal: 14

Time point:

24 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: next 24 h

Irritation parameter:

conjunctivae score

Basis:

animal: 14

Time point:

24/48 h

Score:

1

Max. score:

3

Irritation parameter:

chemosis score

Basis:

animal: 14

Time point:

24/48 h

Score:

1

Max. score:

4

Irritation parameter:

cornea opacity score

Basis:

animal: 14

Time point:

other: 7th day - 21th day

Score:

1

Max. score:

4

No alterations of control eyes were observed during the whole study.

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

see Executive summary

Executive summary:

The test item, 5-aminotetrazole, was tested for the assessment of eye irritation/corrosion effects using albino rabbit (New Zealand Albino breed).

The test was performed according the following documents:

- Method B.5 – Acute Eye Irritation/Corrosion, Commission Regulation (EU) 2017/735, Published in O.J.L. 112, 2017

- The OECD Test Guideline No. 405 Acute Eye Irritation/Corrosion. Adopted October 9, 2017

Before in vivo testing, the sequential testing strategy as it is recommended in supplement to TG 405 (2012) was respected. The results did not allow the classification of test item with regard to its potential to cause eye irritation or serious eye damage.

Therefore in vivo test was started. The test was performed initially using one animal (No. 13). Because no corrosive or severe irritating effects were observed in the initial test, the response should be confirmed in additional animals. Only one additional animal (No. 14) was used, because an irritant effect was observed in the initial test.

The following changes were observed on eye during the 72 hours observation period after administration of the test item:

cornea - diffuse areas of opacity or easily discernible translucent area with details of iris slightly obscured, conjuctivae - some blood vessels hyperaemic, chemosis – swelling above normal, iris – markedly deepened rugae, were observed in rabbit No. 13.

Cornea - easily discernible translucent area with details of iris slightly obscured or nacrous area, no details of iris visible, conjuctivae – some blood vessels hyperaemic, chemosis – swelling above normal, iris - markedly deepened rugae, were observed during the 72 hours observation period after administration of the test item in rabbit No.14.

Easily discernible translucent area, details of iris slightly obscured in the cornea were not fully reversible during the first 72 hours in rabbit No. 13 and No. 14. Diffuse areas of opacity were observed till the 14th day after administration of the test item in rabbit No. 13. The lesions of the cornea have not disappeared during the whole observation period in rabbit No.14 (21th days).

No clinical signs of systemic intoxication were detected.

Evaluation of results after single application demonstrated, that the test item, 5-aminotetrazole, is irritating for the eye of the rabbit. The lesions of the cornea were irreversible.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on in vitro/ex vivo and in vivo tests the test substance 5-aminotetrazole was non-irritating for skin and irritating for eye.