2H-Tetrazol-5-amine, potassium salt (1:1)

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis.

Details on animal used as source of test system:

Epi-200 tissues and MTT-100 assays diluent were procured from MatTek Corporation in Ashland, USA. Day of delivery: 17. Dec. 2008 batch: 121108TTA, 120408WDA, 11219

Vehicle:

water

Details on test system:

Commercially available Epi-200-Kit.
The EpiDermTM tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell cultures inserts.Four 6-well-plates were prepared with 0.9 mL assay medium in each well. The inserts containing the tissues were transferred to the wells using sterile forceps and the 6-well-plates were set into the incubator at 37°C and 5% CO2 for one hour (pre-incubation). For each experiment (“three minutes” and “one hour”), one 24-well-plate was prepared as holding plate. 12 wells of each plate were filled with 300 μL assay medium, the other 12 with 300 μL MTT medium. One additional plate was left empty. The plates were stored in the incubator. For each experiment (“three minutes” and “one hour”), two 6-well-plates were used. After pre-incubation, the assay medium was replaced by fresh assay medium and the test was started, using two wells as negative control with 50 μL H2O demin., two wells as positive controls with 50 μL potassium hydroxide solution and two other wells for testing the test item. Solid test items were ground, and 25 mg test item were applied together with 25 μL H2O. At the begin of each experiment (application of negative controls), a stop watch was started. After the respective incubation time, the inserts were removed from the plates using sterile forceps. The inserts were thoroughly rinsed with PBS, blotted with sterile cellulose tissue and set into the respective holding plate, using the wells containing assay medium.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg test item were applied together with 25 μL Water.

Duration of treatment / exposure:

3 minutes and 1 hour

Number of replicates:

2

Vehicle:

water

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes

Value:

ca. 87.8

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour

Value:

ca. 76.8

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

After treatment with the test item, the relative absorbance values were reduced to 87.8% after three minutes treatment. This value is above the threshold for corrosivity (50%). After one hour treatment, the relative absorbance values were reduced to 76.8%. Lying, above the threshold for corrosivity (15%). Therefore the test item is considered as not corrosive. The negative control was within the normal range (criterion OD > 0.8), showing an OD of
2.134 (3 min.) resp. 2.050 (1 hour). The positive control showed a clear corrosive effect, with value of the three-minuteexperiment being 23.2% and value of the one-hour-experiment being 11.3%.

Executive summary:

One valid experiment was performed. Two tissues of the human skin model EpiDermTM were treated with the test item for three minutes and one hour, respectively. 25 mg of the solid test item were applied to each tissue and spread to match the tissue size. Deionised water was used as negative control, 8m KOH was used as positive control. After treatment with the negative control the absorbance values were well above the required acceptability criterion of mean OD > 0.8 for both treatment intervals thus showing the quality of the tissues. After treatment with the test item, the relative absorbance values were decreased to 87.8 % after three minutes treatment. This value is well above the threshold for corrosion

potential (50%). After one hour treatment, relative absorbance values were reduced to 76.8 %. This value, too, is well above the threshold for corrosion potential (15%). Therefore, 5-Aminotetrazol Potassium is considered as “not corrosive in the Human Skin Model Test”.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

yes

Remarks:

The eggs were 10 days in the heating chamber, because the ninth day was a Sunday.

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Details on test animals or tissues and environmental conditions:

Freshly laid Lohmann Leghorn chicken eggs were obtained from the LSL Rhein Main Geflügelvermehrungsbetrieb (Dieburg, Germany) ten days before the start of the test. Then the eggs were incubated at 37.0 ± 0.5 °C for ten days (arrived on 23. Jan. 2009). During incubation, the eggs were gently rotated to prevent an attachment of the embryo to one side of the egg.

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.2986 g of the test item was given on the membrane in such a manner that 50% of the surface of the membrane were covered with test item.

Number of animals or in vitro replicates:

6 for test item, 3 for controls

Irritation parameter:

in vitro irritation score

Value:

ca. 11.84

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Interpretation of results:

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

Conclusions:

This in vitro study was performed to assess the irritating potential of 5-Aminotetrazol Potassium by detection of damages in blood vessels under the chorionallantoic membrane of incubated chicken eggs (ten days old). Observation time was 5 minutes at room temperature. Physiological sodium chloride solution was used as negative control, sodium dodecyl sulfate (1% solution ) and sodium hydroxide (0.1-n solution) were used as positive controls. The positive controls induced a severe irritation on the blood vessels. A mean irritation score of 19.69 was calculated for the positive control NaOH. This value is considered as valid, because the positive control showed a clear severe irritation. The upper threshold of 19 is not mandatory in the guideline. The negative control showed no irritation, the irritation score for the positive control SDS lay within the demanded range. The test item was tested pure. A mean irritation score of 11.84 was calculated, corresponding to a classification as “severely irritant”. No observations were made which might cause doubts concerning the validity of the study outcome. The test is considered valid.

Executive summary:

One valid experiment was performed. Hen’s eggs with an age of ten days were used. The test item 5-Aminotetrazol Potassium was brought onto the surface of the CAM of a hen’s egg which had been incubated at 37 oC for ten days. Observation time was 5 minutes at room temperature. Physiological sodium chloride solution was used as negative control. The negative control showed no irritating effect on the blood vessels under the membrane. Solutions of Sodium Dodecyl Sulfate (1%) and NaOH (0.1 n) were used as positive controls. The positive controls induced a severe irritation on the blood vessels. The test item 5-Aminotetrazol Potassium showed severe effects on the blood vessels of the CAM. The calculated mean irritation score is 11.84. In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item 5-Aminotetrazol Potassium possesses severe irritation potential.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Eye Irritation/corrosion:

The available study HED-CAM Study is a test methods currently with limited application under REACH (ECHA Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7a: Endpoint specific guidance, Version 5.0 ,December 2016). Although the test shows positive results and it can be stated that in this study and under the experimental conditions reported, the test item 5-Aminotetrazol Potassium possesses severe irritation potential. Following the ECHA Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7a: Endpoint specific guidance, Version 5.0 ,December 2016 which states that ”The Manual of Decisions of the Competent Authorities (EC, 2009) concluded that there is enough evidence available to conclude that the test methods are able to detect substances causing severe damage to eyes. Positive results can therefore be used for classification purposes i.e. leading to a classification of Category 1 for serious eye damage and labelling with H318 “Causes serious eye damage” according to CLP” it is decided to perform a worst-case classification as Category 1 serious eye damage.

 

Skin irritation/corrosion:

The available skin corrosion study considered that 5-Aminotetrazol Potassium is “not corrosive in the Human Skin Model Test”. But due to the experimental results in the eye irritation study and the classification as Category 1 serious eye damage, it is decided to perform a worst-case classification as Category 2 skin irritation.