2H-Tetrazol-5-amine, N-2H-tetrazol-5-yl-, ammonium salt (1:1)

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to an internationally accepted guideline. All study parameters are based on the specific guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

485-280-6

EC Name:

-

Cas Number:

303749-96-4

Molecular formula:

Hill formula: C2H6N10 CAS formula: C2H3N9.H3N

IUPAC Name:

N-(1H-1,2,3,4-tetrazol-5-yl)-1H-1,2,3,4-tetrazol-5-amine amine

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Test animals

Species:

rat

Strain:

CD-1

Sex:

male/female

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

The vehicle and test article were administered once per day for 28 consecutive days during the study via oral gavage. The dose levels for the treated groups were 100, 300, and 1000/600 mg/kg/day at a dose volume of 10 mL/kg, with the exception of Week 2 when the 1000/600 mg/kg/day group was administered the test article at a dose volume of 6 mL/kg. Prior to the animals being dosed, the test article was allowed to reach room temperature for at least 30 minutes. The formulations were stirred continuously using a magnetic stir bar and plate from acquisition throughout dosing. Animals were dosed using a 3 or 5 cc Becton Dickinson syringe and a gavage tip. A new syringe was used for each group and discarded as necessary. The gavage tip was wiped prior to dosing each animal. Individual doses were based on the most recent body weights.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300, 1000/600 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

Cageside Observations
All animals were observed for morbidity, mortality, injury, and the availability of food and water twice daily throughout the duration of the study.
Detailed Clinical and Neurobehavioral Examinations
A detailed clinical examination of each animal was performed daily except on days when detailed neurobehavioral examinations were conducted. On one occasion, a clinical observation was recorded at an unscheduled interval. The observations included, but were not limited to, evaluation of the skin, fur, eyes, ears, nose, oral cavity, thorax, abdomen, external genitalia, limbs and feet, respiratory and circulatory effects, autonomic effects such as salivation, and nervous system effects including tremors, convulsions, reactivity to handling, and atypical behavior.
Detailed neurobehavioral examinations of each animal were performed once prior to initiation of test article administration and weekly during the study (Days 4, 11, 18, and 25). Observations were made outside the home cage in a standard arena using an applicable scoring system at approximately the same time of day each week. Observations were carefully recorded, and an effort was made to ensure that variations in the test conditions were minimal. The observations included, but were not limited to, changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture, and reactivity to handling, as well as the presence of clonic or tonic movements, stereotypy (e.g., excessive grooming, repetitive circling), difficult or prolonged parturition, or bizarre behavior (e.g., self-mutilation, walking backwards) was also recorded.
Functional Observation Battery (FOB) Evaluations
FOB evaluations were conducted without knowledge on the part of the testers of the treatment groups. FOB evaluations, including those conducted in the home-cage, during handling, in the open field, and others, were conducted on all animals on Day 24 during the last week of test article administration. During open-field evaluations, each animal was observed for a minimum of 3 minutes in a black plexiglass, observation box measuring 20 x 20 x 8 in. The parameters evaluated in the FOB were based on those outlined in Moser, et al. The observations included, but were not limited to, evaluation of activity and arousal, posture, rearing, bizarre behavior, clonic and tonic movements, gait, mobility, stereotypy, righting reflex, response to stimulus (approach, click, tail pinch, and touch), palpebral closure, pupil response, piloerection, exophthalmus, lacrimation, salivation, and respiration. The amount, qualitative and/or quantitative measures, of defecation and urination were also recorded. Forelimb and hindlimb grip strength was measured using the procedure described by Meyer, et al. , and hindlimb splay was quantitatively measured as described by Edwards and Parker . Pain perception was assessed by measuring the latency of response to a nociceptive (thermal) stimulus when each animal was placed on a hot plate apparatus set to
n
52°C as described by Ankier . Body weight and temperature were also measured. A glossary of FOB terms used in this study is included in this report.
Monitoring of Locomotor Activity
Locomotor activity monitoring was conducted on Day 25 during the last week of test article administration on all surviving animals. Testing was conducted using Hamilton-Kinder motor monitoring system. The duration of monitoring was 40 minutes with the data summarized into 10 minute segments. A range of different activities were recorded but only the following were used in comparisons between treated and control animals as the most representative activity parameters: basic movement, fine movement, rearing, and distance.
Body Weights
Body weights for all animals were measured and recorded at receipt, prior to randomization, and weekly during the study. The body weights recorded at receipt and prior to randomization are not reported, but are maintained in the study file.
Food Consumption
Food consumption was measured and recorded weekly during the study.
Clinical Pathology
Clinical pathology evaluations were conducted on all animals at termination. The animals had access to drinking water but were fasted overnight prior to sample collection. Blood samples (approximately 4.8 mL) were collected via the vena cava after carbon dioxide inhalation. Samples were collected into tubes containing K3EDTA for evaluation of hematology parameters and citrate for evaluation of coagulation parameters. No anticoagulant was used for the clinical chemistry samples. The order of bleeding was by alternating one animal from each dose group, then repeating to reduce handling and time biases.
The animals were housed in stainless steel metabolism cages and urine was collected for at least 16 hours.

Sacrifice and pathology:

Macroscopic
Necropsy examinations were performed under procedures approved by a veterinary pathologist on animals found dead and all surviving animals at the scheduled necropsy. Euthanasia was by carbon dioxide inhalation followed by exsanguination via the abdominal vena cava. The animals were examined carefully for external abnormalities including masses. The skin was reflected from a ventral midline incision and any abnormalities were identified and correlated with antemortem findings. The abdominal, thoracic, and cranial cavities were examined for abnormalities and the organs removed, examined, and, where required, placed in fixative. The pituitary was fixed in situ. All designated tissues were fixed in neutral buffered formalin, except for the testes, which were fixed using a modified Davidson's fixative . Formalin was infused into the lung via the trachea and into the urinary bladder. A full complement of tissues and organs was collected from all animals.
Organ Weights
Body weights and protocol-designated organ weights were recorded for all surviving animals at the scheduled necropsy and appropriate organ weight ratios were calculated (relative to body and brain weights). Paired organs were weighed together. The thyroid/parathyroid gland and pituitary gland were weighed following fixation.
Microscopic
Microscopic examination of fixed hematoxylin and eosin-stained paraffin sections was performed on protocol-designated sections of tissues for animals at 0 and 1000/600 mg/kg/day. The slides were examined by a board-certified veterinary pathologist. A four-step grading system was utilized to define gradable lesions for comparison between dose groups.
A glossary of macroscopic and microscopic terms used in this study is included in this report. A full list of organs and tissues collected, weighed, and examined for this study is presented in Appendix P.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight was lower (7%) among males at 1000 mg/kg/day during the first week of treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was lower than control values during Week 1 among males at 300 mg/kg/day (9%) and in both sexes at 1000 mg/kg/day (males, 25%; females, 49%). This decrease was transient in nature as values were comparable to control values by Week 2.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Effect levels

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, where 1 H-Tetrazol-5- amine-, N-IH-tetrazol-5-yl-, monoammonium salt as administered orally once daily for
28 consecutive days at 0, 100, 300, or 1000/600 mg/kg/day, the no-observed-adverse-effect- level (NOAEL) was 300 mg/kg/day.

Executive summary:

This study was conducted for to evaluate the oral toxicity of the test article after administration for 28 days. Three treatment groups of ten male and ten female CD® [Crl:CD®(SD)] rats were administered the test article at respective dose levels of 100, 300, and 1000 mg/kg/day. During Week 2 (Day 9), the 1000 mg/kg/day dose was reduced to 600 mg/kg/day due to death of two females at 1000 mg/kg/day. One additional group of ten animals/sex served as the control and received the vehicle, distilled water. The test article or vehicle was administered to all groups via oral gavage once a day for 28 consecutive days. The dose volume was 10 mL/kg with the exception of Week 2 when the 1000/600 mg/kg/day group was administered the test article at a dose volume of 6 mL/kg to lower the dose level for that week.

Observations for morbidity, mortality, injury, and the availability of food and water were conducted twice daily for all animals. Clinical observations were conducted daily except for on the day of the weekly neurobehavioral observation for each animal (Days 4, 11, 18, and 25). Functional Observational Battery (FOB) evaluations were conducted on Day 24 and locomotor activity was conducted on Day 25. Body weights and food consumption were measured and recorded weekly. Blood and urine samples for clinical pathology evaluations were collected from all surviving animals at study termination. At study termination, necropsy examinations were performed and organ weights were recorded. Selected tissues were microscopically examined for animals at 0 and 1000/600 mg/kg/day.

Two females at 1000 mg/kg/day died during the study, one each on Days 5 and 6. Acute lymphoid necrosis of the thymic cortex and lymphoid depletion in the spleen was present in both rats and was most likely stress-associated and not test article related. The cause of death for either animal could not be determined microscopically. Most females at 1000 mg/kg/day lost weight during the first week of treatment. Food consumption was considerably lower as well. As a result, the 1000 mg/kg/day dose was lowered to 600 mg/kg/day during Week 2. No further deaths occurred following the dose reduction.

No test article-related clinical observations were noted at 100 or 300 mg/kg/day. At 1000/600 mg/kg/day, clinical observations relating to gastrointestinal distress were noted in both sexes. These signs began on Day 2 and continued periodically through the treatment period, even after lowering the dose. The signs included: soft/watery feces with associated brown discharge from the anus and brown stained anogenital areas.

There were no clear test article-related findings in the weekly neurobehavioral observations or in the FOB examinations. In the locomotor activity parameters, total distance, basic and fine movement was statistically significantly lower at 1000/600 mg/kg/day during the 20-30 and 30-40 minute intervals. Fine movement was also lower over the 0 to 40 minute interval as well.

Mean body weight at 1000 mg/kg/day in males was lower than controls, and females at this level generally lost weight during Week 1. However, body weight gain in both sexes was comparable to or exceeded control values beginning in Week 2 once the dose was reduced to 600 mg/kg/day. A corresponding transient reduction in food consumption was noted as well in both sexes at 1000 mg/kg/day. A slight reduction in food consumption was also noted in males at 300 mg/kg/day during Week 1.

There were no adverse, test article-related effects on hematology or clinical chemistry parameters up to and including 300 mg/kg/day. There were no test article-related effects on coagulation parameters. At 1000/600 mg/kg/day, erythrocytes, hemoglobin and hematocrit were decreased 7, 11, and 11%, respectively, in males. Hemoglobin and hematocrit were decreased 5 and 6% in the females. At 1000/600 mg/kg/day, phosphorus was increased 1.12- fold in males and females, and ALT was increased 1.98 and 1.90-fold in males and females, respectively. Potassium was increased 1.18 and 1.12-fold in males and females, respectively, but did not reach statistical significance in the females. This increase was compatible with a mild potential acidosis. Urine pH decreased in a dose-dependent response in males and females at 300 and 1000/600 mg/kg/day however this effect was not considered adverse.

There were no test article-related macroscopic or microscopic findings. Mean kidney weights (absolute and relative to body and brain weights) were statistically significantly increased in males and females at 300 and 1000/600 mg/kg/day when compared to controls. Mean absolute kidney weights were increased 14% in both male groups and 17% and 23% in females at 300 and 1000/600 mg/kg/day, respectively. Increased kidney weights are likely test article related; however, there were no correlating microscopic findings. Mean spleen weights (absolute and relative to body and brain weights) were statistically significantly decreased in females at 300 mg/kg/day and in males and females at 1000/600 mg/kg/day when compared to controls. Mean absolute spleen weights were decreased 15% in females at 300 mg/kg/day and 22% and 20% in males and females at 1000/600 mg/kg/day, respectively. Decreased spleen weights are likely test article related; however, there were no correlating microscopic findings.

In summary, under the conditions of this study, where 1 H-Tetrazol-5- amine-,N-IH-tetrazol-5-yl-,monoammonium salt administered orally once daily for 28 consecutive days at 0, 100, 300, or 1000/600 mg/kg/day, the no-observed- adverse-effect-level (NOAEL) was 300 mg/kg/day.