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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-10-07 - 2007-11-28

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:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The vehicle and test article were administered once daily from GD 6 through 19 during the study via oral gavage. The dose levels for the treated groups were 50, 200, and 800 mg/kg/day at a dose volume of 10 mL/kg. The control animals received the vehicle, distilled water, at the same frequency and dose volume as the treated animals. Due to notable body weight loss between GD 6 to 9 at 800 mg/kg/day, the dose level was reduced to 600 mg/kg/day beginning on GD 9. Due to the stagger-started study design and for consistency of the data, the first subset of animals (animal numbers 289 through 300) were dosed twice on GD 8. The animals were first dosed at 600 mg/kg/day using the prepared formulation at 800 mg/kg/day at a reduced dose volume of 7.5 mL/kg. The animals received an additional dose at 2.5 mL/kg with the same formulation to achieve a total daily dose of 800 mg/kg/day. This allowed both subsets of animals to begin the lowered dose level of 600 mg/kg/day on the same day, GD 9.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

gestation day 6 to 19

Frequency of treatment:

once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
50, 200, and 800(600) mg/kg
Basis:

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Examinations

Maternal examinations:

Cageside Observations, Detailed Clinical Observations, Body Weights and Body Weight Changes, Food Consumption

Ovaries and uterine content:

On GD 20, each surviving female was euthanized by carbon dioxide inhalation, followed by exsanguination via the abdominal vena cava and immediately subjected to a laparohysterectomy.
The skin was reflected from a ventral midline incision to examine mammary tissue and locate any subcutaneous masses. The abdominal cavity was then opened and the uterus was exposed. The uterus was excised, and the gravid uterine weight was recorded. Beginning at the distal end of the left uterine horn, the location of viable and nonviable fetuses, early and late resorptions for each uterine horn, position of the cervix, and the total number of implantations were recorded. The number of corpora lutea on each ovary was also recorded.
Each implant was categorized according to the following criteria. Viable fetuses responded to touch. Nonviable fetuses did not respond to touch and had no signs of autolysis. Late resorptions were characterized by recognizable fetal form, but undergoing autolysis. Early resorptions were characterized as implantation sites that had no recognizable fetal characteristics. The fetuses were removed by making a dorsal incision longitudinally along both uterine horns. The embryonic membrane of each fetus was gently removed, and each fetus was pulled away from the placenta, fully extending the umbilical cord. The placentae were examined grossly.
Uteri from females that appeared nongravid were opened and placed in 10% ammonium sulfide solution for detection of implantation sites . The foci, if detected, were considered early resorptions, and data from these females were included in mean calculations. If no foci were detected, the female was considered to be nonpregnant.

Fetal examinations:

Each fetus was individually weighed, sexed, tagged, and examined for external malformations and variations. Each fetus was euthanized by intraperitoneal injection of sodium pentobarbital solution.
Approximately one-half of the fetuses in each litter were placed in Bouin's solution and the remaining fetuses were fixed in alcohol. All fetuses fixed in Bouin's solution were examined
for soft tissue defects using the Wilson razor-blade sectioning technique5. The fetuses fixed in alcohol were macerated in potassium hydroxide, stained with Alizarin Red S and Alcian Blue, and cleared with glycerin for subsequent skeletal examination. Fetal findings were classified as malformations or developmental variations under procedures approved by a developmental toxicologist. On occasion, additional information to clarify or identify a visceral or skeletal observation was documented. These comments are not reported, but are maintained in the study data. Mechanical artifacts (i.e., tail removed and discarded) occurred during examination and processing of the fetuses. These artifacts are not reported, but are maintained in the study data.
During the skeletal staining process, there were quite a few fetuses from each group (control and treated) that experienced disarticulation and subsequent loss of ossifications from the fore- and/or hind paws prior to examination. The overall incidence of fetuses [litters] affected with a loss of all or some of these ossifications was about 47% [61%], 40% [64%], 43% [56%], and 22% [20%] in the control, 50, 200, and 800/600 mg/kg/day groups, respectively. The cause for this disarticulation was likely related to removal of too much tissue from the paws of these fetuses prior to staining. The loss of these skeletal structures (metatarsals, metacarpals, and/or phalanges) from evaluation was unfortunate but was not considered to have compromised the integrity of the study. With the exception of forelimb flexure seen in four fetuses from a single litter in the 50 mg/kg/day group, there were no external findings involving the fore-or hind-paws or the digits. Additionally, no malformations of these structures were seen at examination of the intact fetal skeletal specimens that remained in these groups. The latter represented over 50% of the fetuses in each group and approximately 40% of the litters.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
No maternal toxicity was seen at 50 mg/kg/day. At 200 mg/kg/day, the lower weight gain and lower food consumption seen early in treatment (GD 6-9) were not considered adverse since the responses were transient and not apparent over the remainder of gestation. No other maternal toxicity was seen at this dose level. At 800/600 mg/kg/day, four animals died and at necropsy were noted with depleted fat reserves and/or small thymus glands. Clinical signs of toxicity at this dose level included decreased activity, changes in fecal appearance and amounts, red or black material around the mouth and/or nose, hunched posture, thin appearance, hair/fur discolored in the abdominal and anogenital regions, and unkempt appearance. These animals lost weight early in the treatment period (GD 6-9 and 9-12) and gained less weight for the remainder. Food consumption was also reduced.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
The pregnancy rate was 96% in controls and 100% in each of the treated groups. One control female was not pregnant. All fetuses were resorbed in three females at 800/600 mg/kg/day at GD 20. Additionally, there were 13 females in this high-dose group (800/600 mg/kg/day) whose pregnancy was confirmed only on the basis of stained uterine foci. Exclusive of these animals, there were 23, 25, 25, and 5 litters with viable fetuses for evaluation on GD 20 in the control, 50, 200, and 800/600 mg/kg/day groups, respectively. No effects of treatment at 50 and 200 mg/kg/day were seen from uterine implantation data, fetal body weight, fetal sex ratios, or fetal external, visceral, or skeletal evaluations. At 800/600 mg/kg/day, there was a high incidence of postimplantation loss as most of the pregnancies were comprised either of all resorptions or stained uterine foci suggestive of early fetal death and resorption. From the
five high-dose litters with viable fetuses on GD 20, fetal weights were lower, ossification delayed, and the incidence of litters with at least one malformed fetus increased. Thus, the maternal No-Observable-Adverse-Effect Level (NOAEL) and the No-Observable-Effect Level (NOEL) for developmental toxicity in this developmental toxicity study in rats with ^Jwas 200 mg/kg/day.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The pre-natal developmental toxicity of 1 -H-Tetrazol-5-amine-,N-1 H-tetrazol-5- yl-,monoammonium salt was investigated according to US-EPA Guideline OPPTS 870.3700.
The maternal No-Observable-Adverse-Effect Level (NOAEL) and the No-Observable-Effect Level (NOEL) for developmental toxicity in this developmental toxicity study in rats with 1 -H-Tetrazol-5amine-,N-1 H-tetrazol-5- yl-,monoammonium salt was 200 mg/kg/day.

Executive summary:

The pre-natal developmental toxicity of 1 -H-Tetrazol-5-amine-,N-1 H-tetrazol-5- yl-,monoammonium salt was investigated according to US-EPA Guideline OPPTS 870.3700.

Three treatment groups of 25 female CD® [Crl:CD®(SD)] rats/group were administered the test article at respective dose levels of 50, 200, and 800 mg/kg/day. Beginning on Gestation Day (GD) 9, the dose level for the high dose group was reduced to 600 mg/kg/day. One additional group of 25 females 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 from GD 6 to 19, at a dose volume of 10 mL/kg/dose. All time-mated females arrived at the laboratory on GD 0, the day evidence of mating was observed.

Observations of the animals included clinical signs, gestation body weights and body weight change, and food consumption. On GD 20, all surviving animals were euthanized and subjected to a complete necropsy, including a uterine examination in which the total number of implantations, early and late resorptions, live and dead fetuses, and the position of the cervix were recorded. The total number of corpora lutea on each ovary was also recorded. Gravid uterine weights were recorded and adjusted GD 20 body weights and body weight changes (GD 0 to 20) calculated. All fetuses were given an external examination, and processed for either visceral or skeletal examination. Malformations and developmental variations were recorded.

Analyses of the formulations used on study confirmed that these were homogeneous at the batch size prepared and the appropriate concentration levels for dosing.

No maternal toxicity was seen at 50 mg/kg/day. At 200 mg/kg/day, the lower weight gain and lower food consumption seen early in treatment (GD 6-9) were not considered adverse since the responses were transient and not apparent over the remainder of gestation. No other maternal toxicity was seen at this dose level. At 800/600 mg/kg/day, four animals died and at necropsy were noted with depleted fat reserves and/or small thymus glands. Clinical signs of toxicity at this dose level included decreased activity, changes in fecal appearance and amounts, red or black material around the mouth and/or nose, hunched posture, thin appearance, hair/fur discolored in the abdominal and anogenital regions, and unkempt appearance. These animals lost weight early in the treatment period (GD 6-9 and 9-12) and gained less weight for the remainder. Food consumption was also reduced.

The pregnancy rate was 96% in controls and 100% in each of the treated groups. One control female was not pregnant. All fetuses were resorbed in three females at 800/600 mg/kg/day at GD 20. Additionally, there were 13 females in this high-dose group (800/600 mg/kg/day) whose pregnancy was confirmed only on the basis of stained uterine foci. Exclusive of these animals, there were 23, 25, 25, and 5 litters with viable fetuses for evaluation on GD 20 in the control, 50, 200, and 800/600 mg/kg/day groups, respectively. No effects of treatment at 50 and 200 mg/kg/day were seen from uterine implantation data, fetal body weight, fetal sex ratios, or fetal external, visceral, or skeletal evaluations. At 800/600 mg/kg/day, there was a high incidence of postimplantation loss as most of the pregnancies were comprised either of all resorptions or stained uterine foci suggestive of early fetal death and resorption. From the

five high-dose litters with viable fetuses on GD 20, fetal weights were lower, ossification delayed, and the incidence of litters with at least one malformed fetus increased. Thus, the maternal No-Observable-Adverse-Effect Level (NOAEL) and the No-Observable-Effect Level (NOEL) for developmental toxicity in this developmental toxicity study in rats with

1 -H-Tetrazol-5amine-,N-1 H-tetrazol-5-yl-,monoammonium salt

was 200 mg/kg/day.