Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007-07-17 - 2007-09-23
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 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
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
Details on test material:
Purity: 98.7%

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals or test system and environmental conditions:
ICR mice were obtained from Harlan, Frederick, MD and were received on 17 My 2007 (pilot toxicity study), 31 July 2007 (toxicity study) and on 21 August 2007 (definitive micronucleus study). At the time of dose administration for each study, the mice were approximately 6 to 8 weeks old. The mice were housed in an AAALAC-accredited facility with a controlled environment of 72 ± 3°F temperature, 50 ± 20% relative humidity, and a 12 hour light/dark cycle. Mice of the same sex were housed up to five per rodent Micro-Barrier cage. Cages were placed on the racks equipped with Micro-VENT full ventilation, HEP A filter«! system. The purpose of this system is to supply uninterrupted positive air to each individual rodent Micro-Barrier cage and to capture the effluent air from each cage and re-filter the air (HEPA) prior to introducing the air back into the room. Heat-treated hardwood chips were used for bedding. Mice had free access to tap water and a certified laboratory rodent chow (Harlan 2018C Certified Global Rodent Diet), which has been analyzed for environmental contaminants. There were no contaminants in the feed that were considered to have influenced the results of the study. The water used in the study met USEPA drinking water standards and was monitored at least annually for levels of organophosphorus pesticides, metals, coliform bacteria, and other contaminants.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
sterile water for injection
Details on exposure:
In the definitive micronucleus study, mice were exposed to BTA-1NH3 at a dose of 100, 200 or 400mg/kg, the vehicle control or Ihe positive control article. All dose formulations were administered by a single IP administration and at a dose volume of 20 mL/kg. The IP route of administration has been routinely used and is widely-accepted for use in the mammalian bone marrow erythrocyte micronucleus assay. All mice in the experimental and control groups were weighed immediately before dose administration, and the administered volume was based on individual body weight. Mice were observed after dose administration and throughout the course of the study for clinical signs of toxicity.
Frequency of treatment:
once
Post exposure period:
48 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
100 mg/kg, 200 mg/kg, 400 mg/kg),
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide

Examinations

Tissues and cell types examined:
, at the scheduled bone marrow collection time, five mice per sex per treatment were euthanized by CO2 asphyxiation. Immediately following euthanasia, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing a small volume (about 0.1 mL) of serum. The bone marrow cells were transferred to a labeled centrifuge tube containing approximately 1 mL serum. The tubes were identified by labels containing the study, group and animal numbers. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. Hie cells were resuspended by aspiration with a capillary pipette and a small drop of bone marrow suspension was spread onto a clean glass slide. Two slides were prepared from each mouse. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.
Evaluation criteria:
The test article was considered to induce a positive response if a dose-responsive increase in the incidence of micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control (p < 0.05, Kastenbaum-Bowman Tables) at any sampling time.
Values that were statistically significant but did not exceed the range of historical negative controls were judged as not biologically significant/relevant.
The test article was judged negative if no statistically significant increase in the incidence of micronucleated polychromatic erythrocytes above the concurrent vehicle control values and no evidence of dose response were observed at any sampling time.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The genetic toxicity of 1 -H-Tetrazol-5amine-,N-1 H-tetrazol-5- yl-,monoammonium salt was examined according to OECD Guideline 474 in vivo.
Under the conditions of the study, a single intraperitoneal (IP) administration of BTA-1NH3 at doses up to and including 400 mg/kg (the maximum tolerated dose) did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. Therefore, BTA-1NH3 was concluded to be negative in the micronucleus test using male and female ICR mice.
Executive summary:

The genetic toxicity of 1 -H-Tetrazol-5amine-,N-1 H-tetrazol-5- yl-,monoammonium salt was examined according to OECD Guideline 474 in vivo.

The test article, BTA-1NH3, was tested in the mouse micronucleus assay. The assay was performed in two phases. The first phase, the dose range-finding phase, was designed to assess the toxicity of the test article and to set dose levels for die definitive micronucleus study. The dose range-finding phase consisted of a pilot toxicity study followed by a toxicity study. The second phase, the definitive micronucleus study, was designed to evaluate the potential of the test article to increase the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female ICR mice.

In the pilot toxicity study, two male mice each were exposed to BTA-1NH3, at 1, 10, 100 or 1000 mg/kg body weight while five male and five female mice were exposed to 2000 mg/kg. Mortality occurred following dose administration in all animals administered with the 1000 mg/kg dose level (2/2 males) and with the 2000 mg/kg dose level (5/5 males and 5/5 females). Before being found dead, irregular breathing and lethargy were observed in all animals exposed to the 1000 mg/kg dose level. In order to further assess toxicity of the test article, a toxicity study was performed.

In the toxicity study, male and female mice (5/sex/group) were exposed to BTA-1NH3 at 200,400, 600 or 800 mg/kg. Mortality occurred on the day of dose administration and thereafter as follows: 2/5 females at 600 mg/kg and 4/5 males and 4/5 females at 800 mg/kg. Lethargy was observed at all dose levels, piloerection at ^ 600 mg/kg, and diarrhea at the 800 mg/kg dose level. Based upon these results, the high dose for the micronucleus test was set at 400 mg/kg for male and female mice, which was estimated to be the maximum tolerated dose (MTD).

The definitive micronucleus study consisted of seven groups, each containing 5 male and 5 female mice. Mice in five of these groups were treated either with the controls (vehicle or positive) or with BTA-1NH3 at 100,200, or 400 mg/kg and were euthanized 24 hours after treatment Mice in the other two groups were treated either with the vehicle control or test article at a dose of400 mg/kg and were euthanized 48 hours after treatment. An additional group of 5 male and 5 female mice were treated with BTA-1NH3 at a dose of 400 mg/kg to be used as replacement animals for the high dose in the event of mortality. At the time of euthanasia, femoral bone marrow was collected and bone marrow smears (slides) were prepared and stained with May-Gruenwald-Giems a stain. Bone marrow cells [polychromatic erythrocytes (PCEs)] were examined microscopically for the presence of micronuclei (micronucleated PCEs; MPCEs). A statistically significant difference in the incidence of micronucleated PCEs in the test article-treated groups relative to the concurrent vehicle control groups was determined based on the Kastenbaum-Bowman Tables (binomial


distribution) for p <0.05. The incidence of micronucleated PCEs and the ratio of PCEs to total erythrocytes (PCEs/ECs ratio) served as indication of test article clastogenicity and cytotoxicity, respectively.

No mortality occurred at any dose level during the course of the micronucleus study. Lethargy was observed in most animals exposed to 200 mg/kg dose level. At termination of study however, this clinical sign was not observed. All other mice treated with test article (100 mg/kg) or control articles appeared normal during the course of the study.

Based on bone marrow analysis, the following was observed:

       Reductions in the ratio of polychromatic erythrocytes to total erythrocytes of up to 20% were observed in some of the test article-treated groups relative to the respective vehicle controls. Since no dose dependent reductions were not dose-dependent suggesting that the test article did not inhibit erythropoiesis.

       The incidence of micronucleated PCEs per 10,000 PCEs in test article-treated groups was not statistically increased relative to their respective vehicle controls in either male or female mice, regardless of dose level or bone marrow collection time (p > 0.05, Kastenbaum-Bowman Tables).

       CP, the positive control, induced a statistically significant increase in micronucleated PCEs in both male and female mice (p < 0.05, Kastenbaum-Bowman Tables). The mean incidence of micronucleated PCEs did not exceed the historical vehicle control range in the vehicle control groups. The vehicle and positive controls were consistent with the historical control data, indicating that all criteria for a valid test were met as described in the protocol and that there was no problem with the test system or the quality of the test.

Under the conditions described in this report, a single intraperitoneal (IP) administration of BTA-1NH3 at doses up to and including 400 mg/kg (the maximum tolerated dose) did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. Therefore, BTA-1NH3 was concluded to be negative in the micronucleus test using male and female ICR mice.

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