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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 December 2008 to 19 March 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to a reliable method and in compliance with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material : 3-NITRO-1 ,2,4-TRIAZOL-5-0NE (NTO)
- Substance type: energetic explosive
- Physical state: light green to white crystalline solid with no odor
- Purity ca.99%
Specific details on test material used for the study:
Supplied by Ordnance Systems, Inc., Kingsport, Tennessee
Lot no.:BAE 07B 305001
Purity:99.6%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: well-known breeder
- Age at study initiation: 8 weeks old
- Weight at study initiation:
- Fasting period before study:
- Housing: housed individually in suspended polycarbonate boxes with Harlan Sani-Chip® bedding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26°C
- Humidity (%): 30-70% relative humidity
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
(PEG)-200
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
For each dosing suspension, the calculated amount of NTO was weighed and placed in a ceramic mortar. The NTO was then wetted with a measured amount of PEG-200 and ground with a mortar and pestle to a fine consistency. The slurry was transferred to a volumetric flask and the mortar was rinsed with a measured amount of PEG-200 to remove any remaining slurry. The remaining PEG-200 was then added to the suspension to achieve the calculated concentration.
All solutions/suspensions for the 90-day repeated-dose studies were made approximately 1 week prior to use to allow time for chemical analysis.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity:
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysed via LC/MS
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 30, 100, 315 and 1000 mg/kg/day
Basis:

No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes / No / No data
- Time schedule:
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once weekly

BODY WEIGHT: Yes
- Time schedule for examinations: First day of dosing, day 7 of dosing and weekly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmic examinations were performed prior to the scheduled start of the 90-day study and within 2 weeks of the scheduled necropsies.
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: No data
- How many animals:
- Parameters checked: White blood cell count, %lymphocytes, %monocytes, %eosinophils, %basophils, red blood cell count, hemoglobin. hematocrit, mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, red blood cell distribution width, platelets and mean platelet volume

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Yes / No / No data
- How many animals:
- Parameters checked: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, calcium, cholesterol, creatinine kinase, creatinine, glucose (fasted), lactate dehydrogenase, total bilirubin, total protein, triglycerides, sodium, potassium, chlorine

URINALYSIS: Yes
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked: volume. colour, appearance, pH, specific gravity, glucose, bilirubin, urobilinogen, ketone, blood, protein, nitrite, leukocytes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Week 11
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes, adrenal glands. aorta, urinary bladder, femur, bone marrow, brain, esophagus, eyes, harderian gland, heart, kidneys, lacrimal gland, duodenum, jejunum, ileum, cecum, colon, rectum, liver, lungs, lymph nodes, macroscopic lesions, pancreas, pituitary gland, cervix/uterus,
ovaries, epididymides, prostate, semina vesicles, testes, salivary gland, sciatic nerve, skin, spinal cord, spleen, stomach, thymus, thyroid/parathyroid, tongue and trachea (only control and highest dose group). Kidneys, liver and testes from all dose groups examined.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
none compound related
Mortality:
mortality observed, treatment-related
Description (incidence):
none compound related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
see below for details
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see below for details
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
see below for details
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see below for details
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see below for details
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see below for details
Details on results:
CLINICAL SIGNS AND MORTALITY : 7 deaths throughout study, non considered to be compound related. Transient clinical signs noted, considered to be treatment related: changes in arousal, chromodacryorrhea, crusty eyes, yellow stains on the ventral surface and face, dried red material on the nose, soft feces, and vocalization. The soft feces, low arousal, and yellow staining on the ventral surface signs occurred with greater frequency and duration in the higher dosage groups.

BODY WEIGHT AND WEIGHT GAIN: No toxicologically meaningful differences in male or female rats exposed to any dosage of NTO compared to
controls. However mean male body weight significantly greater for 315 mg/kg/day dose group compared to the control and 1000 mg/kg/day dose groups. Body weight changes in the male 100 mg/kg/day dose group significantly greater than controls for days 34-41.

FOOD CONSUMPTION:

OPHTHALMOSCOPIC EXAMINATION:

HAEMATOLOGY: Statistically significant dose group differences were observed in the male rats for red blood cells, mean corpuscular volume, mean
corpuscular hemoglobin, and red cell distribution width and in female rats for mean corpuscular volume. Red blood cell counts of male rats receiving 1000 mg/kg/day were significantly lower than the controls as well as the 30 and 100 mg/kg-day dose groups. There was a general decrease in male red blood cells with increasing dosage. The male mean red cell distribution width was significantly lower in the 30 mg/kg-day dose group compared to controls and the 1000 mg/kg-day group. The mean male and female mean corpuscular volumes were significantly elevated in the 1000 mg/kg/day dose groups compared to controls and generally increased with dosage.

CLINICAL CHEMISTRY: For albumin and total protein, the male 1000 mg/kg/day dose group had a significantly lower mean than the PEG-200 control
group.

URINALYSIS: Animals at higher dose groups had significantly more animals with bright yellow urine compared to controls. Test substance solutions/suspensions were bright yellow in colour, therefore urine colouring was likely a result of the elimination of test compound.

NEUROBEHAVIOUR:

ORGAN WEIGHTS: Toxicologically meaningful differences observed primarily for the male reproductive organs. Mean testes and epididymides weights and weight ratios (body and brain) were significantly lower in the 1000 and 315 mg/kg/day dose groups compared to controls. Slight reductions in testes and epididymides weights and weight ratios were observed in the 30 and 100 mg/kg/day groups but were not statistically significant versus controls.

GROSS PATHOLOGY

HISTOPATHOLOGY: NON-NEOPLASTIC Microscopic evaluation of a full tissue list for control and 1000 mg/kg/day animals as well as specific target organs for the lower dose groups indicated that the target organs were the liver and testes for NTO-treated animals. Trace to mild toxicologically relevant hepatic lesions were present in the livers of male and female 1000 mg/kg/day dose groups. The main finding indicative of NTO-induced hepatotoxicity was centrilobular hyperplasia. See below for further information.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)

HISTORICAL CONTROL DATA (if applicable)

OTHER FINDINGS

Effect levels

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
315 mg/kg bw/day (actual dose received)
System:
male reproductive system
Organ:
testes
seminiferous tubules
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Moderate to severe testicular hypoplasia was observed in all male rats in the 1000 mg/kg/day dose group. The finding was characterized by interstitial degeneration and loss of spermatogenic epithelium in the seminiferous tubules with practically no cells or spermatogenesis in the spermatic tubules except for some remnant Sertoli cells on the basement membrane. Corresponding aspermia was present in the epididymides of these high-dose males. At lower doses, these lesions were less frequent and severe and consisted of interstitial degeneration rather than tubular degeneration. Table 1 below shows the incidence and severity of the lesions as evaluated by Wallace, 2011.

Table 1 Incidence and Severity of Testicular Lesions in Male Rats

 

Control (PEG-200)

30 mg/kg

100 mg/kg

315 mg/kg

1000 mg/kg

Normal

10/10

9/10

8/9

0/7

0/9

Minimal Tubular Degeneration/Atrophy

 

 

 

 

 

Mild Tubular Degeneration/Atrophy

 

 

 

1/7

 

Moderate Tubular Degeneration/Atrophy

 

 

1/9

 

9/9

Severe Tubular Degeneration/Atrophy

 

1/10

 

6/7

 

Applicant's summary and conclusion

Conclusions:
The NOAEL for the 90-day oral toxicity study in rats with NTO was 100 mg/kg/day based on effect on testes at 1000 and 315 mg/kg bw/day.