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Description of key information

NOAEL for 90-day repeat dose toxicity study in rats was 100 mg NTO/kg/day based on effects on testes. This key study result is supported by the OECD 422 combined repeated dose toxicity study and screening for reproduction/developmental toxicity study. In this study doses of 31.25, 125, and 500 mg/kg bw/day NTO in corn oil were administered to Sprague-Dawley rats for four weeks.

Although the dose of 500 mg/kg bw/day resulted in testes degeneration, reduced sperm counts with no motile sperm observed, this dose did not affect reproduction or development. Therefore the NOAELs for reproduction and developmental toxicity are 125 and 500 mg/kg/day, respectively.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
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
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Supplied by Ordnance Systems, Inc., Kingsport, Tennessee
Lot no.:BAE 07B 305001
Purity:99.6%
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:
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
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
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.
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
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
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
315 mg/kg bw/day (actual dose received)
System:
male reproductive system
Organ:
seminiferous tubules
testes
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

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

 

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.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A 90-day repeat oral dose toxicity study in rats was undertaken according to the U.S EPA guidelines, OPPTS 870.1100 and in accordance with GLP (U.S. Army Public Health Command, 2010). NTO was administered by oral gavage in PEG-200, once daily, to five groups of 10 male and 10 female rats at dosage levels of 0, 30, 100, 315 and 1000 mg/kg/day. The main evidence of toxic effects were primarily found in the male reproductive organs. The absolute 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 also observed in the 30 and 100 mg/kg/day dose groups but were not statistically significant versus controls. The reduction in mean testes and epididymides weights and weight ratios was dose dependent. A further histopathological evaluation of the samples from this study was carried out (Wallace, 2011). Based on tissues examined, testes in the 315 and 1000 mg/kg-day treatment groups consistently demonstrated moderate to severe seminiferous tubular degeneration and atrophy and were different from controls. Incidences of adverse testicular pathology in the animals from the 30 and 100 mg/kg/day treatments were not different from controls. Therefore, based on these updated histopathology findings, the NOAEL was identified as 100 mg/kg/day.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

Study carried out to international guidelines and to GLP.

Repeated dose toxicity: via oral route - systemic effects (target organ) urogenital: testes

Justification for classification or non-classification

Results of the 90 day study shows that the target organs are the liver and testes for NTO-treated animals. However effects are significant at doses >100 mg/kg/day therefore NTO is not classified in accordance with Regulation (EC) No 1272/2008

Liver: Albumin, total protein, and red blood cell counts in the 1000 mg/kg/day dose male rat group were significantly reduced compared to controls while mean corpuscular volume and mean corpuscular hemoglobin concentrations were significantly elevated. The reduced albumin levels correlated with microscopic changes observed in the liver at the highest dose; however, it is unclear if the reduced albumin levels were a result of liver dysfunction. The significantly reduced red blood cell counts and increased mean corpuscular volumes, along with minor decreases in hemoglobin, could be indicative of slight macrocytic anemia at higher doses. Microscopically, liver changes were present in male and female rats exposed to 1000 mg/kg/day NTO. Trace to mild hepatic lesions characterized by centrilobular hepatocellular hyperplasia were observed in the livers of male and female rats given 1000 mg/kg/day NTO.

Testes:Significant reductions in mean testes and epididymides weights and weight ratios were observed in male rats given 315 and 1000 mg/kg/day NTO. Smaller, nonsignificant reductions in testes and epididymides weights were observed in the 30 and 100 mg/kg/day dose groups. Microscopic examination revealed the presence of moderate to severe testicular hypoplasia, characterized by interstitial degeneration and loss of spermatogenic epithelium in the seminiferous tubules, in the 315 and 1000 mg/kg/day dose groups. Trace to moderate testicular hypoplasia consisting of interstitial degeneration was present in the 30 and 100 mg/kg/day dose groups. The macro- and microscopic changes observed in the testes were only significant through the 315 mg/kg/day dose group. The microscopic changes observed in the 30 and 100 mg/kg/day dose groups (up to moderate severity) were part of a dose-related trend. Sperm counts of male rats exposed to 315 and 1000 mg/kg/day NTO were significantly reduced following 90 days of exposure compared to controls. However, a re-evaluation of testicular changes determined that the incidences of adverse testicular pathology in the animals from the 30 and 100 mg/kg-day treatments were not different from controls (p > 0.27).