Registration Dossier

Administrative data

Description of key information

Oral toxicity - NOAEL in rats was found to be 1.25 mg/kg bw/day

Inhalation toxicity - NOAEC in rats was found to be 165 mg/m3.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

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:
September 2010 - March 2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
There are some deviations listed in the report. However, they were not considered to have compromised the integrity or validity of the study.
Deviations:
not applicable
Remarks:
6 deviations listed
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BAE Systems, Ordnance System, 4509 West Stone Drive, Kingsport, TN 37660.
- Expiration date of the lot/batch: lot BAE10H281-008
- Purity test date: 100%

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity:
- Specific activity:
- Locations of the label:
- Expiration date of radiochemical substance:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test material was dried in a vacuum oven at approximately 70°C for 12-48 hours to remove moisture.
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel: Dosing solutions/suspensions were prepared by grinding DNAN using a mortar and pestle to a fine consistency, and mixing with a measured volume of cron oil.
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Crl:CD(SD) CD IGS
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation: 8-week old
- Weight at study initiation: 297,1+/-10.88 for males and 214,1+/-9.14g for females
- Fasting period before study: No
- Housing: individually housed in suspended polycarbonate cages
- Diet : ad libitum)
- Water : ad libitum
- Acclimation period: 14 days

DETAILS OF FOOD AND WATER QUALITY: Certified pesticide-free rodent chow and quality drinking water.

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

IN-LIFE DATES: From: To:
Route of administration:
oral: gavage
Details on route of administration:
Oral dosing was performed using a stainless steel 16 gauge *2 inch gavage needle.

Vehicle:
corn oil
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
4 dosing solutions/suspensions were prepared : 0,25, 1, 4 and 16mg/mL. Dosing solutions/suspensions were prepared in volumes sufficient for approximately 2-3 weels of dosing, resulting in preparation of 5 sets of dosing solutions.


- DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

- 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:
A 1 mL sample was taken from each dosing dolutions/suspensions for analytical verification of concentration of each preparation using a gas chromatograph equipped with an electron capture detector.
Duration of treatment / exposure:
90 days
Frequency of treatment:
All animals were administered at approximately the same time daily, 7 days per week.
Dose / conc.:
1.25 mg/kg bw/day (nominal)
Dose / conc.:
5 mg/kg bw/day (nominal)
Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
80 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 rats/sex/concentration
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
- Rationale for animal assignment : random
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups:
- Section schedule rationale (if not random):
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily : once in the morning and one in the afternoon excepts on weekends when observations occured only in the morning
- Cage side observations checked in tables in the attached study report were included.

DETAILED CLINICAL OBSERVATIONS: Ye
- Time schedule: once in the morning and one in the afternoon excepts on weekends when observations occured only in the morning

BODY WEIGHT: Yes
- Time schedule for examinations: twice pretest, weekly during the study and the morning of the necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determine: Feedes were reweighted weekly and the mass of the empty feeder was sustracted from the mass of the full feeder to determine the grams of food consumed for each animal
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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 / Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-study for all animals and animals from the control and the highest dose were examinated within one week of the conclusion of the study.
- Dose groups that were examined:

HAEMATOLOGY/ CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood abtained from CO2 anesthetized animals via intracardiac puncture at the termination of the study
- Anaesthetic used for blood collection: Yes CO2
- Animals fasted: Yes overnight prior to bllod collection
- How many animals: All animals
- Parameters checked in tables in the attached study report were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: During the last 2 weeks
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, for an overnight period of approximately 12 hours
- Parameters checked in tables in the attached study report were examined

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the same time each morning prior to dosing
- Dose groups that were examined: on each animal prior to initiation of dosing and weekly thereafter with one subset of animals being assessed per day. (animals were divided into 2 subsets for each sex)
- Battery of functions tested: FOB (Functional Observation Battery) and motor activity assessment

IMMUNOLOGY: Not specified
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:
Sacrifice and pathology:
Rats that died during the course of the study were submitted for gross necropsy and histophatological evaluation of possible.
Otherwise, all surviving rats after the 90-day treatment were anesthetized using CO2.

GROSS PATHOLOGY: Yes, on all terminal animals noting all lesions and abnormal observations.

HISTOPATHOLOGY: Yes, the prevalence and severity of findings were graded as compared to controls.
Other examinations:
SPERM ANALYSIS :
The number of sperm, number of motile sperm, and number of progressive sperm were determined in duplicate for each animal.
Statistics:
Details of the statistical analyses can be found in Appendix T of the study report.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Mortality or morbidity was observed in three males and one female in the 80 mg/kg-d dose group.
The female was euthanized on day 19 following observations of lethargy, labored breathing, prostrate posture, ataxia, partial hind limb paralysis, and complete front limb paralysis. The males were found dead on days 51, 64, and 79. Clinical signs of toxicity, including lethargy prostrate or recumbent posture, rapid/labored respiration, ataxia, irregular gait patterns (walking on toes, creeping, dragging hind end, hind end raised, pulling legs up, stiff legs, limping, dragging leg, walking backwards in circles, hind end wobbling, movements jerky, stiff/locked muscle/limb), dark urine, squinting, hunched posture, pulling ears back, twitching ears, twitching tail, body twitching, head shaking, leaning to left, straubbed tail, curled tail, barbering, rough hair coat, piloerection, low arousal, red discharge from nose, chromodacryorrhea were noted in the 80 mg/kg-d dose group. Although the overall signs noted for males and females were similar, there was an apparent gender difference in the pattern of clinical signs. Males were consistently observed in dorsal or lateral recumbency starting approximately three hours after the DNAN was administered and lasting approximately three hours. Males in this posture were alert and responsive, immediately righting themselves when disturbed. Females were observed in this posture relatively infrequently. Males and females both demonstrated gait irregularities, however, males tended to creep with lowered hind quarters while females tended to walk on their toes and pull up their legs and high step while walking; both genders had difficulty moving the hind limbs due to what appeared to be muscle stiffness/tetany. Lethargy, dark urine, dorsal and lateral recumbency, prostrate posture, congestedbreathing, and labored breathing were noted in males in the 20 mg/kg-d dose group. Clinical signs in females in the 20 mg/kg-d group included dark urine, low arousal, irregular gait (walking on toes), and lateral recumbency. Males in lower dose groups exhibited dark urine, curled tail, dorsal and lateral recumbency, irregular gait (creeping, hind end lowered, stiff muscles), chromodacryorrhea, and barbering. Clinical signs in females in lower dose groups were limited to barbering, alopecia and congested breathing, with the exception of one female in both the 1.25 and 5 mg/kg-d groups with hind limb ataxia. Clinical signs in control animals were limited to barbering, chromodacryorrhea, alopecia, scab, and congested breathing, with the exception of one male which was euthanized on day 68 due to a dosing error
Mortality:
mortality observed, treatment-related
Description (incidence):
Mortality or morbidity was observed in three males and one female in the 80 mg/kg-d dose group. The female was euthanized on day 19 following observations of lethargy, labored breathing,
prostrate posture, ataxia, partial hind limb paralysis, and complete front limb paralysis. The males were found dead on days 51, 64, and 79. Clinical signs of toxicity, including lethargy prostrate or
recumbent posture, rapid/labored respiration, ataxia, irregular gait patterns (walking on toes, creeping, dragging hind end, hind end raised, pulling legs up, stiff legs, limping, dragging leg, walking backwards in circles, hind end wobbling, movements jerky, stiff/locked muscle/limb), dark urine, squinting, hunched posture, pulling ears back, twitching ears, twitching tail, body twitching, head shaking, leaning to left, straubbed tail, curled tail, barbering, rough hair coat, piloerection, low arousal, red discharge from nose, chromodacryorrhea were noted in the 80 mg/kg-d dose group.
Although the overall signs noted for males and females were similar, there was an apparent gender difference in the pattern of clinical signs. Males were consistently observed in dorsal or lateral recumbency starting approximately three hours after the DNAN was administered and lasting approximately three hours. Males in this posture were alert and responsive, immediately righting themselves when disturbed. Females were observed in this posture relatively infrequently. Males and females both demonstrated gait irregularities, however, males tended to creep with lowered hind quarters while females tended to walk on their toes and pull up their legs and high step while walking; both genders had difficulty moving the hind limbs due to what appeared to be muscle stiffness/tetany. Lethargy, dark urine, dorsal and lateral recumbency, prostrate posture, congested breathing, and labored breathing were noted in males in the 20 mg/kg-d dose group. Clinical signs in females in the 20 mg/kg-d group included dark urine, low arousal, irregular gait (walking on toes), and lateral recumbency. Males in lower dose groups exhibited dark urine, curled tail, dorsal and lateral recumbency, irregular gait (creeping, hind end lowered, stiff muscles), chromodacryorrhea, and barbering. Clinical signs in females in lower dose groups were limited to barbering, alopecia and congested breathing, with the exception of one female in both the 1.25 and 5 mg/kg-d groups with hind limb ataxia. Clinical signs in control animals were limited to barbering, chromodacryorrhea, alopecia, scab, and congested breathing, with the exception of one male which was euthanized on day 68 due to a dosing error
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body mass increased with time for all dose groups throughout the study; with the exception of week 11 when animals were fasted overnight while in metabolism cages.
Females in the 80mg/kg dose group additionally lost weight during week 10.
Body mass of male rats in the 80 mg/kg dose group was significantly reduced relative to the corn oil control from day 7 to day 90.
Body mass of female rats did not differ significantly between treated and control groups at any time during the study.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Body mass increased with time for all dose groups throughout the study, with the exception of week 11 when animals were fasted overnight while in metabolism cages. Females in the 80 mg/kg-d dose group additionally lost weight during week 10. Body mass of male rats given 80 mg/kg-d DNAN was significantly reduced relative to the corn oil control from day 7 through day 90 (P=0.043, P=0.031, P=0.003, P<0.001, P<0.001, P<0.001, P<0.001, P<0.001, P<0.001, P<0.001, P<0.001, P<0.001, P<0.001 ). Body mass of female rats did not differ significantly between treated and control groups at any time during the study. Food consumption was increased in males 20 mg/kg-d and females given 20 and 80 mg/kg-d; however this increase was only significant in females in the 80 mg/kg-d group (week 2, P=0.004; week 3, P=0.029; week 4, P=0.001; week 7, P=0.024; week 9, P=0.026). Males in the 80 mg/kg-d dose group initially exhibited an increase in food consumption similar to that observed in the 20 mg/kg-d group; however, this increase dissipated by week four (see Appendices J-L). Feed conversion efficiency was reduced in male rats given 80 mg/kg-d at weeks one thro1.1gh six (P<0.001, P=0.001, P<0.001, P=0.024, P=0.005, P=0.033, respectively) and overall (P<0.001 ). Feed conversion efficiency did not differ among treated and control groups for female rats.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Feed conversion efficiency was reduced in male rats given 80mg/kg at week 1 to 6 abd overall. Feed conversion efficiency did not differ among treated and control groups for female rats.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
White blood cell count (WBC) increased in a dose dependent manner in female rats and was significantly higher (1.5 and 1.7 fold, respectively) in the 20 and 80 mg/kg-d groups relative to the control group (P=0.023 and P=0.002, respectively). WBCs were above normal ranges in female rats in all dose groups of 5 mg/kg-d and greater in all dose groups for male rats. The increase in white blood cell count observed in female rats was due to an increase in lymphocytes in the 80 mg/kg-d group (1.6 fold) (P=0.0074) and monocytes in the 20 and 80 mg/kg-d groups (2.2 and 3.2 old) (P=0.014 and P<0.001, respectively) relative to the control group.
Lymphocyte counts demonstrated a dose dependent increase, with all dose groups of 5 mg/kg-d and greater having counts above reported normal ranges. Lymphocyte counts in male rats exceeded normal ranges in all dose groups; treated and control groups did not differ. Monocyte counts increased in a dose dependent manner in female rats. All monocyte and basophil counts were above normal ranges in both male and female rats.

Red blood cell counts (RBC) in female rats were significantly (P<0.001) reduced (0.8 fold) in the 80 mg/kg-d dose group compared to the corn oil control group. The RBC counts in the 80 mg/kg-d group were also outside of the normal range. In male rats, RBC counts were increased (1.1 fold) in the 5 and 20 mg/kg-d groups relative to the control group (P=0.037 and P<0.001, respectively). RBC counts increased in a dose dependent manner from the control group throl.1gh the 20 mg/kg-d group, with groups at and above 1.25 mg/kg-d exceeding normal values. RBC counts in the 80 mg/kg-d group, however, approximated those of the control group. Hemoglobin (HGB) and hematocrit (HCT) were significantly reduced (0.88 and 0.91 fold) in female rats in the 80 mg/kg-d group (P<0.001 and P=0.007, respectively) relative to the corn oil control group; both measures were below normal ranges in the 80 mg/kg-d group. In male rats, HCT and HGB did not differ among treated and control groups. Mean cell hemoglobin (MCH) was reduced (0.93) in males in the 20 mg/kg-d group (P=0.002) relative to the control. MCH did not differ among treated and control groups for female rats. MCH values were below normal ranges for both males and females with the exception of the females in the 80 mg/kg-d group. Mean cell hemoglobin concentration
(MCHC) was significantly reduced in both males (0.97 fold) and females (0.96 fold) in the 80 mg/kgd group (P<0.001 and P<0.001, respectively); however, MCHC remained within the normal range. Mean cell volume (MCV) was increased (1.1 fold) in female rats in the 80 mg/kg-d group (P<0.001 ). In male rats, MCV was reduced (0.93 fold) in the 20 mg/kg-d group (P=0.003). In both males and females, MCV values were below reported normal ranges with the exception of the females in the
80 mg/kg-d group. Red cell distribution width (ROW) was increased in the 20 and 80 mg/kg-d dose groups in both males (1.2 and 1.4 fold; P<0.001 and P<0.001, respectively) and females (1.1 and 1.3 fold; P=0.033 and P<0.001, respectively). Platelet count did not differ between treated and control groups for either male or female rats. Mean platelet volume (MPV) was slightly increased (1.1 fold) in the 80 mg/kg-d groups; however, this increase was only significant in females
(P=0.048).

Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Cholesterol levels decreased in a dose dependent manner in DNAN treated males, were significantly lower (1.4 and 1.5 fold, respectively) in the 20 and 80 mg/kg-d groups than in the control group (P=0.007 and P=0.005), and were outside of normal ranges.
Blood chloride levels were higher in the 80 mg/kg-d group than in the control group (P=0.007); however, these values were within normal ranges.
Blood urea nitrogen (BUN) in males in the 80 mg/kg-d group exceeded the normal range and was elevated relative to the control (1.2 fold); however, the 80 mg/kg-d group did not differ from the control group. Similarly, in females. BUN did not differ significantly between treated and control groups, but was outside of normal ranges and was increased relative to the control in all groups, particularly the 80 mg/kg-d group (1.4 fold).
Alanine aminotransferase (AL T) levels were elevated in both males and females in the 80 mg/kg-d groups relative to the control group; however, AL T levels did not differ significantly between treated and control groups. AL T levels were above reported normal levels in both treated and control groups, up to two-fold in the
80 mg/kg-d group.
Alkaline phosphatase (ALKP) levels were slightly above reported normal levels in male rats in the 80 mg/kg-d group. ALKP levels in the 80 mg/kg-d group were not elevated with respect to the control group which also had ALKP levels above reported normal levels.
Lactate dehydrogenase levels in males were below the reported normal range in all groups (1.1-1.5 fold).
Total bilirubin (TBIL) levels in females were below reported normal ranges (1.56 to 2 fold) in all groups except the 5 mg/kg-d group.
Sodium and potassium levels were above reported normal ranges for both males and females in all dose groups.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Urine color, volume, specific gravity, and protein concentration differed significantly between DNAN treated and control groups in both males and females. Urine was significantly darker in the 20 and 80 mg/kg-d dose groups than in the control groups in males (P=0.003 and P<0.001, respectively) and females (P<0.001 and P<0.001, respectively). In males, urine color increased from dark yellow in controls to gold and brown in the 20 and 80 mg/kg-d groups, respectively. Urine color in females
increased from yellow in controls to dark yellow and gold/amber in the 20 and 80 mg/kg-d groups, respectively. Urine volume increased in a dose dependent manner in females and was significantly higher (3.2 fold) in the 80 mg/kg-d group than the control group (P<0.001 ).
Urine volume was also increased (1.7 fold) in the 20 mg/kg-d group, however, this increase was not statistically significant.
In males, urine volume was significantly higher (2.0 fold) in the 20 mg/kg-d group than in the control (P=0.022). Urine volume was also increased (1.7 fold) in the 80 mg/kg-d group, however, this increase was not statistically significant. Specific gravity decreased in a dose dependent manner in female rats and was significantly reduced in the 80 mg/kg-d group relative to the control group (P=0.006). In males, specific gravity was significantly lower in the 20 mg/kg-d dose group than in
the control group (P=0.039).
Protein concentration was significantly lower in females in the 80m g/kg-d group (P=0.037) and males in the 20 mg/kg/d group (P=0.027) than in the control groups.
Bilirubin concentration was increased (2.7 fold) in males in the 80 mg/kg-d group relative to the control group (P=0.009).
Urine appearance, glucose, ketones, pH, urobilinogin, and leukocytes did not differ between DNAN treated and control groups.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
See neuropathological findings
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Kidney, liver, and spleen mass, organ to body mass ratios, and organ to brain mass ratios differed significantly between DNAN treated and control groups in both males and females. In males, absolute kidney mass did not differ significantly between treated and control groups; however, kidney mass to body mass ratios were increased in the 20 and 80 mg/kg-d groups relative to the control (P=0.001 and P<0.001, respectively). Kidney mass to brain mass ratios were increased in males in the 20 mg/kg-d group only (P=0.036). In females. kidney mass increased in a dose dependent manner and was significantly higher in the 20 and 80 mg/kg-d groups than in the control group (P=0.035 and P=0.035, respectively).
Kidney mass to body mass and brain mass ratios were increased, relative to the control, in female rats given 80 mg/kg-d DNAN (P<0.001 and
P=0.019).
Liver mass to body mass ratios were increased, relative to the control, in males (P<0.001) and females (P=0.001) in the 80 mg/kg-d dose group.
Spleen mass to body mass ratios were higher in males and feniales given 80 mg/kg-d DNAN relative to controls (P<0.001 and P<0.001, respectively).
Absolute spleen mass and spleen mass to brain mass ratios were also increased, relative to the control, in females in the 80 mg/kg-d group (P<0.001 and P<0.001, respectively).
In males, testes mass, testes to body mass, and testes to brain mass ratios were reduced in the 80 mg/kg-d group (P<0.001, P<0.001 and P<0.001, respectively).
Epididymides mass and epididymides to brain mass ratio were also reduced, relative to the control, in males given 80 mg/kg-d DNAN (P<0.001 and P<0.001, respectively).
Thymus mass and thymus to brain mass ratio were reduced, relative to the control, in males in the 80 mg/kg-d group (P=0.001 and P<0.001, respectively).
Adrenal mass was reduced, relative to the control, in females in the 80 mg/kg-d group (P=0.042)
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The gross pathological examination of the pre-term mortality of the female in the 80 mg/kg-d dose group indicated an enlarged dark spleen, enlarged submandibular lymph nodes, and dark kidneys.
Macroscopic findings in the pre-term mortalities in males in the 80 mg/kg-d group included dark spleen (1/3), enlarged kidneys (2/3), dark red focus on kidney (1/3), small testes (1/2), red focal area on jejuna serosa (1/3).
The macroscopic findings in the pre-term mortality in the control male, esophageal rupture proximal to thoracic inlet and subcutaneous edema were consistent with a dosing error. Additional macroscopic findings included enlarged heart in two male rats, one control and one in the 5 mg/kg-d group. Pale and/or mottled kidney were noted in nine females, one
control, one 1.25 mg/kg-d, three 5 mg/kg-d, one 20 mg/kg-d, and three 80 mg/kg-d animals. One female from the 80 mg/kg-d group was noted as having dark kidneys. In males, kidneys were noted to be mottled and/or pale in three controls, one 1.25 mg/kg-d, and three 5 mg/kg-d animal.
Hydronephrosis was noted in one control and one 20 mg/kg-d males. Two males in the 80 mg/kg-d group had enlarged kidneys. Ovarian findings were noted in two females in the 5 mg/kg-d, an adhesion and a hemorrhagic cyst. Three female rats were noted as having pale areas in the liver, one each from the control, 1.25 and 5 mg/kg-d groups. In male rats, liver findings included six rats with diffusely pale livers (three controls, one 1.25 mg/kg-d, and two 5 mg/kg-d), three with mottled livers (one each from control, 1.25 and 5 mg/kg-d), and two 1.25 mg/kg-d rats with enlarged livers.
All of the females and eight of the males in the 80 mg/kg-d group were noted as having dark spleens. Small testes were noted in six of the males in the 80 mg/kg-d group. Hydrouterus was noted in five females: one 5 mg/kg-d, two 20 mg/kg-d, and two 80 mg/kg-d. Two control females and four males (one control and three 80 mg/kg-d) had enlarged mesenteric lymph nodes. Six females: one control, one 1.25 mg/kg-d, one 5 mg/kg-d, one 20 mg/kg-d and two 80 rng/kg-d had enlarged submandibular lymph nodes.
Neuropathological findings:
effects observed, treatment-related
Description (incidence and severity):
Handheld Observation :
Males
There were no differences among treatment groups for lacrimation, salivation, piloerection, palpebral closures, exopthalmos and pupillary status. The 80 mg/kg-day dose group had fewer rats that were classified as very easy to remove from the cage at weeks one (P = 0.0154), five (P = 0.0046), seven (P = 0.0497) and eight (P <0.001). To help show the drastic difference in observed responses between the 80 mg/kg-day group and the other four dose groups, an average ease of removal score was calculated for each rat. The 80 mg/kg-day group had the seven highest average scores, pointing towards more difficult ease of removal for this dose group. Reactivity to handling also differed among treatment groups at weeks two (P = 0.0485), three (P = 0.0495), six (P = 0.0218), seven (P < 0.001), nine (P = 0.0422) and ten (P = 0.0061). The 80 mg/kg-day group
had fewer low and more moderately high reactivity to handling observations than the other dose groups. The 80 mg/kg-day group had six of the seven highest 11 week average reactivity to handling scores, indicating that reactivity was higher, in general, for rats in this dose group. For both ease of removal and reactivity to handling, responses for rat 102 differed from the remaining rats in the 80 mg/kg-day, with rat 102 appearing less affected by the treatment than the other rats.
Females
There were no differences among treatment groups for ease of removal, reactivity to handling, lacrimation, salivation, piloerection, palpebral closures, exopthalmos and pupillary status. At week 6, the 80 mg/kg-day dose group had more barbering (P = 0.0289) observations than the control group. Barbering was, however, present in all dose groups (80 = 5, 20 = 2, 5 = 5, 1.25 = 4, control = 1 rat) during the 11 week study and did not differ between dose groups at any other time point.

Open Arena Observation :
Males
There were no differences found in grooms, rears, arousal, fecal boli, fecal description, and urine. The 80 mg/kg-day group had fewer normal gait observations than other dose groups at weeks five (P = 0.002), nine (P = 0.011) and 11 (P = 0.015). Generally rats in the 80 mg/kg-day dose group had too little movement to determine gait; however, at week 11 more hunched body position was observed in this group. Additional gait observations included ataxia, hind limb impairment, and walking on toes. If a rat was recorded as either having hind limb impairment, walking on toes or hunched body position, the rat usually displayed all three characteristics.
Females
There were no differences found in grooms, arousal, fecal boli, fecal description, and urine. Females in the 80 mg/kg-day dose group had fewer normal gait observations at weeks nine (P < 0.001 ), ten (P = 0.050) and 11 (P = 0.013). Similar to the males, females in the 80 mg/kg-day had fewer normal observations and more hunched body position was observed at weeks ten and 11. As with males, hind limb impairment, walking on toes or hunched. body position, when observed, typically occurred together. Rats in the 80 mg/kg-day group reared less often than those in the other dose groups at weeks six (P = 0.004), seven (P = 0.020) and ten (P = 0.040).

Sensory Motor :
Males
There were no differences among treatment groups in any of the sensory motor responses: approach, auditory startle response, tail pinch, pinna response, pupillary response, righting reflex, aerial righting, landing foot splay, forelimb grip strength, and hindlimb grip strength.
Females
There were no differences for auditory startle response, pinna response, pupillary response, righting reflex, aerial righting, landing foot splay, forelimb grip strength, and hindlimb grip strength. Tail pinch and approach differed among the five dose groups for females (P = 0.020 and P = 0.024, respectively). The 80 mg/kg-day dose group had fewer response observations for tail pinch and fewer slow approach observations for the approach variable. Nine of the ten animals in the 80 mg/kg-day dose group had no reaction responses for the approach variable. There were three, two, three, and four no reaction responses in the control, 1.25, 5, 20 mg/kg-day groups, respectively. Three animals in the 80 mg/kg-day group had no response to the tail pinch whereas all animals in all other dose groups showed a response with the exception of one animal in the 20 mg/kg-day group.

Motor Activity:
Males
There were no differences among treatment groups in basic movement, immobility, X and Y ambulation. Mean number of nose pokes was lower (P = 0.009) in the 80 mg/kg-day group than the other dose groups.
Females
There were no differences among treatment groups in basic movement, immobility, X and Y ambulation. Mean number of nose pokes was lower (P = 0.014) in the 80 mg/kg-day group than the other dose groups.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Mortality occurred in three males and one female dosed at 80 mg/kg-d. Selected tissues were collected from these animals; no cause of death was identified based on microscopic examination.
Test-article related microscopic findings were noted in the testes, epididymides, spleen, liver, brain, and kidney.
Degeneration and atrophy of testicular seminiferous tubules (moderate to severe) was present in nine of nine males examined in the 80 mg/kg-d group. No test article-related changes were noted in the testes in the control and 20 mg/kg-d groups. Seminiferous tubules of the 80 mg/kg-d group retained only Sertoli cells, spermatogonia and early spermatocytes. Absent germ cell layers included all spermatid and late spermatocyte stages resulting in the absence of mature sperm in seminiferous tubules. Testes additionally demonstrated moderate to numerous numbers of atrophic tubules. Aspermia with eosinophilic cellular tubular debris (moderate to severe) was present in the epididymis of nine of nine males examined in the 80 mg/kg-d group. Few sperm were noted in the cauda of individual animals in the 80 mg/kg-d group.
In the spleen, extramedullary hematopoiesis (minimal to severe) was present in 6/10 males and 0/10 females in the control group, 3/10 males and 3/10 females at 1.25 mg/kg-d, 6/10 males and 3/1 0 females at 5 mg/kg-d, 6/1 0 males and 4/10 females at 20 mg/kg-d and 7/1 0 males and 9/1 0 females at 80 mg/kg-d. Severity was greater in females than in males and increased with dose in females. Hemosiderin, excess iron deposited in the spleen in normal rats due to the breakdown of old erythrocytes, was present in all rats on study. An increase in hemosiderin deposits can result from hemolytic crisis or hematotoxic insult. A dose related increase in the severity of hemosiderosis was apparent in males. Hemosiderosis of greater severity than observed in the control (minimal) group was present in 3/10 (mild) males at 1.25 mg/kg-d, 4/10 (mild) at 5 mg/kg-d, 7/10 (mild) at 20 mg/kg-d and 10/10 (mild to severe) at 80 mg/kg-d. Hemosiderosis (mild to moderate) was present in 10/10 females at 1.25 mg/kg-d, 7/10 at 5 mg/kg-d, 10/10 at 20 mg/kg-d and 10/10 at 80 mg/kg-d; however the incidence 10/10 and severity (mile to moderate) was similar in control females.
In the liver, lymphohistiocytic infiltrates were observed in treated control groups. Although these aggregates are often considered to be background lesions, the frequency may be increased by treatment. A slight increase in severity with increasing dose was noted in females which may have been treatment related. Lymphohistiocytic infiltrates were present in 10/10 (minimal to mild) control females, 10/10 (mild to moderate) females at 5 mg/kg-d, 9/10 (minimal to moderate) at 20 mg/kg-d, and 9/10 (minimal to severe) at 80 mg/kg-d. Livers from animals in the 1.25 mg/kg-d group were not examined. Focal hepatic biliary hyperplasia (minimal to mild) was present in 1/10 males in the 80 mg/kg-d group and 1/10 females in the 5 mg/kg-d group. Due to the isolated incidence and minimal severity, these lesions are considered incidental and not treatment related.
Cerebellar or brain stem gliosis was noted in 3/10 males and 1/10 females in the 80 mg/kg-d dose group. Two of the males and the female were pre-term mortalities. Microscopically, lesions appeared as spongiotic grey or white matter with increased glial cells and astrocytes occasionally with macrophages (gitter cells). These lesions were considered to be compound related. Renal mineralization at the corticomedullary junction was present in 3/10 (minimal to mild) control females, 6/10 (minimal to mild) females at 5 mg/kg-d, 10/10 (minimal to moderate) at 20 mg/kg-d, and 7/10 (minimal to mild) at 80 mg/kg-d. Kidneys from the 1.25 mg/kg-d group were not examined. The prevalence of mineralization was higher in DNAN treated females than in the controls; however, a clear dose response was not apparent in either prevalence or severity. Renal mineralization was not noted in males. Other kidney lesions including, basophilic tubules, pelvic
dilatation (hydronephrosis), and lymphocytic interstitial infiltrates occurred at similar rates in control and treated animals and were considered background lesions. Additional lesions noted but determined to be background or incidental due to low frequency of occurrence or comparable occurrence in control and treated groups included: prostatic, epididymal and coagulating gland lymphocytic infiltrates, harderian gland lymphocytic infiltrates, rare lymphoid hyperplasia of submandibular or mesenteric lymph nodes, adrenal gland vacuolation, plasmacytosis of the submandibular lymph node, pancreatic acinar atrophy or degeneration, myocardial necrosis, cardiac lymphocytic infiltrates, and ultimobranchial cysts of the thyroid .
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Sperm Analysis
The number of sperm per gram in the cauda epididymis in male rats in the 80 mg/kg-d group was reduced to 4.5 percent of the number of sperm per gram found in controls (P=0.038). No motile sperm were found in any of the animals in the 80 mg/kg-d group. No significant reductions insper m per gram, percent motile sperm, or percent progressively motile sperm were observed in the1.25 , 5, or 20 mg/kg-d dose groups.
Key result
Dose descriptor:
LOAEL
Effect level:
ca. 20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
food efficiency
haematology
clinical biochemistry
urinalysis
behaviour (functional findings)
organ weights and organ / body weight ratios
gross pathology
neuropathology
histopathology: non-neoplastic
Key result
Dose descriptor:
BMDL10
Effect level:
2.3 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
haematology
Remarks on result:
other:
Remarks:
BMDL10 calculated based on the extramedullary hematopoiesis (EMH) in females (please see Any other information on results incl. tables)
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
20 mg/kg bw/day (nominal)
System:
haematopoietic
Organ:
blood
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
80 mg/kg bw/day (nominal)
System:
male reproductive system
Organ:
testes
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
80 mg/kg bw/day (nominal)
System:
nervous system
Organ:
brain
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Extramedullary hematopoeisis (EMH) was identified as the critical endpoint in this study based on the increased incidence in female rats in the DNAN treated groups (Barnes and Dourson 1988, EPA 2002). Benchmark Dose Software (BMDS v.2.1.2) was used to fit mathematical models to the EMH incidence dose response data and calculate a lower-bound confidence limit on a dose corresponding to a 10 percent response rate (BMDL10) (EPA 1995, EPA 2000). The Gamma, quantal-linear, and Weibull models were selected based on goodness-of-fit and statistical parameters (p>0.1, lowest AIC values and residuals) (Appendix X). A mean BMDL of 4.08 mg/kgday and a BMDL10 of 2.3 mg/kg-day were calculated based on the results of these three models.

Conclusions:
Mortality occurred in three male rats (days 50, 63, and 77) and one female rat (day 26) all from the 80 mg/kg-day dose group. Rats in the highest dose group (80 mg/kg-day) experienced lethargy, labored/rapid respiration, prostrate and/or recumbent posture, hunched posture, ear twitching, squinting, curled tail, and gait irregularities. A functional observation battery (FOB) and analysis of motor activity at week 13 indicated that rats given 80 mg/kg-day had altered neuromuscular function and decreased activity levels. In the 80 mg/kg-day group, female rats also had reduced sensorimotor responses while male rats had increased excitability responses.
Although food intake was similar among groups for male rats, animals from the 80 mg/kg-day dose group exhibited reduced body mass and a reduced food efficiency ratio. Females in the 80 mg/kg-day dose group also had a reduced food efficiency ratio, but had elevated food consumption at several time points during the study. Body mass did not differ among dose groups for female rats.
Female rats in the 80 mg/kg-day dose group and male rats in the 20 mg/kg-day group produced higher volumes of urine with lower specific gravity. Despite the increase in volume, urine color was darker in the 20 and 80 mg/kg-day dose groups for both sexes.
Increased mean kidney, liver, and spleen mass were observed in male and female rats given 80 mg/kg-day DNAN. In male rats, increased mean kidney and liver mass were also noted in the 20 mg/kg-day dose group; however, the changes were not associated with treatment related microscopic abnormalities or alterations in clinical chemistry parameters. Decreased mass of the testes and epididymides as well as degeneration and atrophy of the testicular seminiferous tubulesand aspermia were also observed in rats from the 80 mg/kg-day group.
In females, changes in hematology indicative of anemia, including decreased red blood cell count, hematocrit, and hemoglobin, and increased red cell distribution width were observed in the 80 mg/kg-day group. A dose related increase in extramedullary hematopoeisis was noted in spleens of female rats at 20 and 80 mg/kg-day. Glial lesions within the cerebellum were noted in four rats (1 female/3 males) in the 80 mg/kg-day group.
This study, the first repetitive oral dosing conducted with DNAN, demonstrated a steep dose response curve, with most effects occurring only in the highest doses and occurring at or near lethal doses. Likely owing to its conversion to 2,4-DNP, an inhibitor of mitochondrial energy homeostasis, DNAN treatment resulted in an apparent increase in metabolism leading to reduced feed conversion efficiency and ultimately reduced body mass gain in males. Changes in hematology parameters indicative of anemia, splenic enlargement, hemosiderosis, and
extramedullary hematopoeisis indicate that the blood is a target organ for DNAN, with female rats being more sensitive to these effects than males. DNAN demonstrated testicular toxicity that, combined with the documented reproductive/developmental effects of its metabolite, 2,4-DNP, raises concern about the reproductive/developmental toxicity of DNAN. DNAN treatment resulted in progressive development of behavioral neurotoxicity as well as associated brain lesions.
Extramedullary hematopoeisis in female rats was identified as the critical endpoint in this study and was used to derive a BMDL10 of 2.3 mg/kg-day. This BMDL10 may be used for development of safe exposure levels.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
1.25 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Correct
System:
haematopoietic
Organ:
blood

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 - 25 July, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
Some rats have been sacrified with carbon dioxide inhalation instead of carbon dioxide/oxygen inhalation which is not considered to have affected the integrity of the study because insignificant difference between the 2 anesthesics.
3 animals inadvertently had their organs weighed at the time of sacrifice.Not considered have affected the integrity of the study because more data than necessary was recorded and these values were not used in the statistical evaluation of mean organ weights.
1 animal inadvertently had a macroscopic skin lesion. This was not required tissue. Not considered have affected the integrity of the study because more data
than required was recorded and approved by the Sponsor.
Deviations:
not applicable
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
Supplier : Thiokol Propulsion, 9160 North Hwy 83 Bldg M3, Corine, Utah 84307
Lot : 99-99-01 (trials), 114054 (trials), JH-1744-88 (exposures)
- Expiration date of the lot/batch: Not available
- Purity test date: >99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Kept tightly closed; stored in a cool dry place
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Albino rats Vaf/Plus
Sprague-Dawley-derived (CD)
Crl:CD (SD) IGS BR

The rat is an animal model commonly utilized in toxicity studies. In addition, a historical database is available for comparative evaluation.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Kingston, New York, 12484
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6 weeks
- Weight at study initiation:
Males = mean 187; Female = mean 149; Individual weights of animals were within +/-20% of the mean weight
- Fasting period before study: No
- Housing:
Housing during non-exposure period : in elevated, stainless steel, wire mesh cages, doubly-housed at receipt and during the first week of the acclimation period and all other non-exposure period
Housing during exposure period : Individually-housed in polycarbonate nose-only tubes attached to a cast aluminium and alloy 40 liter exposure chamber
- Diet and Water ad libitum during non-exposure period and not provided during exposure
- Acclimation period: 2 weeks from reception and 2 days prior to the first exposure and the day preceding the first exposure to the test substance, animals were acclimated to the nose-only cones and chambers for at least 2 hours and for at least 4 hours, respectively.

DETAILS OF FOOD AND WATER QUALITY:
Analytical certification of each feed lot used during the study was performed by the manufacturer.
Water analysis are conduted by Elizabethtown Water Company, to ensure that water meets standards specified under the EPA Federal Safe Drinking Water Act Regulations. In addition, water samples are collected biannually from representative rooms in the Testing facility; chemical and microbiological water analyses are conduted on these samples by a subcontratct laboratory.
No known contaminants in the feed or water were expected to have interfered with the results of this study.

ENVIRONMENTAL CONDITIONS
During non-exposure period, desired temperature = 18-26°C, Actual = 16-26°C
During exposure periods; desired temperature = 20-24°C, Actual = 16-30°C
During non-exposure period, desired Relative Humidity = 30-70%, Actual = 50-78%
During exposure periods; desired Relative Humidity = 40-60%, Actual = 10-61%

- Photoperiod (hrs dark / hrs light): 12-hour light/dark
Route of administration:
inhalation: mixture of vapour and aerosol / mist
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 1.6 - <= 2.8 µm
Geometric standard deviation (GSD):
1.8
Remarks on MMAD:
These results indicated that the test substance atmospheres were respirable in size to rats.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Tes test substance was mixed in an appropirately sized Erlenmeyer flask on a stirplate with a magnetic stir br with the vehicle (acetone).
The test substance mixture or the vehicle was fed directly via 1/8'' Teflon tubing from the flask, through a FMI Fluid Metering Pump with a 1/8'' piston and into an air atomizing nozzle. as a vapor/aerosol. House-line air was delivered from a regulator and backpressure gauge, via 1/4'' tubing and connected to a plastic Y tube taht split the airflow into the generation and dilution systems. The generation air (20Lpm) was directed, via 1/4" tubing, through a flowmeter regaulated by a metering valve, and a blackpressure gauge, to the inlet of the air atomizing nozzle to produce the aerosol.

- Temperature, humidity, pressure in air chamber:
During exposure periods; desired temperature = 20-24°C, Actual = 16-30°C
During exposure periods; desired Relative Humidity = 40-60%, Actual = 10-61%

- Method of particle size determination: TSI Aerodynamic Particle Sizer
- Treatment of exhaust air: The chamber were exhausted through a system consisting of a coarse filter, a HEPA filter and an activated charcoal bed.

TEST ATMOSPHERE
- Brief description of analytical method used: Gravimetric analysis of the airborne aerosol concentration for each test substance exposure level and HPLC analysis

VEHICLE: Acetone
- Lot/batch no. of vehicle (if required): 2079, T06253 and N23293
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Check table Doses/Concentrations below.
Exposure levels were determined analytically 4 times per exposure.

The analytically measured levels of airbone test substance were reasonably close to the target exposure levels.
These exposures were essentially an aerosol exposure with a minor vapor component.
Duration of treatment / exposure:
2 weeks
Frequency of treatment:
6 hours per day, 5 day per week, total of 11 exposures
Dose / conc.:
150 mg/m³ air
Remarks:
Target concentration
Dose / conc.:
165 mg/m³ air (analytical)
Dose / conc.:
500 mg/m³ air
Remarks:
Target concentration
Dose / conc.:
545 mg/m³ air (analytical)
Dose / conc.:
1 500 mg/m³ air
Remarks:
Target concentration
Dose / conc.:
1 313 mg/m³ air (analytical)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Exposure levels were Sponsor specified based on the results of an Acute Inhalation Study. The high exposure level was intented to exceed the limit test of 1000 mg/m3 of the US EPA in OPPTS 870.3465 for subchronic inhalation toxicity study
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: NA
Positive control:
A group of rats received Acetone only.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS and VIABILITY: Yes
- Time schedule: Once in the morning and once in the afternoon
Animals that were in poor health or in a possible moribund condition were identified for further monitoring and possible euthanasia.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once during each exposure
Observations for signs of toxic or pharmacological effects.
- Cage side observations checked in table were included in the attached report of the whole study.

BODY WEIGHT: Yes
- Time schedule for examinations: Twice pretest, weekly during the study and fasted jsut prior necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

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: Not specified

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND COAGULATION : Yes
- Anaesthetic used for blood collection: A lightly anesthesized (CO2/O2) via puncture of the orbital sinus (retrobulbar).
- Animals fasted: Yes overnight
- How many animals: All animals surviving to terminal necropsy
- Parameters checked in table in that attached study report were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: A lightly anesthesized (CO2/O2) via venipuncture of the dorsal aorta
- Animals fasted: Yes overnight
- How many animals: All animals surviving to terminal necropsy
- Parameters checked in table in the attached study report were examined.

URINALYSIS: Yes
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes, overnight, 16 hours
- Animals fasted: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: twice pretest and once weekly
- Dose groups that were examined: All animals
- Battery of functions tested: general condition, skin and fur, eyes, nose, oral activity, abdomen and external genitalia, occurence of secretions and excretions and autonomic activity; changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypy or bizarre bahavior.

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
A complete macroscopic postmortem examination was performed on all animals, including animals euthanized prior to study termination of found dead.
Animals were fasted prior to the scheduled sacrifice.

HISTOPATHOLOGY: Yes
Animals showing signs of severe debility, particularly if death appeared imminent, were euthanized to prevent loss of tissue through autolysis.
Terminal necropsies were performed on 5 animals/sex for Groups 1 and 2 and 1 animal/sex for Group 3.

TISSUE/MICROSCOPIC EVALUATION : Yes for all animals in Group 1 and 2 and for all animals in Group 3 that survived until terminal sacrifice. Slides of tissues indicated in the table in the attached study report were prepared and examinated.

METHOD OF EUTHANASIA : Animals were exsanguinated following carbon dioxide/oxygen inhalation except animals euthanized on 13 July 2000 were exsanguinated carbon dioxide inhalation.
Other examinations:
ORGAN WEIGHTS
Organs indicated in the table in the attached study report were taken from all survivors at the scheduled necropsy, weighted, recorded and organ/body and organ/brain weight ratios calculated.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The only-treatment related responses during the exposures were labored breathing and decreased activity noted in the 500 mg/m3 exposure group beginning with the third exposure.

Signs of toxicity noted during the morning or afternoon viability checks during the 2-week exposure period included decreased fecal volume, decreased feed consumption, prostration, yellow stains on the ventral surface, red nasal discharge, irregular gait, lethargy, head bobbing, poor conditions, pale, blackwards walking and labored breathing. These signs were seen most frequently in the 500 and 1500 mg/m3 exposed animals preceding the animals being euthanized or found dead.
Yellow stains on the ventral surface were also frequently noted in the 150 mg/m3 exposed animals.
Irregular gait was also occasionally noted during the afternoon viability checks in the acetone contro and 150 mg/m3 exposed animals; this was probably a vehicle (cns depression) effect.
Clinical signs seen in the 150 mg/m3 group were considered to be of no toxicological importance.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were 18 unscheduled deaths during the study.
1 of the 1500 mg/m3 exposed female was found dead on the morning after the second exposure and ALL of the remaining animals of this group were sacrified in poor health on that day thus terminating this exposure level early.
4 of 5 males and 4 of 5 females exposed to 500 mg/mg3 were euthanized in a moribund conditions or found dead during the 2 weeks of exposure.
2 animals (1 by sex) from this test group survived to study termination.
All animals in the control and 150 mg/m3 groups survived to study termination


Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically meaningful differences in BW in the 150 mg/m3 exposed animals compared to the Acetone control animals.
The 500 mg/m3 exposed animals showed significant less weight gain than the Acetone control animals during the first week of exposure.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A significant decrease in feed consumption (grams/animals/day and grams/kg/day) was noted in the 500 mg/m3 exposed female animals during the first week of exposure.
A statistically significant increase in feed consumption was noted in the 150 mg/m3 exposed male animals during the second week of exposures. However an increase in feed consumption is not considered of toxicological significance
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
There were statistically significant decreases in 150 mg/m3 exposed females for hemoglobin concentration, mean copuscular volume and mean corpuscular hemoglobin and a statistically significant increase un absolute monocytes. HOWEVER, the absolute differences were minimal in the males and were not seen as statistically significant in the males.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
A possible toxicologically meaningful decrease (statistically significant in males) in blood urea nitrogen was noted in the 150 mg/m3 exposed animals.
Urinalysis findings:
no effects observed
Description (incidence and severity):
A possible toxicologically meaningful difference unrine color was noted in the 150 mg/m3 exposed animals. Most of these animals were noteed with yellow urine compared to Acetone control animals which were mostly noted with straw colord urine.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Signs of toxicity included irregular gait, decreased activity, lethargy, head bobbing and labored breathing.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were statistically significant increases in absolute kidney weight for the 150 mg/m3 exposed males and for absolute kidney weights for the V=150 mg/m3 exposed females. HOWEVER, the absolute differences were minimal, were not seen as statistically significant in the other sex and there were no microscopic findings associated.
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The only test-related microscopic finding was in the LARYNX at the level of the ventral seromucous glands.
This finding considered to be a non-specific direct local effect of the test substance.
Other findings in the larynx and in the other tissues and organs occurred with comparable incidence and severity in the Acetone and 150 mg/m3 groups or they occurred sporadically..
These incidental findings have been seen in the rats of this strain and age used in similar studies conducted in this facility. Squamous metaplasia is a common finding in the rat larynx since the area is very sensitive to insult and is usually an adaptive response and reversible
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 165 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/m³ air
System:
other: Clinical signs and mortality
Organ:
other: clinical signs and mortality
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

NA

Conclusions:
165 mg/m3 was determined to be the NOAEL (analytically determined concentration, whereas the target concentration was 150 mg/m3)
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
165 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
Correct
System:
other: clinical signs and mortality
Organ:
not specified

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 - 25 July, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
Some rats have been sacrified with carbon dioxide inhalation instead of carbon dioxide/oxygen inhalation which is not considered to have affected the integrity of the study because insignificant difference between the 2 anesthesics.
3 animals inadvertently had their organs weighed at the time of sacrifice.Not considered have affected the integrity of the study because more data than necessary was recorded and these values were not used in the statistical evaluation of mean organ weights.
1 animal inadvertently had a macroscopic skin lesion. This was not required tissue. Not considered have affected the integrity of the study because more data
than required was recorded and approved by the Sponsor.
Deviations:
not applicable
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
Supplier : Thiokol Propulsion, 9160 North Hwy 83 Bldg M3, Corine, Utah 84307
Lot : 99-99-01 (trials), 114054 (trials), JH-1744-88 (exposures)
- Expiration date of the lot/batch: Not available
- Purity test date: >99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Kept tightly closed; stored in a cool dry place
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Albino rats Vaf/Plus
Sprague-Dawley-derived (CD)
Crl:CD (SD) IGS BR

The rat is an animal model commonly utilized in toxicity studies. In addition, a historical database is available for comparative evaluation.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Kingston, New York, 12484
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6 weeks
- Weight at study initiation:
Males = mean 187; Female = mean 149; Individual weights of animals were within +/-20% of the mean weight
- Fasting period before study: No
- Housing:
Housing during non-exposure period : in elevated, stainless steel, wire mesh cages, doubly-housed at receipt and during the first week of the acclimation period and all other non-exposure period
Housing during exposure period : Individually-housed in polycarbonate nose-only tubes attached to a cast aluminium and alloy 40 liter exposure chamber
- Diet and Water ad libitum during non-exposure period and not provided during exposure
- Acclimation period: 2 weeks from reception and 2 days prior to the first exposure and the day preceding the first exposure to the test substance, animals were acclimated to the nose-only cones and chambers for at least 2 hours and for at least 4 hours, respectively.

DETAILS OF FOOD AND WATER QUALITY:
Analytical certification of each feed lot used during the study was performed by the manufacturer.
Water analysis are conduted by Elizabethtown Water Company, to ensure that water meets standards specified under the EPA Federal Safe Drinking Water Act Regulations. In addition, water samples are collected biannually from representative rooms in the Testing facility; chemical and microbiological water analyses are conduted on these samples by a subcontratct laboratory.
No known contaminants in the feed or water were expected to have interfered with the results of this study.

ENVIRONMENTAL CONDITIONS
During non-exposure period, desired temperature = 18-26°C, Actual = 16-26°C
During exposure periods; desired temperature = 20-24°C, Actual = 16-30°C
During non-exposure period, desired Relative Humidity = 30-70%, Actual = 50-78%
During exposure periods; desired Relative Humidity = 40-60%, Actual = 10-61%

- Photoperiod (hrs dark / hrs light): 12-hour light/dark
Route of administration:
inhalation: mixture of vapour and aerosol / mist
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 1.6 - <= 2.8 µm
Geometric standard deviation (GSD):
1.8
Remarks on MMAD:
These results indicated that the test substance atmospheres were respirable in size to rats.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Tes test substance was mixed in an appropirately sized Erlenmeyer flask on a stirplate with a magnetic stir br with the vehicle (acetone).
The test substance mixture or the vehicle was fed directly via 1/8'' Teflon tubing from the flask, through a FMI Fluid Metering Pump with a 1/8'' piston and into an air atomizing nozzle. as a vapor/aerosol. House-line air was delivered from a regulator and backpressure gauge, via 1/4'' tubing and connected to a plastic Y tube taht split the airflow into the generation and dilution systems. The generation air (20Lpm) was directed, via 1/4" tubing, through a flowmeter regaulated by a metering valve, and a blackpressure gauge, to the inlet of the air atomizing nozzle to produce the aerosol.

- Temperature, humidity, pressure in air chamber:
During exposure periods; desired temperature = 20-24°C, Actual = 16-30°C
During exposure periods; desired Relative Humidity = 40-60%, Actual = 10-61%

- Method of particle size determination: TSI Aerodynamic Particle Sizer
- Treatment of exhaust air: The chamber were exhausted through a system consisting of a coarse filter, a HEPA filter and an activated charcoal bed.

TEST ATMOSPHERE
- Brief description of analytical method used: Gravimetric analysis of the airborne aerosol concentration for each test substance exposure level and HPLC analysis

VEHICLE: Acetone
- Lot/batch no. of vehicle (if required): 2079, T06253 and N23293
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Check table Doses/Concentrations below.
Exposure levels were determined analytically 4 times per exposure.

The analytically measured levels of airbone test substance were reasonably close to the target exposure levels.
These exposures were essentially an aerosol exposure with a minor vapor component.
Duration of treatment / exposure:
2 weeks
Frequency of treatment:
6 hours per day, 5 day per week, total of 11 exposures
Dose / conc.:
150 mg/m³ air
Remarks:
Target concentration
Dose / conc.:
165 mg/m³ air (analytical)
Dose / conc.:
500 mg/m³ air
Remarks:
Target concentration
Dose / conc.:
545 mg/m³ air (analytical)
Dose / conc.:
1 500 mg/m³ air
Remarks:
Target concentration
Dose / conc.:
1 313 mg/m³ air (analytical)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Exposure levels were Sponsor specified based on the results of an Acute Inhalation Study. The high exposure level was intented to exceed the limit test of 1000 mg/m3 of the US EPA in OPPTS 870.3465 for subchronic inhalation toxicity study
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: NA
Positive control:
A group of rats received Acetone only.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS and VIABILITY: Yes
- Time schedule: Once in the morning and once in the afternoon
Animals that were in poor health or in a possible moribund condition were identified for further monitoring and possible euthanasia.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once during each exposure
Observations for signs of toxic or pharmacological effects.
- Cage side observations checked in table were included in the attached report of the whole study.

BODY WEIGHT: Yes
- Time schedule for examinations: Twice pretest, weekly during the study and fasted jsut prior necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

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: Not specified

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND COAGULATION : Yes
- Anaesthetic used for blood collection: A lightly anesthesized (CO2/O2) via puncture of the orbital sinus (retrobulbar).
- Animals fasted: Yes overnight
- How many animals: All animals surviving to terminal necropsy
- Parameters checked in table in that attached study report were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: A lightly anesthesized (CO2/O2) via venipuncture of the dorsal aorta
- Animals fasted: Yes overnight
- How many animals: All animals surviving to terminal necropsy
- Parameters checked in table in the attached study report were examined.

URINALYSIS: Yes
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes, overnight, 16 hours
- Animals fasted: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: twice pretest and once weekly
- Dose groups that were examined: All animals
- Battery of functions tested: general condition, skin and fur, eyes, nose, oral activity, abdomen and external genitalia, occurence of secretions and excretions and autonomic activity; changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypy or bizarre bahavior.

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
A complete macroscopic postmortem examination was performed on all animals, including animals euthanized prior to study termination of found dead.
Animals were fasted prior to the scheduled sacrifice.

HISTOPATHOLOGY: Yes
Animals showing signs of severe debility, particularly if death appeared imminent, were euthanized to prevent loss of tissue through autolysis.
Terminal necropsies were performed on 5 animals/sex for Groups 1 and 2 and 1 animal/sex for Group 3.

TISSUE/MICROSCOPIC EVALUATION : Yes for all animals in Group 1 and 2 and for all animals in Group 3 that survived until terminal sacrifice. Slides of tissues indicated in the table in the attached study report were prepared and examinated.

METHOD OF EUTHANASIA : Animals were exsanguinated following carbon dioxide/oxygen inhalation except animals euthanized on 13 July 2000 were exsanguinated carbon dioxide inhalation.
Other examinations:
ORGAN WEIGHTS
Organs indicated in the table in the attached study report were taken from all survivors at the scheduled necropsy, weighted, recorded and organ/body and organ/brain weight ratios calculated.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The only-treatment related responses during the exposures were labored breathing and decreased activity noted in the 500 mg/m3 exposure group beginning with the third exposure.

Signs of toxicity noted during the morning or afternoon viability checks during the 2-week exposure period included decreased fecal volume, decreased feed consumption, prostration, yellow stains on the ventral surface, red nasal discharge, irregular gait, lethargy, head bobbing, poor conditions, pale, blackwards walking and labored breathing. These signs were seen most frequently in the 500 and 1500 mg/m3 exposed animals preceding the animals being euthanized or found dead.
Yellow stains on the ventral surface were also frequently noted in the 150 mg/m3 exposed animals.
Irregular gait was also occasionally noted during the afternoon viability checks in the acetone contro and 150 mg/m3 exposed animals; this was probably a vehicle (cns depression) effect.
Clinical signs seen in the 150 mg/m3 group were considered to be of no toxicological importance.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were 18 unscheduled deaths during the study.
1 of the 1500 mg/m3 exposed female was found dead on the morning after the second exposure and ALL of the remaining animals of this group were sacrified in poor health on that day thus terminating this exposure level early.
4 of 5 males and 4 of 5 females exposed to 500 mg/mg3 were euthanized in a moribund conditions or found dead during the 2 weeks of exposure.
2 animals (1 by sex) from this test group survived to study termination.
All animals in the control and 150 mg/m3 groups survived to study termination


Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically meaningful differences in BW in the 150 mg/m3 exposed animals compared to the Acetone control animals.
The 500 mg/m3 exposed animals showed significant less weight gain than the Acetone control animals during the first week of exposure.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A significant decrease in feed consumption (grams/animals/day and grams/kg/day) was noted in the 500 mg/m3 exposed female animals during the first week of exposure.
A statistically significant increase in feed consumption was noted in the 150 mg/m3 exposed male animals during the second week of exposures. However an increase in feed consumption is not considered of toxicological significance
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
There were statistically significant decreases in 150 mg/m3 exposed females for hemoglobin concentration, mean copuscular volume and mean corpuscular hemoglobin and a statistically significant increase un absolute monocytes. HOWEVER, the absolute differences were minimal in the males and were not seen as statistically significant in the males.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
A possible toxicologically meaningful decrease (statistically significant in males) in blood urea nitrogen was noted in the 150 mg/m3 exposed animals.
Urinalysis findings:
no effects observed
Description (incidence and severity):
A possible toxicologically meaningful difference unrine color was noted in the 150 mg/m3 exposed animals. Most of these animals were noteed with yellow urine compared to Acetone control animals which were mostly noted with straw colord urine.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Signs of toxicity included irregular gait, decreased activity, lethargy, head bobbing and labored breathing.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were statistically significant increases in absolute kidney weight for the 150 mg/m3 exposed males and for absolute kidney weights for the V=150 mg/m3 exposed females. HOWEVER, the absolute differences were minimal, were not seen as statistically significant in the other sex and there were no microscopic findings associated.
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The only test-related microscopic finding was in the LARYNX at the level of the ventral seromucous glands.
This finding considered to be a non-specific direct local effect of the test substance.
Other findings in the larynx and in the other tissues and organs occurred with comparable incidence and severity in the Acetone and 150 mg/m3 groups or they occurred sporadically..
These incidental findings have been seen in the rats of this strain and age used in similar studies conducted in this facility. Squamous metaplasia is a common finding in the rat larynx since the area is very sensitive to insult and is usually an adaptive response and reversible
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 165 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/m³ air
System:
other: Clinical signs and mortality
Organ:
other: clinical signs and mortality
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

NA

Conclusions:
165 mg/m3 was determined to be the NOAEL (analytically determined concentration, whereas the target concentration was 150 mg/m3)
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
subacute
Species:
rat
Quality of whole database:
Correct

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

Justification for classification or non-classification

In a 2 -week inhalation toxicity study, performed according to OECD guidelines 402, Sprague-Dawley CD rats (5/sex/group) were exposed nose-only 6 hours by day, 5 days per week for 2 weeks, to 2,4 -Dinitroanisole. The test substance was administrated (using Acetone as vehicle) at target concentrations of 150, 500 and 1500 mg/m3 of airborne vapor/aerosol. In addition, a control group (5/sex) received Acetone only.

These exposures were essentially an aerosol exposure with a minor vapor component. The test substance aerosol atmospheres were of a respirable size for the test animals with an average MMAD of 2,1um and an average GSD of 1,8. The nominals concentrations were generally comparable between groups thus indicating that each group was exposed to a similar level of Acetone.

In conclusion, a 2 -week nose-only inhalation exposure of rats to 150, 500 and 1500 mg/m3 of DNAN resulted in test substance-related mortality at the higher exposure levels, clinical effects predominantly at the higher exposure levels. With regards to the laryngeal changes seen at 150 mg/m3   - Squamous metaplasia is a common finding in the rat larynx since the area is very sensitive to insult and is usually an adaptive response and reversible.  The one male from the 500 mg/m3group did not have the finding (animal reported as NAD) and in the female examined there was no increase in severity.  This finding is not considered to be adverse.  In addition, since this type of response is adaptive, and the rat larynx is very sensitive it is usually not considered to be indicative of significant human risk.

 The NOAEC is considered to be 165 mg/m3.

In a 90-day oral repeated dose toxicity study, performed according to the OPPTS 870.3100 guidelines and GLP-compliance, 10 rats Sprague-Dawley of each sex were dosed via oral gavage at 0, 1, 2.5, 5, 20 and 80mg/kg of bw of DNAN, approximately the same time daily, 7 days per week, for 90 days. A vehicle control group of animals were dosed with corn oil at the same volume (5mL/kg) per body mass as the DNAN exposed animals.

Likely owing to its conversion to 2,4 -DNP, an inhibitor of mitochondrial energy homeostasis, DNAN treatment caused an apparent increase in metabolism, leading to reduced feed conversion efficiency and reduced body mass gain in males.

Anemia, splenic enlargement, hemosiderosis and extramedullary hematopoeisis (EMH) indicated that the blood as a target orgna for DNAN, with females being more sensitive than males.

DNAN was a testicular toxicant, causing decreased mass of the testes and epididymides, as well as degeneration and atrophy of the testicular seminiferous tubules and epididymal aspermia.

Stereotypical behavior in males, gait irregularities, and cerebellar glial lesions indicated that DNAN is neurotoxic.

LOAEL in rats was found to be 20 mg/kg bw/day.