Registration Dossier

Administrative data

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
September 2010 - March 2011
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
other: OPPTS 870.3100
GLP compliance:
yes (incl. certificate)
Type of method:
in vivo

Test material

Reference
Name:
Unnamed
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:

Test animals

Species:
rat
Strain:
Sprague-Dawley
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:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on 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.
Doses / concentrationsopen allclose all
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:
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: 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.

Results and discussion

Effect levels

Dose descriptor:
conc. level:
Effect level:
>= 80 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic

Observed effects

Organ weight findings
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)

Gross pathology findings:
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

Histopathological findings :
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.

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 in sperm per gram, percent motile sperm, or percent progressively motile sperm were observed in the 1.25 , 5, or 20 mg/kg-d dose groups.

Applicant's summary and conclusion

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.
Decreased mass of the testes and epididymides as well as degeneration and atrophy of the testicular seminiferous tubulesand aspermia were observed in rats from the 80 mg/kg-day group.
As discussed in the repeated dose toxicity study endpoint, 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.
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.
Executive summary:

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, 25, 5, 20 and 80mg/kg of bw of DNAN, approximately the same time daily, 7 days per week, for 90 days. A vehicule control group of animals was 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 os 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.

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

A BMDL10 of 2,3 mg/kg-day was derived based on the EMH in females.