Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant study conduct in accordance with international guidelines.
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproductive/Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Principles of method if other than guideline:
PROTOCOL DEVATIONS:
The following protocol deviation occurred during the course of the study. All deviations were
considered minimal and had no impact on the integrity of the study.
1. Careful clinical observations were not obtained for female Number 401 in the
100 mg/kg/day group on Test Day 21 (8/23/05).
2. Careful clinical observations for all animals were performed 1 hour later than required by
the protocol on Test Day 24 (8/26/05).
3. At one observation period during the acclimation period, the humidity in the animal room
was 72%, which was 2% higher than the protocol-required range.
4. Female Number 604 in the 300 mg/kg/day group was dosed from gestation day 0 to 6
with 0.1 mL more per day than required (i.e., received 1.0 mL/day instead of
0.9 mL/day).
5. Careful clinical observations were recorded at the time of dosing instead of
approximately 2 hours postdosing on Test Day 8 (8/10/05) and Test Day 25 (8/27/05).
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
N,N-dimethyl-3-oxobutyramide
EC Number:
218-059-8
EC Name:
N,N-dimethyl-3-oxobutyramide
Cas Number:
2044-64-6
Molecular formula:
C6H11NO2
IUPAC Name:
N,N-dimethyl-3-oxobutyramide
Constituent 2
Reference substance name:
N,N-dimethylacetoacetamide (DMAA)
IUPAC Name:
N,N-dimethylacetoacetamide (DMAA)
Test material form:
other: liquid
Details on test material:
Test material name: N,N-dimethylacetoacetamide (DMAA)

Test animals

Species:
rat
Strain:
other: CrI:CD(SD)IGS BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
On July 26, 2005, 45 male and 46 female Crl:CD®(SD)IGS BR rats, with an assigned birth date of June 3, 2005, were received from
Charles River Laboratories, Inc., Raleigh, North Carolina for use on this study. The rats were approximately 53 days old upon
arrival and weighed in the range of 207.6 - 232.8 grams (males) and 167.7 - 199.0 grams (females) the day after arrival.
Male and female rats were nonsiblings; females were nulliparous.
The rats were approximately 59 days old at the start of treatment (August 3, 2005), and weighed in the range of 263.7 – 305.4 grams (males)
and 190.9 – 227.5 grams (females).
HOUSING:
All male rats were housed individually during non-mating periods in stainless steel, wire-mesh cages suspended above cageboards.
TEMPERATURE:
Animal rooms were maintained at an acceptable temperature of 18-26ºC.
HUMIDITY:
Animal rooms were maintained at an acceptable relative humidity of 30%-70%.
LIGHTING:
Animal rooms were artificially illuminated (fluorescent light) on a 12-hour light/dark cycle (approximately 0600-1800 hours).
FEED AND WATER:
All rats were fed pelleted PMI® Nutrition International, Certified Rodent LabDiet® 5002 ad libitum, except when fasted.
Tap water from United Water Delaware was provided ad libitum.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
NanoPure water
Details on oral exposure:
TEST FORMULATION PREPARATION:
Formulations of the test substance in the vehicle (NanoPure® water) were prepared daily until 7-day test formulation stability
was confirmed; thereafter, dose preparation was performed weekly and stored refrigerated until used.
The method of mixing the test substance with the vehicle was documented in the study records.
ADMINISTRATION:
The test substance was administered once daily by oral intubation (gavage) the route recommended by test guidelines.
The volume of test substance or vehicle given to each rat was based on the most recently recorded body weight.
The dose volume was 4 mL/kg. Male and female control rats were similarly treated with the vehicle at the same dose volume as used in the
other groups.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test Substance Stability/Analytical Content:
---------------------------------------
The stability of the neat test substance was confirmed by analysis of the bulk sample for concentration of DMAA in the aqueous solution
conducted near the beginning of the study by Exygen Research. A sample from the bulk test substance received at Haskell Laboratory was
shipped from the Laboratory to Exygen Research for analysis. The Certificate of analysis provided by Exygen indicated that the Analytical
content of DMAA in the aqueous solution was reported to be 78.5%. [Note: although identified as “purity” in the attached documents, the
value obtained is the analytical content of DMAA in the aqueous solution].

Test Substance Formulation Sampling:
----------------------------------
Two sets of samples were collected from test substance formulations, at all concentration levels, on the first day of dosing formulation
preparation. Analyses addressed concentration, uniformity of mixing, and stability.
• The first set of samples was analyzed immediately to verify mixing uniformity/concentration and 5-hour room temperature stability
of the test substance in the formulation.
• The second set of samples was held refrigerated for 7 days and analyzed for 7-day refrigerated stability and 7-day refrigerated,
5-hours at room temperature stability.
One set of samples was collected from test substance formulations, at all concentration levels, during the second week on test (test day 10 [8/12/05]) and then again near the end of the study (test day 31 [9/2/05] and test day 52 [9/23/05]). Analyses addressed concentration verification/
uniformity of mixing.
Duration of treatment / exposure:
DOSING SCHEDULE:
• All animals were dosed once daily by gavage for at least 2 weeks (14 days) prior to cohabitation and during the cohabitation period (up to 2 weeks).
• Male rats and female rats showing no evidence of copulation were dosed after the end of the cohabitation period until the day before sacrifice.
• Females showing evidence of copulation were dosed throughout gestation.
• Pregnant females in the process of delivery or showing signs of delivery were not dosed.
• Females were dosed after delivering litters, until day 3 postpartum.
• Females that did not deliver a litter continued to be dosed until the day before sacrifice.
Frequency of treatment:
Animals were dosed daily 7 days a week.
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg b.w./day
Basis:
actual ingested
No. of animals per sex per dose:
Ten males and ten females per dose level.
Control animals:
yes, concurrent vehicle
Details on study design:
In a previous 14-day range-finding study,(1) groups of male and female rats (5/group) were administered the test substance
once daily by gavage for 14 consecutive days at a volume of 4 mL/kg at dosages of 0, 100, 300, or 1000 mg/kg bw/day.
There were no test substance-related effects on body weight, food consumption, clinical signs of toxicity, mortality, or gross
morphology. Based on these results, the selected doses are 0, 100, 300, and 1000 mg/kg bw/day for the current study.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
MORTALITY/VIABILITY OBSERVATIONS:
Cage-site examinations to detect moribund or dead animals and abnormal behavior and/or appearance were performed on all
animals at least once daily during quarantine and pretest, and twice daily thereafter.

CLINICAL OBSERVATIONS:
After initiation of dosing, careful clinical observations to detect acute/systemic toxicity were performed on all animals daily at
approximately the same time of day after dosing (+ 2 hours).

BODY WEIGHT:
All selected P1 rats were weighed during Functional Observational Battery (FOB) assessments. All P1 rats were weighed at terminal
sacrifice.

FOOD CONSUMPTION:
Each feeder was weighed at the beginning and end of the weekly food consumption interval and the final weight of the feeder and
the amount of spillage from the feeder during the interval wassubtracted from the initial feeder weight. From the food consumption
and body weight data, the mean daily food efficiency was calculated.

NEUROBEHAVIOURAL EXAMINATION:
On test day 13, an abbreviated functional observational battery (FOB) and motor activity were conducted on the last 5 rats/sex/group.
The FOB and MA assessments were conducted in 2 replicates over a one-day period. Assignment to a given replicate was counterbalanced
across all groups. Replicate designation was not reported in the final report, but was recorded in the study records.

HAEMATOLOGY AND COAGULATION:
Complete blood counts, including reticulocytes, were determined on a Bayer® Advia 120 hematology analyzer or determined from
microscopic evaluation of the blood smear. Wright’s stained blood smears from all hematology samples were examined microscopically
for confirmation of automated results and evaluation of cellular morphology. Blood smears, stained with new methylene blue, were prepared
from each animal undergoing a hematology evaluation, but were not needed for examination. Coagulation times were determined on a
Sysmex® CA-1000 Coagulation Analyzer.

CLINICAL CHEMISTRY:
Serum clinical chemistry parameters were determined on an Olympus® AU640 clinical chemistry analyzer.


Sacrifice and pathology:
Clinical Pathology Evaluation:
--------------------------
A clinical pathology evaluation was conducted on 5 rats/sex/group once before the cohabitation period on test day 14
(hematology and clinical chemistry) and test days 55 and 42 – 55 (coagulation for males and females, respectively).
The day before collection of samples for the clinical pathology evaluation (hematology, clinical chemistry), the animals were
fasted after 3 p.m. for at least 15 hours. Blood samples for hematology and clinical chemistry measurements were collected from the orbital
sinus of each animal while the animal was under carbon dioxide anesthesia. Blood samples for coagulation parameters were collected at sacrifice
from the abdominal vena cava of each animal while the animal was under carbon dioxide anesthesia. Additional blood collected from the vena cava
was placed in a serum tube, processed to serum, and frozen at approximately -80°C. Serum was not analyzed because further tests were not
required to support experimental findings. Bone marrow smears were prepared at sacrifice from all surviving animals. Bone marrow smears were
stained with Wright’s stain, but analysis was not necessary to support experimental findings. All blood samples were evaluated for quality by
visual examination. Results were maintained in the study records and reported only if the sample was analyzed.
Other examinations:
No other examinations.
Statistics:
Male and female parental data were evaluated separately. If the preliminary test was not significant, then a repeated measures ANOVA was performed followed by Linear contrasts. If the preliminary test was significant, then the sequential application of the Jonckheere-Terpstra trend test was performed.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There was no test substance-related mortality during the study. There were no test substancerelated increased incidences of any clinical observations.
Mortality:
no mortality observed
Description (incidence):
There was no test substance-related mortality during the study. There were no test substancerelated increased incidences of any clinical observations.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related reductions in body weight and body weight gain were observed at 300 and 1000 mg/kg bw/day and were considered adverse.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no test substance-related effects on food consumption or food efficiency at any dose level tested.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Some general increases in hematologic parameters (reticulocytes and echinocytes) were observed, but were considered not to be treatment related as there was no accompanying increase in red cell mass parameters.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Cholesterol was minimally to mildly increased in males and females dosed with 1000 mg/kg bw/day (statistically significant only in males). This change was considered to be potentially adverse in male rats.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no test substance-related effects on any neurobehavioral parameter evaluated in either males or females.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
A test substance-related increase in mean absolute and relative (% body weight) liver (1000 mg/kg bw/day) and kidney (≥100 mg/kg bw/day) weights was observed in male rats. Organ weight effects were not observed in female rats.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Daily gavage administration of 1000 mg/kg bw/day of the test substance to male and female rats resulted in adverse increases in serum cholesterol concentrations in male rats. Increased hyaline droplets in males.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Non-adverse histopathological effects were limited to minimal to mild hepatocellular hypertrophy in males at > 300 mg/kg/day which was attributed to enzyme induction.
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
Male and Female rats: There was no test substance-related mortality during the study. There were no test substance related
increased incidences of any clinical observations.

BODY WEIGHT AND WEIGHT GAIN
Male rats: Test substance-related reductions in body weight and body weight gain were observed at 300 and 1000 mg/kg bw/day and were
considered adverse. Body weight gain for the entire dosing period (days 1-55) and the post-cohabitation period (days 15-55) at 300
and 1000 mg/kg bw/day were 15%-20% lower than controls, respectively; this was due to lower weekly body weight gain during
most of the weekly intervals, beginning during the third week of dosing (days 15-22). Accordingly, weekly body weights at 300 and 1000 mg/kg bw/day were 6%-8% lower than controls on test day 29 and thereafter.
For female rats, there were no adverse test substance-related effects on body weight during premating, gestation or lactation at any dose level.

FOOD CONSUMPTION
Male and Female rats: There were no test substance-related effects on food consumption or food efficiency at any dose level tested.

HAEMATOLOGY
There were no adverse changes in hematologic parameters in male or female rats. The following changes in mean hematologic parameters
were not adverse or not related to treatment:
Reticulocytes were minimally increased in males dosed with 1000 mg/kg bw/day (statistically significant). However, red cell mass parameters
(red blood cell count, hemoglobin, and hematocrit) were similar across all groups. In addition, there were no histologic changes in bone
marrow or spleen and all individual reticulocyte counts were within the 95% reference interval for similarly aged rats. The increased reticulocyte
counts were unlikely to be due to treatment. Since there was no primary effect on red cell mass, this change, if treatment-related, was considered to
be non-adverse.
Echinocytes (red blood cells with small surface projections) were observed in greater numbers per blood smear in blood from males dosed with
1000 mg/kg bw/day, and from females dosed with ≥100 mg/kg bw/day. Because echinocytes were not associated with changes in red
cell mass parameters, they were considered to be non-adverse.
Platelet counts were minimally increased in females dosed with 300 mg/kg bw/day. This change was considered to be unrelated to treatment and
non-adverse because it did not occur in a dose-related pattern.

CLINICAL CHEMISTRY
Cholesterol was minimally to mildly increased in males and females dosed with 1000 mg/kg bw/day (statistically significant only in males).
Means were 181 and 138% of control group means for males and females, respectively. This change was likely to be related to treatment.
Individual concentrations of 4 of 5 males and 2 of 5 females were above the upper limit of the 95% reference interval for similarly aged rats.
Due to the consistency and magnitude of the changes in male rats, this change was considered to be potentially adverse in male rats.
There were no other treatment-related changes in clinical chemistry parameters in male or female rats. The following statistically significant
change in the mean clinical chemistry parameter was not adverse or not related to exposure to the test substance because it did not occur in
a doserelated pattern:
• Increased sodium concentration in males dosed with 300 mg/kg bw/day.

NEUROBEHAVIOUR
There were no test substance-related effects or statistically significant differences on forelimb or hindlimb grip strength in either males or females administered any dosage of the test substance. Motor Activity, there were no test substance-related effects or statistically differences on duration of
movement or number of movements in males or females administered any dosage of the test substance.There were no test substance-related effects or statistically significant differences for any behavioral parameter evaluated in males or females administered any dosage of the test substance.

ORGAN WEIGHTS
A test substance-related increase in mean absolute and relative (% body weight) liver (1000 mg/kg bw/day) and kidney (≥100 mg/kg bw/day) weightswas observed in male rats. Organ weight effects were not observed in female rats.
LIVER:
In high-dose males (1000 mg/kg bw/day), mean absolute and relative (% body weight) liver weights were increased 21% and 32%, respectively, as
compared to the control values. The increases were statistically significant. Liver weight parameters were not increased in females.
The increased liver weights correlated with minimal to mild centrilobular hepatocellular hypertrophy observed microscopically in the high-dose
males. A lesser degree of hypertrophy was observed in the 300 mg/kg bw/day males and the 1000 mg/kg bw/day females, without any
corresponding liver weight increases. Both the organ weight and morphological changes were regarded as indicative of non-adverse enzyme
induction.
KIDNEYS:
In males, mean absolute kidney weights were increased 9%, 6%, and 15% in the 100, 300, and 1000 mg/kg bw/day dose groups, respectively, as
compared to control values. Mean relative (% body weight) kidney weights were increased 14%, 15%, and 25% in the 100, 300, and
1000 mg/kg bw/day dose group, respectively. All increases were statistically significant except for the 9% and 6% increases in absolute weight.
Kidney weights were not increased in females. The increase in male kidney weights at all doses was interpreted to likely be the result of both
pharmacological enzyme induction (≥100 mg/kg bw/day) and the increased in tubular hyaline droplets (≥300 mg/kg bw/day). Due to the lack of a
dose response at 100 and 300 mg/kg bw/day and the minimal magnitude of change, a clear effect of treatment could not be established and the
differences were considered not adverse. As in liver hepatocytes, xenobiotics may induce P450 microsomal enzymes in the renal proximal
tubular epithelium. Increased kidney weights often precede morphological changes (i.e., hypertrophy) in non-adverse enzyme induction.
All other individual and mean organ weight differences were considered to be either the result of decreased body weights or spurious and
unrelated to test substance administration.

GROSS PATHOLOGY
There were no test substance-related gross observations in any of the P1 adults.

HISTOPATHOLOGY
Under the conditions of this study, the only test substance-related adverse pathology effect was the microscopic increase in hyaline droplets
(≥300 mg/kg bw/day) and secondary chronic progressive nephropathy (1000 mg/kg bw/day) in male rats. No adverse effects were observed in
female rats at any dose.

Effect levels

Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Due to decreased body weights and body weight gains and increased hyaline droplets in males at ≥ 300 mg/kg/day. Hyaline droplets are, however, species/sex-specific, occurring only in male rats. Systemic toxicity

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

There were no statistically significant or treatment-related changes in coagulation parameters in male or female rats.

Mean Absolute and Relative (% body weight) Liver and Kidney Weights in Male and Female Rats

Sex:

Male

Female

Dose (mg/kg bw/day):

  0        100          300            1000

0           100           300            1000

Weight (grams)

 

 

Final Body Weight

 

Liver

% body wt

 

Kidney

% body wt

535.9    511.4    493.7*    493.0*

 

18.261  17.817 18.176  22.154*

  3.394    3.483    3.679    4.485*

 

 

3.563    3.883    3.768    4.090*

 0.666   0.759*   0.763*  0.831*

318.1    317.2       325.8       310.5

 

13.340  12.260     14.093   13.818

 4.177    3.866        4.325     4.439

 

 2.473    2.375        2.542     2.497

 0.778    0.749        0.781     0.804

Underlined values were interpreted to be test substance-related effects.

* Statistically significant (alpha = 0.05) by Dunnett/Tamhane-Dunnett pair-wise test (parametric).

Incidences of Test Substance-Related Microscopic Findings in Male and Female Rats

Sex:

Male

Female

Dose (mg/kg bw/day):

  0       100        300           1000

0          100          300          1000

Number of Rats in Group

 9a        10          10                10

10          10            10                9b

Liver

Hypertrophy, hepatocellular, centrilobular

 

Kidney

Hyaline droplets, increased

 

Nephropathy, chronic progressive

 

 0           0            2                  8

 

 

 

1            1             3                  8

 

1            1             2                  7

 

0             0               0                 4

 

 

 

0              -               -                  0

 

1              -               -                  0

aOne animal (101) was sacrificed in extremis following rupture of an eye during blood collection on test day 22.

bOne animal (804) was sacrificed in extremis following rupture of an eye during blood collection on test day 22.

Underlined values were interpreted to be test-substance related increases in microscopic observations.

A dash indicates not examined.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the no-observed-effect level (NOEL) for systemic toxicity was 100 mg/kg bw/day due to decreased body weights
and body weight gains and increased hyaline droplets in males at ≥ 300 mg/kg bw/day. Hyaline droplets are, however, species/sex-specific,
occurring only in male rats. Female rats were not affected up to the highest dose tested (1000 mg/kg bw/day).
Executive summary:

A combined repeated dose toxicity study with reproduction/developmental toxicity screening test was conducted according to OECD 422 and EPA OPPTS 870.365. The test material was determined in a 28 day oral study performed in line with GLP. Rats (10/sex/dose level) were dosed once daily by gavage at dose levels of 0, 100, 300, or 1000 mg/kg bw/day. Under the conditions of this study, the no-observed-effect level (NOEL) for systemic toxicity was 100 mg/kg bw/day due to decreased body weights and body weight gains and increased hyaline droplets in males at >= 300 mg/kg bw/day. Hyaline droplets are, however, species/sex-specific, occurring only in male rats. Female rats were not affected up to the highest dose tested (1000 mg/kg bw/day).

Categories Display