N,N-dimethyl-3-oxobutyramide

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant study conducted 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 guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

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: yellow liquid

Details on test material:

Test item 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
45 male and 46 female Crl:CD®(SD)IGS BR rats were received from Charles River Laboratories, Inc.
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.

ANIMAL HUSBANDRY:
HOUSING:
All male rats were housed individually during non-mating periods in stainless steel, wire-mesh cages suspended above cageboards.

ENVIRONMENTAL CONDITIONS:
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).

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

NanoPure® water

Details on exposure:

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.

Details on mating procedure:

1. Start of Cohabitation:
Animals were cohoused after approximately 2 weeks of exposure to the test substance. The day animals were first cohoused was
designated as day 1 of cohabitation.
2. Duration of Cohabitation Period:
Animals were cohoused until evidence of copulation was observed or until 2 weeks had elapsed. The cohabitation period ended in the
morning of day 15 of cohabitation.
3. Evidence of Copulation:
Once daily, each female was examined for the presence of an intravaginal copulation plug or sperm in the vaginal lavage sample,
either of which was considered evidence of copulation. The presence of an intravaginal plug and/or sperm was recorded.
The day evidence of copulation was observed was designated as day 0 of gestation.
4. Cohousing:
Each female was continually housed on a 1:1 basis with a randomly selected, nonsibling male of the same dietary concentration level,
in the male's cage. On the day copulation was confirmed, the female was transferred back to individual cage housing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the neat test substance was confirmed by analysis of the bulk sample for concentration of
DMAA in the aqueous solution. Data from the analysis of the samples during the study indicate that the test substance was at the
targeted concentrations, mixed uniformly, and stable under the conditions of the study.The test substance was stable when held for
5 hours at room temperature in the vehicle and stable for 7 days of refrigeration and 7 days of refrigeration followed by 5 hours at room
temperature in the vehicle. The data for samples submitted from all test formulation preparations show that the test substance was at the
targeted concentrations and uniformly mixed.

Duration of treatment / exposure:

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).

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
100 mg/kg bw/day
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
300 mg/kg bw/day
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

10

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
doses for the main study are 0, 100, 300, and 1000 mg/kg bw/day.

Examinations

Parental animals: Observations and examinations:

Daily Animal Health 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.

DETAILED CLINICAL OBSERVATIONS:
Once daily during dosing; detailed clinical observations were recorded once during pretest and weekly
thereafter.

BODY WEIGHT AND FOOD CONSUMPTION:
All selected P1 rats were weighed during Functional Observational Battery (FOB) assessments. All P1 rats were weighed at terminal sacrifice.
Body weights and food consumption were recorded weekly for P1 males and females
(premating), on days 0, 7, 14, and 21 of gestation and on days 0 and 4 of lactation;

An abbreviated neurobehavioral evaluation consisting of a functional
observational battery and motor activity was conducted in P1 rats (5/sex/group) on test day 15.
Clinical pathology parameters were measured in P1 rats (5/sex/group) at the end of the premating
period (hematology, clinical chemistry) and at terminal sacrifice (coagulation).

All P1 rats were given a gross pathological examination on test day 55 (males and females that
did not deliver a litter) and on postpartum day 4 (lactating females).

Sperm parameters (parental animals):

Once daily, each female was examined for the presence of an intravaginal copulation plug or sperm in the vaginal lavage sample, either of which
was considered evidence of copulation. The presence of an intravaginal plug and/or sperm was recorded. The day evidence of copulation
was observed was designated as day 0 of gestation.

Litter observations:

Lactation Procedures:
--------------------
The day when delivery was complete was designated day 0 postpartum. At each examination period (days 0 and 4 postpartum),
offspring were individually handled and examined for abnormal behavior and appearance; any dead or abnormal pups were recorded.

Day 0 Postpartum:
-----------------
Live and dead pups in each litter were counted by sex as soon as possible after delivery was completed. Live pups in each litter were
individually weighed.

Day 4 Postpartum:
-----------------
Live pups in each litter were counted by sex and individually weighed and a gross external examination was performed. All pups were euthanized
(by decapitation).

Postmortem examinations (parental animals):

Anatomic Pathology:
------------------
All P1 adult rats that survived until the scheduled sacrifice were euthanized by carbon dioxide anesthesia and exsanguination on days 55 (males)
or 42 - 55 (females). The order of sacrifice for scheduled deaths was random among all treatment groups within a sex.
One male rat (Animal Number 101) and one female rat (Animal Number 804) were similarly euthanized on day 22 following rupture of an eye
during blood collection.
Gross examinations were performed on all adult rats. The presence and number of uterine implantation sites and ovarian corpora lutea were
evaluated for all cohabited females. Final body weights and organ weights were recorded.

Postmortem examinations (offspring):

F1 pup pathology data was limited to the gross examination of individuals that did not survive until day 4 of lactation.
No organ weight data was collected and no microscopic evaluation was conducted.

Statistics:

The following parameters were analyzed by Levene's test for homogeneity and Shapiro-Wilk's test for normality: body weight and gain, food consumption and efficiency, gestation length, number of implantation sites and corpora lutea, implantation efficiency, mean number of pups per litter, percent born alive, 0-4 day viability, clinical pathology and organ weights. The following parameters were analyzed by the Cochran-Armitage test for trend: incidence of clinical observations and functional observational battery (FOB) descriptive parameters, and mating, fertility and gestation indices.

Reproductive indices:

There were no test substance-related effects on mating, fertility, gestation length, number of implantation sites, implantation efficiency,
or number of corpora lutea at any dose level.

Offspring viability indices:

There were no test substance-related effects on the number of pups born, born alive, and alive on day 4, nor were there any effects on sex ratio,
or survival indices during lactation days 0-4 at any dose level.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test substancerelated increased incidences of any clinical observations. The signs reported were unremarkable and generally occurred with low frequency across all groups, including the control group.

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

There was no test substance-related mortality during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Decreased body weights and body weight gains in males.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Decreased body weights and body weight gains in males.Test substance-related reductions in body weight and body weight gain were observed at 300 and
1000 mg/kg/day and were considered adverse. The statistically significant decrease in body weight gain at 100 mg/kg/day on days 36-43 was considered spurious since it was transient and had no appreciable effect on overall body weight
gain.

Food efficiency:

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.

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not specified

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Under the conditions of the study, there were no test substance-related effects on any neurobehavioral parameter evaluated in either males or
females administered dosages of 1000 mg/kg bw/day or below.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Increased hyaline droplets in males.

Histopathological findings: neoplastic:

not specified

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on mating, fertility, gestation length, number of implantation sites, implantation efficiency, or number of corpora lutea at any dose level.

Details on results (P0)

Clinical Observations and Mortality:
--------------------------------
There was no test substance-related mortality during the study. There were no test substance related increased incidences of any clinical
observations. The signs reported were unremarkable and generally occurred with low frequency across all groups, including the control group.

Body Weights and Body Weight Gains:
---------------------------------
Test substance-related reductions in body weight and body weight gain were observed at 300 and 1000 mg/kg bw/day for male rats and were
considered adverse. 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 and Food Efficiency:
------------------------------------
There were no test substance-related effects on food consumption or food efficiency at any dose level tested for male and female rats.

Reproductive Indices:
-------------------
There were no test substance-related effects on mating, fertility, gestation length, number of implantation sites, implantation efficiency, or number
of corpora lutea at any dose level.

Neurobehavioral Evaluation:
--------------------------
Under the conditions of the study, there were no test substance-related effects on any neurobehavioral parameter evaluated in either males or
females administered dosages of 1000 mg/kg bw/day or below.

Clinical Pathology:
-----------------
There were no adverse changes in hematologic parameters in male or female rats. There were no statistically significant or treatment-related changesin coagulation parameters in male or female rats.
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.

Organ Weight:
-------------
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.
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 Observations:
-------------------
There were no test substance-related gross observations in any of the P1 adults.

Microscopic Findings:
--------------------
Test substance-related microscopic findings were observed in the liver (hepatocellular hypertrophy) of males (≥300 mg/kg bw/day) and females
(1000 mg/kg bw/day) and the kidneys (increased hyaline droplets (≥300 mg/kg bw/day) and chronic progressive nephropathy (1000 mg/kg bw/day) of males. All other microscopic observations in this study were consistent with normal background lesions in rats of this age and strain.

Effect levels (P0)

1

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

2

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

Remarks on result:

other:

Remarks:

Decreased body weights and body weight gains in males.Test substance-related reductions in body weight and body weight gain were observed at 300 and 1000 mg/kg/day and were considered adverse. The statistically significant decrease in body weight gain at 100 mg/kg/day on days 36-43 was considered spurious since it was transient and had no appreciable effect on overall body weight gain. Increased hyaline droplets in males at ≥ 300 mg/kg/day.

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test substance-related clinical observations at any dose level; all offspring appeared normal.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no test substance-related effects on the number of pups born, born alive, and alive on day 4, nor were there any effects on sex ratio, or survival indices during lactation days 0-4 at any dose level.
The statistically significantly lower number of pups born and number of pups born alive at 300 mg/kg/day was not considered test substance-related since these reductions did not demonstrate a dose response relationship.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on pup weights at birth or on day 4 of lactation at any dose level tested.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not specified

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Description (incidence and severity):

Organ weight data was not collected from any F1 pups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test substance-related gross observations determined from the gross examination of those F1 pups that did not survive until lactation day 4.

Histopathological findings:

not examined

Other effects:

not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Litter Size, Sex Ratio and Pup Survival:
----------------------------------
There were no test substance-related effects on the number of pups born, born alive, and alive on day 4, nor were there any effects on sex ratio,
or survival indices during lactation days 0-4 at any dose level. The statistically significantly lower number of pups born and number of pups born
alive at 300 mg/kg/day was not considered test substance-related since these reductions did not demonstrate a dose response relationship.

Clinical Observations:
--------------------
There were no test substance-related clinical observations at any dose level; all offspring appeared normal.

Pup Weights:
------------
There were no test substance-related effects on pup weights at birth or on day 4 of lactation at any dose level tested.

Organ Weight:
-------------
Organ weight data was not collected from any F1 pups.

Gross Observations:
-------------------
There were no test substance-related gross observations determined from the gross examination of those F1 pups that did not survive until lactation day 4.

Microscopic Findings:
--------------------
Microscopic evaluation of tissues was not conducted on any F1 pups.

Effect levels (F1)

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reproductive toxicity and effects on F1 offspring growth and survival was 1000 mg/kg bw/day, the highest dose level tested.

Results: F2 generation

General toxicity (F2)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the no-observed-effect level (NOEL)a for systemic toxicity was 100 mg/kg/day 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. The NOEL for reproductive toxicity and F1 offspring was
1000 mg/kg/day, the highest dose level tested.

Executive summary:

A combined repeated dose toxicity study with reproduction/developmental toxicity screening test was conducted in year 2005/2006 according to OECD 422 and GLP. Crl:CD®(SD)IGS BR rats (10/sex/dose level) were dosed with DMAA once daily by gavage at dose levels of 0, 100, 300, or 1000 mg/kg bw/day. Following a 2 week premating period, parental (P1) males and females were cohoused for up to 2 weeks within their respective treatment groups to produce F1 litters. The NOEL for reproductive toxicity and F1 offspring was 1000 mg/kg bw/day, the highest dose level tested.