Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 3, 2002 - April 15, 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was done according to EPA OPPTS 870.3700 with no listed deviations, and the test substance is adequately defined.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Principles of method if other than guideline:

according to EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Confidential

Test animals

Species:
rabbit
Strain:
other: Hra:(NZW)SPF
Details on test animals and environmental conditions:
The rabbit (Hra:(NZW)SPF) was selected for this study to provide data in a non-rodent species and
because it is a preferred species for developmental toxicity testing. The Hra:(NZW)SPF strain
was chosen because extensive background information is available from the literature, the
supplier, and previous studies with other compounds at Haskell Laboratory.
A total of 110 nulliparous, time-mated female rabbits were received from COVANCE, Denver,
Pennsylvania. Sixty-six female rabbits were received on June 19, 2002 and 44 female rabbits
were received on June 21, 2002. Body weights on the day of mating defined as day 0 of
gestation (G) were supplied by the vendor. The rabbits for this study were approximately 6
months old and were at either day 1, 2, or 3G upon arrival.

Animal rooms were maintained at an acceptable temperature of 17-23°C (targeted at 20°C) and a
targeted relative humidity of 50% ± 10%. Animal rooms were artificially illuminated
(fluorescent light) on a 12-hour light/dark cycle (approximately 0600-1800 hours). Animals were
housed individually in suspended, wire-mesh, stainless steel cages. Nesting material was not
provided because the does were euthanized prior to parturition.
To address logistical considerations, the rabbits on this study were housed in 4 animals rooms
near the exposure chambers. There was a significant aerosol component in the high level
chamber; therefore, to prevent unintentional exposure of control or lower level animals to the
residual aerosol, the control group rabbits were housed separately in one room. The rabbits
exposed to 1000 mg/m³ were housed separately within a hood so that the higher air flow in the
hood promoted evaporation of aerosol deposits on the fur. The remaining 3 groups of rabbits
were housed in 2 other rooms.

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Spraying Systems Nebulizer
- Method of holding animals in test chamber: individually restrained in wire mesh cages
- Source and rate of air: house supply
- Method of conditioning air: Cole Palmer Masterflex® Console Drive pump
- System of generating particulates/aerosols: Brooks model 5851i Mass Flow Controller
- Temperature, humidity, pressure in air chamber: 17-23°C at 50 ± 10% humidity
- Air flow rate:
- Air change rate:
- Method of particle size determination: Sierra® Series 210 Cyclone Preseparator/Cascade Impactor and Sierra® Series 110 Constant Flow Air Sampler
- Treatment of exhaust air:

TEST ATMOSPHERE
- Brief description of analytical method used: GC
- Samples taken from breathing zone: yes

VEHICLE (if applicable)
- Justification for use and choice of vehicle:
- Composition of vehicle: air
- Type and concentration of dispersant aid (if powder):
- Concentration of test material in vehicle: 0, 30 100, 300, 1000 mg/m3
- Lot/batch no. of vehicle (if required):
- Purity of vehicle: NA
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For all test exposure chambers, the vapor component of the atmospheric concentration of DMG
was determined by gas chromatography. For the test exposure chambers, known volumes of
chamber atmosphere were drawn, at approximately 90-minute intervals, from the breathing zone
of the animals through glass impingers containing acetone as the collection medium. The control
chamber was similarly sampled once daily for measurement of atmospheric concentration.
Aliquots of impinger solution were injected on a Restek® fused silica glass (inside diameter
0.53 mm) with RTX-502.2 coating column in a Hewlett-Packard model 6890 Series Gas
Chromatograph equipped with a flame ionization detector. The atmospheric concentration of the
test substance was determined from a standard curve derived from liquid standards. Standards
were prepared weekly by the quantitative dilution of DMG liquid in known volumes of acetone.
For the 300 and 1000 mg/m³ exposure chambers, the aerosol component of the atmospheric
concentration of DMG was determined by gravimetric analysis. Known volumes of chamber
atmosphere were drawn, at approximately 30-minute intervals, from the breathing zone of the
animals through a 25 mm filter cassette that contained a pre-weighed Gelman glass fiber (Type
A/E) filter. The filters were weighed on a Cahn model C-31 Microbalance®. The filter weights
were automatically transferred to CITADS, which calculated the chamber concentrations based
on the difference in pre- and post-sampling filter weights divided by the volume of chamber
atmosphere sampled. Gravimetric sample start- and stop-times for each sample were also
controlled and recorded by CITADS. Prior to study start, gravimetric samples collected from the
30 and 100 mg/m³ chambers indicated that no aerosol was present for these 2 concentrations.
A sample to determine particle size distribution (mass median aerodynamic diameter and percent
particles less than 1, 3, and 10 µm diameter) was taken once per week from the 1000 mg/m³
exposure chamber with a Sierra® Series 210 Cyclone Preseparator/Cascade Impactor and Sierra®
Series 110 Constant Flow Air Sampler.(3)
2. Environmental Monitoring
Chamber airflow
Details on mating procedure:
Mating details not provided. A total of 110 nulliparous, time-mated female rabbits were received from COVANCE. Sixty-six female rabbits were received on June 19, 2002 and 44 female rabbits were received on June 21, 2002.
Duration of treatment / exposure:
6 hours per day
Frequency of treatment:
Daily during days 7-28G
Duration of test:
22 days
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 30, 100, 300, 1000 mg/m3 (for the 1000 mg/m3 group the mean daily vapor concentrations ranged from 450-590 mg/m³, and the mean daily aerosol concentrations ranged from 410-580 mg/m³
Basis:
analytical conc.
No. of animals per sex per dose:
22
Control animals:
yes

Examinations

Maternal examinations:
1. In-life Observations of Females
a. Body Weight
Body weights were recorded in the morning on the day after arrival through day 29G.
b. Food Consumption
Food consumption was measured daily on days 4-29G.
c. Clinical Signs
Clinical signs were recorded each morning on days 4-29G and each afternoon following
exposures on days 7-28G. Observations, at approximately hourly intervals, were made during
the exposures to assess, to the extent possible, the overall state of the animals. In addition, the
response of the animals to a sound stimulus while in the chambers was recorded before vapor
generation began, after approximately 3 hours of exposure, and prior to removal of the animals
from the chambers.

2. Postmortem Observations of Females Dying Prior to Scheduled Euthanasia
A gross external and visceral examination was performed within 24 hours after the doe was
found dead or sacrificed in extremis. Pregnancy status was determined and the uterine contents
were described. Dead rabbits were refrigerated until necropsied.

3. Postmortem Observations of Does Aborting or Delivering Early
a. Dam Observations
Dams that aborted (day 27G or earlier) or delivered early (day 28 or 29G) were euthanized within
24 hours of detection and a gross external and visceral examination was performed.
b. Fetal Observations
Fetuses remaining in the uterus or aborted/delivered early were examined to the extent possible.
Data from these litters were maintained with the raw data but were excluded from all relevant
fetal data calculations.

4. Postmortem Observations of Does Surviving to Scheduled Euthanasia
a. External Appearance
Each doe was examined immediately after euthanasia on day 29G.
b. Viscera
Viscera were examined grossly immediately after euthanasia.
c. Uterine Weight
The intact and empty uterus of each doe having at least one viable fetus was weighed to permit
calculation of maternal body weight adjusted to exclude the products of conception.
d. Corpora Lutea
The count for each ovary of dams with viable fetuses was recorded.
e. Implantations
For each female with visible implants, the type (live and dead fetuses, and resorptions) and their
relative positions were recorded. The uterus of each apparently "nonpregnant" female was
stained with ammonium sulfide to detect very early resorptions.
Ovaries and uterine content:
see materal examinations
Fetal examinations:
Fetuses of Does Surviving to Scheduled Euthanasia
a. Number, Location, and Condition
These parameters were recorded for each fetus.
b. Fetal Weight
The body weight for each fetus was recorded.
c. External Alterations
The alterations detected for each live fetus was recorded.
d. Soft Tissue Alterations
For each live fetus, fetal sex and any visceral alterations detected were recorded.(5) A transverse
section, deep enough to permit examination of the brain, was made between the parietal and
frontal bones. On the day of sacrifice, the eyelids of each fetus were removed to examine the
eyes for alterations.
e. Skeletal Alterations
After fixation in alcohol, the alizarin-stained skeletons were examined and alterations detected
were recorded for each live fetus.
Statistics:
Statistical Analyses
Descriptive statistics including mean, standard deviation, and standard error of the mean were
used to summarize exposure concentration and environmental data.
For the parameters listed below, descriptive statistics were performed and sequential trend tests
were applied to the data as tabulated below.(6) If a significant dose-response trend was detected,
data from the top dose group were excluded and the test repeated until no significant trend was
evident. For litter parameters, the proportion of affected fetuses per litter or the litter mean was
used as the experimental unit for statistical evaluation.(7) The level of significance selected was
p < 0.05.
Parameter Trend Test
Maternal weight Linear contrast of means(8)
Maternal weight changes
Maternal food consumption
Live fetuses Jonckheere's test(9)
Dead fetuses
Resorptions
Nidations
Corpora lutea
Incidence of fetal alterations
Incidence of pregnancy Cochran-Armitage test(8)
Clinical observations
Maternal mortality
Females with total resorptions
Abortions/early deliveries
Fetal weight Linear contrast of least square means(10)
Sex ratio
Where the data were tied and the standard large sample version of Jonckheere's test was not
applicable, exact p values for Jonckheere's test were calculated using permutation
methodology.
Indices:
Maternal weight
Maternal weight changes
Maternal food consumption
Live fetuses
Dead fetuses
Resorptions
Nidations
Corpora lutea
Incidence of fetal alterations
Incidence of pregnancy
Clinical observations
Maternal mortality
Females with total resorptions
Abortions/early deliveries
Fetal weight
Sex ratio

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects. Remark: 300 mg/m3

Details on maternal toxic effects:
1. Mortality and Reproductive Outcome
There was no compound-related mortality at exposure levels = 300 mg/m3. At 1000 mg/m3, one
female was found dead on day 13G and another female was sacrificed in extremis on day 22G.
There were no compound-related effects on indicators of reproductive outcome. One control
group rabbit aborted on day 19G and one 100 mg/m3 group rabbit delivered early prior to
euthanasia on day 29G. In addition, one 1000 mg/m3 litter consisted entirely of late resorptions.
The findings for this one litter were not considered to be compound-related because no other
litters were similarly affected and additionally, there was no increase in resorptions for litters also
containing live fetuses. At 1000 mg/m3, there was a significant decrease in the mean number of
nidation sites per doe. This was not considered related to the exposure because implantation is
considered to have occurred prior to the start of exposures on day 7G. Furthermore, this decrease
is consistent with a lower mean corpora lutea count in this group.

2. Body Weights and Weight Changes
There were no compound-related effects on maternal body weight parameters at exposure levels
= 100 mg/m3. There were significant reductions in overall weight change (days 7-29G) at 300
and 1000 mg/m3; these significant effects were evident at the onset of exposures (days 7-9G) and
generally persisted throughout gestation.

3. Food Consumption
There were no compound-related effects on maternal food consumption at exposure levels
= 300 mg/m3. At 1000 mg/m3, mean food consumption was significantly reduced when averaged
over the exposure period (days 7-29G); the reduction was significant and pronounced at the onset
of exposures (days 7-9G) and persisted throughout exposures to some extent as well.

4. Clinical Observations
There were no compound-related increased clinical observations at exposure levels = 100 mg/m3.
At 300 and 1000 mg/m3, compound-related clinical observations included clear ocular discharge;
at 1000 mg/m3, all animals had wet fur.

5. Postmortem Findings
There were no compound-related gross postmortem findings at any level tested.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Remarks:
100 mg/m3
Effect level:
> 0 - <= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects. Remark: 1000 mg/m3

Details on embryotoxic / teratogenic effects:
Fetal Findings
1. Mortality and Sex Ratio
There were no compound-related effects on embryofetal viability or on fetal sex ratio at any level
tested. The mean number of live fetuses per litter appeared to be slightly reduced at 1000 mg/m3,
however, this apparent reduction was not considered compound-related and is believed to have
resulted from previously discussed reductions in mean numbers of corpora lutea and nidations at
this level that were not considered compound-related.

2. Body Weight
There was no compound-related effect on fetal body weight at any level tested.

3. Malformations
There were no compound-related fetal malformations at any level tested.

4. Variations
There were no compound-related fetal variations at any level tested.
There was a significant increase in retarded sternebral ossification at 1000 mg/m3. This increase,
however, was not considered to be compound-related; rather, the statistical significance appears
to be the result of an unusually low control group value relative to relevant historical control
group data.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the conditions of the current study, there was no evidence of compound-related maternal
toxicity at 30 or 100 mg/m3 and there was no evidence of developmental toxicity at any level
tested. Thus, the no-observed-effect level (NOEL) a for maternal toxicity was considered
100 mg/m3 and the NOEL for developmental toxicity was considered 1000 mg/m3.
Executive summary:

Dimethyl glutarate (DMG) was administered by inhalation to groups of 22 time-mated Hra:(NZW)SPF rabbits. The inhalation exposures were whole-body exposures and animals were exposed for approximately 6 hours each day on days 7 through 28 of gestation. Target chamber concentrations were 0, 30, 100, 300, or 1000 mg/m3. During the in-life period, maternal body weight, food consumption, and clinical observation data were collected daily. Animals were euthanized on day 29 of gestation; dams were subjected to a gross external and internal postmortem examination. Gravid uteri were removed, weighed, and dissected and the uterine contents were described. Each live fetus was weighed, sexed, and examined for external, visceral, and skeletal alterations. At 1000 mg/m3, there was compound-related mortality; one animal was found dead on gestation day 13 and one animal was sacrificed in extremis on gestation day 22. Among survivors, there were compound-related reductions in maternal body weight gains and food consumption and there were compound-related clinical observations (ocular discharge and wet fur). At 300 mg/m3, evidence of compound-related maternal toxicity was limited to reduced maternal body weight gains and increased clinical observations (ocular discharge). Under the conditions of the current study, there was no evidence of compound-related maternal toxicity at 30 or 100 mg/m3 and there was no evidence of developmental toxicity at any level tested. Thus, the no-observed-effect level (NOEL)a for maternal toxicity was considered 100 mg/m3 and the NOEL for developmental toxicity was considered 1000 mg/m3.