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EC number: 202-319-2
CAS number: 94-28-0
The objective of this study was to obtain information on the toxicity of
2,2’- ethylenedioxydiethyl bis(2-ethylhexanoate) when administered daily
for 90 days by oral gavage to the Wistar rat at 3 dose levels.
Doses of 40, 120, and 480 mg/kg /day were administered to male and
female Wistar rats according to the experimental design presented in the
Table 1: Experimental design
Dose Level (mg/kg/day)
Dose volume (mL/kg bw/day)
Number of Animals
Rats assigned to Recovery groups, following the completion of 90-day
treatment period were kept for a 28-Day observation period.
Satellite groups were assigned to immuntoxicology T-cell Dependent
Antibody Response (TDAR) investigation.
An additional group of ten male and ten female Wistar rats served as
positive control for TDAR investigation (animals were
treated with cyclophosphamide, a known immunosuppressant, via
The test item was formulated in propylene glycol containing 1% Tween 80
and the control group was treated with the vehicle only. The first day
of the treatment was Day 0 for all animals.
During the study, observations for mortality and clinical signs were
performed daily, detailed clinical examination, body weight and food
consumption measurements were performed at weekly intervals.
Ophthalmoscopy and neurological assessment including a functional
observation battery (FOB) and measurements of the landing foot splay,
grip strength and motor activity were performed during the last week of
treatment. A motor activity measurement was repeated for males groups
during the last week the recovery.
Clinical pathology examinations (haematology, clinical chemistry and
urinalysis) were conducted prior to necropsy following the end of the
treatment and recovery periods (Days 90 and 118), followed by necropsy
with macroscopic examination and selected organ weight measurements.
Full histopathology was performed for Groups 1 (Control) and 4 (High
dose); in addition, the affected organs (kidneys and liver) from females
of the Low and Mid dose and Recovery groups were also examined. Organs
showing macroscopic findings from any animal of any group were also
Analysis of test item formulations for concentration and homogeneity was
performed four times during the treatment period, using a validated GC
method. All formulations were found to be in the range of 90 to 102% of
nominal concentrations and were homogeneous. No test item was detected
in the control samples. Based on these results, the test item
formulations were considered suitable for the study purposes.
No mortality or clinical signs considered related to test item was
observed during the study. There were no toxicologically significant
changes in the animal behaviour, general physical condition, in the
reactions to different type of stimuli in the control or treated groups.
No test item related effects were observed in the landing foot splay
test or grip strength measurements. In motor activity measurements, the
total travelled distance was slightly lower (by approximately 22%), in
males at 480 mg/kg/day. The average velocity and total time moving fast
were also slightly lower, than control mean. As the patterns of activity
were comparable with those of the control and the individual values were
within the physiological range, therefore the effect was not regarded as
adverse. Following the 28-day recovery period, all parameters in the
motor activity test examined in males at 480 mg/kg/day, were comparable
The oestrus cycle of the females was undisturbed by the treatment with
the test item.
No test item related changes compared to pre-treatment were noted at
There were no test item related effects on body weight and body weight
gain or food consumption.
There were no test item related adverse effects in haematology, blood
clotting, bone marrow cellularity, clinical chemistry or urinalysis
parameters at any dose level.
There were no macroscopic findings considered related to test item
For organ weight, minimal differences were measured on Day 90 in females
for liver and kidneys at 480 mg/kg/day and for liver at 120 mg/kg/day.
The absolute and relative weights of liver were higher than control
means by approximately 8-10% (Mid dose) and 8-12% (High dose). The
changes were not considered adverse.
Microscopically a minimal periportal hepatocellular hypertrophy and
minimal to mild periportal mixed cell infiltrate were seen in the liver
in 2-3 of 10 Mid Dose and 3-4 of 10 High Dose females, occasionally
accompanied by proliferation of the Kupffer cells (1 of 10 High Dose
females). The overall microscopic picture in the High dose was
classified as adverse, while findings in the Mid dose rather as adaptive
High dose females had also slightly higher weights of kidneys (no more
than 11%). Microscopically in the kidneys, minimal degeneration of
epithelium of cortical tubule (cytoplasmic vacuolation, cellular
sloughing) associated with the presence of homogenous eosinophilic
content (casts) filled tubular lumen was seen in 2 of 10 High dose
females. Although minimal focal degeneration and eosinophilic content
was observed in right kidney in 1 of 10 Mid dose female, but taking into
account unilateral occurrence and minimal focal degree, the finding was
not regarded as adverse.
Following a 28 -day treatment-free period, complete recovery was
observed in the liver and signs of the recovery were detected in the
kidney. In kidneys, mild bilateral or minimal unilateral multifocal
tubular degeneration/regeneration with the presence of luminal
homogenous eosinophilic casts was observed in 2 of 10 High Dose females.
The tubules also underwent regenerative process accompanied with
increased tubule cells basophilia, transition of cell shape from
flattened to cuboidal and/or mitosis.
During Immuntoxicology investigation, in a T-cell Dependent Antibody
Response (TDAR) assay, no changes were detected in males. Decreased
anti-SRBC IgM levels compared to the negative control group were
detected in the mid and low dose group of the females. However, no dose
response was observed, and the observed values were statistically not
different from negative control values.
There were no test item effects on lymphocyte subpopulation analysis
that could be attributed to the treatment at any of the dose level up to
and including 480 mg/kg/day. Minor differences between groups were
observed which were considered to represent normal variation and of no
toxicological or biological relevance.
A daily oral (gavage) administration of 2,2’-ethylenedioxydiethyl
bis(2-ethylhexanoate) to Wistar rats for 90 consecutive days at 40, 120
and 480 mg/kg/day was associated with the following findings.
There were no toxicologically significant effects on clinical signs,
ophthalmology, body weight, food intake, haematology, blood clotting,
bone marrow cellularity, clinical chemistry or urinalysis parameters.
There were no adverse effects on organ weights or on macroscopic
observations at necropsy. There were no treatment related effects on any
immunotoxicology endpoints including lymphocyte subpopulation analysis
or T-cell Dependent Antibody Response (TDAR) assay.
At 480 mg/kg/day minimal to mild changes were found in the liver and
kidneys of females only. Changes in the liver consisted of periportal
hepatocellular hypertrophy and mixed cell infiltrate and proliferation
of the Kupffer cells, correlated with slightly higher organ weight and
were fully reversible during 28-day recovery period. The renal changes
consisted of the minimal bilateral focal/multifocal degeneration of the
epithelium of cortical tubules (cytoplasmic vacuolation, cellular
sloughing) Associated with the presence of eosinophilic casts in tubular
lumen. Signs of recovery were observed in the cortical tubuli.
A decreased performance was noticed during motor activity measurements
in males dosed at 480 mg/kg/day, however the effect was reversible and
not considered to be adverse. No other adverse effects were noted in
At 120 mg/kg/day minimal changes were found in the liver (minimal
periportal hepatocellular hypertrophy) of few females and were regarded
as adaptive. In one female, unilateral focal degeneration of tubule
epithelium with eosinophilic casts in tubular lumen was seen in kidney,
but taking into account unilateral occurrence and minimal focal degree,
the finding was not regarded to be adverse. No effects were observed for
Treatment at 40mg/kg/day was not associated with any adverse effects or
histopathological changes. In conclusion, the NOAEL of
2,2’-ethylenedioxydiethyl bis(2-ethylhexanoate) administered by oral
gavage to Wistar rats for 90 consecutive days is considered to be 480
mg/kg/day for males and 120 mg/kg/day for females, based on microscopic
changes in the liver and kidneys in females at 480 mg/kg/day. The NOEL
is 120 mg/kg/day for males and 40 mg/kg/day for females. No
immunotoxicity was detected at any dose level up to and including 480
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