Registration Dossier

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Diss Factsheets

Administrative data

Description of key information

Male and female rats were administered vehicle control item or 250, 500, or
1000 mg/kg/day VEHA via oral gavage once daily. Vinyl 2-ethylhexanoate-related
observations in animals administered 1000 mg/kg/day included: nonadverse increased
incidence of ataxia in the first week; nonadverse decreased body weight gain; adverse
reduction in activity at Week 12; pronounced alterations in clinical chemistry and
hematology endpoints of minimally lower total white blood cell count, minimally lower
platelet count, decreased total protein concentration, higher cholesterol, higher highdensity
lipoprotein, and higher triglyceride; microscopic findings of centrilobular
hepatocyte hypertrophy (males), decreased lymphocytes (females), tubular
degeneration/atrophy of testes and luminal cell debris of the epididymis (males) with
correlated decreased organ weights; and a significant decrease in sperm motility (males).
For these reasons 1000 mg/kg/day was considered adverse when administered for
13 weeks. Vinyl 2-ethylhexanoate-related observations in animals administered
500 mg/kg/day included: nonadverse reduction in activity at Week 12, alterations in
clinical chemistry and hematology endpoints seen at 1000 mg/kg/day but at a lower
magnitude, centrilobular hepatocyte hypertrophy in males, tubular degeneration/atrophy
of the testes and luminal cell debris of the epididymis with no correlating organ weight
changes. Due to the mild severity of findings and the lack of an impact on the health and
wellbeing of animals administered 500 mg/kg/day, effects for this dose were considered
nonadverse when administered for 13 weeks. Thus, the no observed adverse effect level
(NOAEL) is 500 mg/kg/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
GLP compliance:
yes
Specific details on test material used for the study:
Identification: Vinyl 2-ethylhexanoate
CAS Number: 94-04-2
EC Number: 202-297-4
Batch: PM9CEH01
Purity: 99.6%
Physical State/Appearance: Clear colorless liquid
Expiry Date: 04 January 2022
Storage Conditions: Room temperature in the dark over silica gel
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Forty-four male and 44 female Sprague Dawley (Hsd:Sprague Dawley SD) rats were
received from Envigo RMS, Inc., Indianapolis, Indiana. Animals were acclimated to the
test facility for 12 (males) or 13 (females) days prior to initiation.
At initiation of dosing, animals were 8 to 9 weeks old, and body weights ranged from
230 to 274 g for males and 178 to 216 g for females. Animals not used on study were
sacrificed and discarded.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Environmental Conditions, Diet, and Water
Housing
Animals were group-housed (up to three animals of the same sex/cage) in polycarbonate
cages with hardwood chip bedding. Animals were individually housed in stainless steel
or polycarbonate cages for study-related procedures.

Water
Water was provided ad libitum.

Diet
Animals were offered Certified Rodent Diet #2014C (Envigo RMS, Inc.) ad libitum,
unless fasted for study procedures.

Environment
Environmental controls were set to maintain a temperature range of 20 to 26C, a relative
humidity range of 30 to 70%, 10 or greater air changes/hour, and a 12-hour light/12-hour
dark cycle. The light/dark cycle was interrupted for study-related activities. Any
variations to these conditions are maintained in the raw data and had no effect on the
study outcome.

Dietary and Environmental Enrichment
Animals were given various cage-enrichment devices and dietary enrichment (which did
not require analyses).

Animal Identification and Assignment to Study
Animals were identified using tail marks, an implantable microchip identification device,
and/or cage card.
Cages were identified with study information, including study number and animal
numbers.
Animals were assigned to the study using a computerized procedure designed to achieve
body weight balance with respect to group assignment. Prior to group assignment,
animals were excluded from the selection pool/sex to produce minimal variation. After
group assignment, the mean body weight for each group/sex was not statistically different
at the 5.0% probability level, as indicated by analysis of variance F probability.
For FOB and locomotor activity testing, up to three extra animals/sex received in the
animal shipment but not assigned to a group underwent FOB testing to establish baseline
values in the event replacement animals were needed.
Animals were individually identified as follows:
Study Animal Numbers
Group Males Females
1 R0001-R0010 R0401-R0410
2 R0101-R0110 R0501-R0510
3 R0201-R0210 R0601-R0610
4 R0301-R0310 R0701-R0710
Route of administration:
oral: gavage
Details on route of administration:
Dose formulations were administered once daily by oral gavage for 13 weeks at a dose
volume of 5 mL/kg. Day 1 of the dosing phase was defined as
the first day of dosing for each sex. Doses were based on the most recently recorded
scheduled body weight.
Dose formulations were allowed to equilibrate to approximately room temperature and
were stirred using a magnetic stir plate and stir bar for at least 30 minutes prior to and
throughout dosing.
Vehicle:
corn oil
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Sample Collection and Handling
Stability
Stability has been evaluated within the bracketed concentrations of 1.04 and 200 mg/mL
in a previous study (Labcorp Study 8461330).
The test item formulation is stable for 5 (1.04 mg/mL) or 24 (100 to 200 mg/mL) hours at
room temperature and protected from light; for up to 8 (1.04 to 100 mg/mL) or
15 (200 mg/mL) days in a refrigerator, set to maintain 2 to 8°C, and protected from light;
and for up to 8 (1.04 mg/mL) or 15 (100 to 200 mg/mL) days in a freezer, set to maintain
-10 to -30°C, and protected from light.

Homogeneity
Two sets of duplicate samples (1.00 mL each) were taken from the top, middle, and
bottom strata of the 50- and 200-mg/mL formulations prepared for administration on
Day 1 of the dosing phase. Additional samples were also collected on Day 6 (males)/
5 (females) of the dosing phase due to a 30% batch size change.
Samples were stored protected from light in a refrigerator, set to maintain 2 to 8C, until
analyzed for test item content or discarded, unless analyzed on the day of collection. If
analyzed on the day of collection, samples were stored at room temperature (15 to 30C)
and protected from light.

Concentration Verification
Duplicate samples (1.00 mL each) were taken from the middle of the vehicle control
item. Two sets of triplicate samples (1.00 mL each) were taken from the middle of each
test item formulation. Samples were collected from vehicle control item and test item
formulations prepared for administration on Days 1 and 91 of the dosing phase. When
samples were collected at the same interval as homogeneity, the mean result from the
homogeneity analysis was used for concentration verification.
Samples were stored protected from light in a refrigerator, set to maintain 2 to 8C, until
analyzed for test item content or discarded, unless analyzed on the day of collection. If
analyzed on the day of collection, samples were stored at room temperature (15 to 30C)
and protected from light.

Sample Analysis and Disposition
Dose formulations were analyzed by Labcorp using a concurrent validated
high-performance liquid chromatography method (Method 12693).
One control group sample was analyzed to confirm the absence of test item content. The
second sample was retained as backup. One set of samples from the test item
formulations (for each stratum, as applicable) was analyzed for test item content. The
second set was retained as backup.
Samples not consumed during analysis were discarded.
Duration of treatment / exposure:
90 days
Frequency of treatment:
once per day
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Vinyl 2-ethylhexanoate was administered orally via gavage because this is an acceptable
and commonly used method for administering test item to rats.

The doses selected for this study were based on findings from a 14-day dose
range-finding study and from a 4-week dose range-finding study. In the 14-day study, the
test item was administered using corn oil as the vehicle, and in the 28-day study, the
vehicle was 0.5% methylcellulose and was used for all groups and an additional
high-dose group formulated in corn oil (Labcorp Study 8461249; Labcorp Early
Development Laboratories Inc., 2021).

In the 14-day dose range finding study (ECHA Dossier, 1995), administration of VEHA
to Fisher 344 rats resulted in mortality for 5 of 6 males and 3 of 6 females following the
second daily dose of 2000 mg/kg/day. Due to this mortality, surviving animals from this
dose group were not dosed for the remainder of the study. Clinical observations noted for
surviving animals included decreased food consumption and body weight, which
resolved following the termination of dosing. Microscopic lesions of the central nervous
system were observed in surviving animals administered 2000 mg/kg/day but not in
animals that died during the study. A slight, nonstatistically significant increase
(approximately 20%) in landing leg splay were observed in females administered
1000 mg/kg/day; however, this change was not associated with other alterations in
neuromotor function or microscopic lesions of the central nervous system and could not
be clearly attributed to the test item. Equivocal hematology changes in females and
increased liver and kidney weights for males were observed the group administered
200 mg/kg/day. The no observed adverse effect level (NOAEL) of VEHA under the
conditions of that study was 200 mg/kg/day.

In the 4-week dose range-finding study, VEHA was administered to Sprague-Dawley rats
at doses of 100, 250, 500, or 1000 mg/kg/day (0.5% methylcellulose vehicle) and at a
dose of 1000 mg/kg/day in corn oil vehicle. In this study, a slight, nonstatistically
significant decrease in body weight gain was observed for animals administered
1000 mg/kg/day formulated in corn oil, which correlated with the nonsignificant
reduction in food consumption. Animals administered 1000 mg/kg/day in corn oil were
noted with observations of ataxia and piloerection on Days 3 to 5 of the dosing phsae.
Liver and kidney weight to body weight percentages were increased for animals
administered 1000 mg/kg/day, independent of vehicle
Observations and examinations performed and frequency:
Clinical Observations
Health Monitoring
Animals were checked once on the days of arrival/termination and twice daily (a.m. and
p.m.) for mortality, abnormalities, and signs of pain or distress. Abnormal findings were
recorded (see Protocol Deviations).

Clinical Examinations
Cageside observations were conducted for each animal once daily during the dosing
phase.
Detailed observations were conducted for each animal once during the predose phase and
prior to dosing once weekly (based on Day 1 of the dosing phase) to Week 13 of the
dosing phase and on Day 91 of the dosing phase. Detailed observations were also
collected for each animal on days of scheduled sacrifice (for animals sacrificed only).
Abnormal findings or an indication of normal was recorded.

Postdose Observations
On each day of dosing, cageside observations were conducted for each animal, except for
the entire room on days of FOB and locomotor activity testing, at approximately 2 hours
postdose (see Protocol Deviations). Observations for each sex and group were based on
the dosing completion time for that sex and group. Abnormal findings were recorded (see
Protocol Deviations).

Ophthalmic Examinations
Ophthalmic examinations were conducted once during the predose phase (including extra
animals) and once during Week 13 of the dosing phase. Pupils were dilated with a
mydriatic agent prior to examination by a Veterinarian using an indirect ophthalmoscope.

Body Weights
Body weights were recorded once during the predose phase, before dosing once weekly
(based on Day 1 of the dosing phase) to Week 13 of the dosing phase, and on Day 91 of
the dosing phase.

Food Consumption
The amount of food consumed by each cage of animals was recorded once weekly (based
on Day 1 of the dosing phase) to Week 13 of the dosing phase and from Days 85 to 91 of
the dosing phase. Wet food was recorded. Consumption was calculated as g/animal/day.

Estrous Cycle Determination
Beginning during Week 13 of the dosing phase, daily vaginal smears were assessed for
the stage of estrous for 4 consecutive days from Days 88 through 91 of the dosing phase
(see Protocol Deviations).

Functional Observational Battery
Animals were examined at least once during the predose phase (including up to three
extra animals/sex received in the animal shipment but not assigned to a group) and once
during Week 12 of the dosing phase (10 animals/sex/group). All examinations were
completed at the same time of day (2 hours) and were completed within 8 hours of
dosing.
At the time of testing, the observer was unaware of the dose level administered to each
animal.
The FOBs (see Attachment 1 of the Protocol), which included a body temperature
assessment, were conducted for 10 toxicity animals/group during the dosing phase. All
observations were assessed and recorded. Each animal was observed in their home cage,
in an arena/open field (open-field observations), and during handling (hand-held
observations) and was assessed for sensory reactivity to stimuli (elicited behaviors) and
for quantitative testing parameters.

Locomotor Activity
Locomotor activity was assessed once during the predose phase (including up to three
extra animals/sex received in the animal shipment but not assigned to a group) and once
during Week 12 of the dosing phase (10 animals/sex/group).
Locomotor activity assessments were conducted in sound-attenuated, dark chambers on
animals that underwent FOB testing.
Animals were placed into an automated photocell activity recording device, and activity
was recorded for 60 minutes. Testing was done to include an approximately equal
distribution of animals/group/device.
The intervals for reporting and data presentation were in twelve, 5-minute bins. The
parameters for presentation and analysis were basic movement, x + y ambulation, fine
movements, and rearing activity.

Thyroid Hormone and Serum Bile Acids Analyses
Sample Collection and Handling
Blood samples (approximately 1.0 mL) were collected via the jugular vein on Day 92 of
the dosing phase. Samples were collected approximately 5.5 to 6.5 hours after the light
cycle began. Animals were fasted for sample collections.
Blood was collected into serum separator tubes (without anticoagulant), allowed to clot
for at least 30 minutes at room temperature, and centrifuged within 1 hour of collection.
Serum was harvested and placed in a freezer, set to maintain -10 to -30°C, until shipped
on dry ice to Labcorp-Madison for analysis.

Sample Analysis
Samples were analyzed by Labcorp-Madison for thyroid hormone and serum bile acids:
Serum bile acids
Total T3
Total T4
Thyroid-stimulating homone

Potential Hormone Analyses
Sample Collection and Handling
Blood sample (approximately 1.0 mL) was collected from fasted Animal R0006 (Group 1
male) via abdominal aorta on Day 92 of the dosing phase. Blood samples (approximately
1.0 mL) were collected from fasted animals via abdominal aorta (males) or vena cava
(females) on Day 93 (remaining males) or 92 (females) of the dosing phase (see Protocol
Deviations). Animals were anesthetized with carbon dioxide prior to blood collection.
Blood was collected into serum separator tubes (without anticoagulant), allowed to clot
for at least 30 minutes at room temperature, and centrifuged within 1 hour of collection.
Serum was harvested into two aliquots, with approximately 300 L in the first aliquot
and any remaining serum in the other, and placed in a freezer, set to maintain
-10 to -30°C, until further directive.
Sacrifice and pathology:
Clinical Laboratory Procedures
Clinical Pathology
Sample Collection and Handling
Blood samples for hematology (approximately 500 L), coagulation (approximately
0.5 mL or 1.3 mL, dependent on tube availability), and clinical chemistry (approximately
1 mL) were collected from fasted animals via abdominal aorta (males) or vena cava
(females). Blood samples were collected on Day 92 of the dosing phase.
The anticoagulants were sodium citrate for coagulation tests and potassium EDTA for
hematology tests. Samples for clinical chemistry were collected without anticoagulant.
Urine samples for urinalysis were collected chilled during the overnight period before
blood collection from animals fasted overnight. Urine samples were collected on Day 92
of the dosing phase.

Hematology Tests
red blood cell (erythrocyte) count
hemoglobin
hematocrit
mean corpuscular volume
mean corpuscular hemoglobin
mean corpuscular hemoglobin concentration
red cell distribution width
absolute reticulocyte count
platelet count
white blood cell (leukocyte) count
absolute neutrophil count
absolute lymphocyte count
absolute monocyte count
absolute eosinophil count
absolute basophil count
absolute large unstained cell count
blood smear

Coagulation Tests
prothrombin time
fibrinogen
activated partial thromboplastin time

Clinical Chemistry Tests
glucose
urea nitrogen
creatinine
total protein
albumin
globulin
albumin:globulin ratio
total cholesterol
triglycerides
total bilirubin
low-density lipoproteins
aspartate aminotransferase
alanine aminotransferase
alkaline phosphatase
gamma glutamyltransferase
creatine kinase
calcium
inorganic phosphorus
sodium
potassium
chloride
high-density lipoproteins

Urinalysis Tests
appearance (clarity and color)
volume
specific gravity
pH
protein
glucose
ketones
bilirubin
blood
microscopic examination of sediment

Bone Marrow Smear Evaluation
Evaluation of bone marrow smears was not warranted based on other findings
(see Necropsy, Organ Weights, and Macroscopic Observations section for bone marrow
smear preparation).

Terminal Procedures
Necropsy, Organ Weights, and Macroscopic Observations
On Day 92 of the dosing phase, Animal R0006 (Group 1 male), having been fasted
overnight, was anesthetized with carbon dioxide, exsanguinated, and necropsied. On
Day 93 (remaining males) or 92 (females) of the dosing phase, all animals, having been
fasted at least 6 hours (males) or overnight (females), were anesthetized with carbon
dioxide, exsanguinated, and necropsied .
Terminal body weights were recorded for sacrificed animals. A macroscopic examination
of the external features of the carcass; external body orifices; abdominal, thoracic,
cranial, pelvic, and oral cavities; organs; and tissues was performed. A Pathologist was
available for consultation during necropsies.
Organ weights, as indicated in the following table, were recorded at each scheduled
sacrifice. Paired organs were weighed together.
Bone marrow smears (two slides) were prepared from the femur of each animal at
scheduled sacrifices.

Histology
As indicated in the previous table (Necropsy, Organ Weights, and Macroscopic
Observations section), the following animals had tissues (unless noted as missing)
embedded in paraffin and sectioned at a nominal 5 μm, and slides were prepared and
stained with hematoxylin and eosin.
 All animals in control and high-dose groups
 Kidney and macroscopic lesions from animals in low- and intermediate-dose groups
 Liver, testis, and epididymis from all males in low- and intermediate-dose groups
 Thymus and mesenteric lymph node from all females in low- and intermediate-dose
groups

Microscopic Observations
Tissues indicated in the previous table (Necropsy, Organ Weights, and Macroscopic
Observations section) from the following animals were examined microscopically by the
Principal Investigator for Anatomic Pathology.
 All animals in control and high-dose groups
 Kidney and macroscopic lesions from animals in low- and intermediate-dose groups
 Liver, testis, and epididymis from all males in low- and intermediate-dose groups
 Thymus and mesenteric lymph node from all females in low- and intermediate-dose
groups

Male Reproductive Assessment
Reproductive assessments were prioritized over histology collections in the case of
missing tissues.
Right vas deferens and right epididymis (unless noted as missing) were collected from
sacrificed males on Day 92 or 93 of the dosing phase; the left vas deferens and left
epididymis may be used, when necessary.

Vas Deferens Collection
For sperm motility evaluations, a single section (approximately 2 cm) of vas deferens was
excised and placed in a petri dish containing a pre-warmed mixture of Media 199 and
1% Bovine Serum Albumin (BSA). The volume of media (Media 199 and 1% BSA) for
analysis was approximately 10 mL.

Sperm Motility Evaluation
Following the dispersal period, a sample of sperm was collected and loaded into a
pre-warmed stage of the Hamilton Thorne IVOS automated sperm analyzer. Five
fields/animal were selected and stored as digital images. Initial parameters for motility
setup and gate settings for motility setup were performed according to SOPs. When gate
settings were changed by the Analyst to improve the accuracy of the analysis, it was
documented in the raw data. A minimum of 15 data points were used for the sort.

Epididymis Collection
After the epididymis was removed and weighed, the epididymis designated for sperm
concentration was placed on dry ice until transferred to a freezer, set to maintain -60 to
-80°C. Samples remained frozen until processed for sperm concentration determination.

Sperm Concentration Determination
Samples for sperm concentration (sperm/gram of epididymal tissue) determination were
prepared according to the processing procedure. A sample of the suspension from the
caudal section of the epididymis was stained with a dye that uniquely stains the sperm
heads. A sample of the stained sperm was placed into a chambered glass slide and loaded
into the Hamilton Thorne IVOS automated sperm analyzer. For each animal, one slide
chamber, with 19 fields/slide chamber, was counted and reported for each caudal
epididymal weight. Initial parameters for density setup and gate settings for density setup
were performed according to SOPs. When gate settings were changed by the Analyst to
improve the accuracy of the analysis, it was documented in the raw data.

Sperm Morphology Evaluation
Two slides of sperm/animal from the total count suspension, were stained with
hematoxylin and eosin stain, shipped to Labcorp-Madison, and evaluated (see Protocol
Deviations).
At least 200 spermatozoa for each sample were classified as normal (head and tailpiece
appeared normal) or abnormal.

Immunohistochemistry
Kidney blocks were shipped to Labcorp-Madison. Hematoxylin and eosin staining (males
and females) and immunohistochemistry (males only) were performed on tissues (see
Protocol Deviations). Tissues were immunolabeled for the detection of Alpha 2uglobulin.
Immunolabeled tissue sections were examined microscopically.
Immunohistochemistry procedures were optimized prior to performing
immunohistochemistry on study slides. Additional slides may be made and used for
method development or controls. Method development slides were not included with the
study data.
Statistics:
See additional information
Description (incidence and severity):
Vinyl 2-ethylhexanoate-related clinical observations for both sexes administered
1000 mg/kg/day included ataxia (one male on Days 6 and 8 and three females on Days 3
to 5) at 2 hours postdose. In addition, one female was noted with piloerection (Day 5),
walking on toes with increased activity (Days 3 and 4), and decreased body tone and
lower lip retraction (Days 3 to 5). Ataxia was limited to individual animals, did not
persist through the entire dosing phase, and did not limit the ability of animals to reach
food and water; thus, this finding was deemed VEHA related but nonadverse.
Other clinical observations included hair loss, scabbing, bent tail, and broken teeth. These
were noted infrequently, were transient, or were noted at comparable incidences as for
the controls; therefore, they were considered not VEHA related.
Description (incidence and severity):
Vinyl 2-ethylhexanoate-related alterations in body weight included nonsignificant but
lower mean body weight (9%) and decreased body weight gain (131 g) for males
administered 1000 mg/kg/day, compared with controls (172 g), over the course of the
13-week study. Decreased body weight gain was observed on Days 1 to 8, with
concomitant (nonsignificant) decreases in food consumption, compared with controls.
Following the first week, decreased body weight gain was observed intermittently from
Day 29 to 91). Vinyl 2-ethylhexanoate-related alterations in body weight for females
included decreased mean body weight for females administered 1000 mg/kg/day (8 to
10%), compared with controls, from Days 78 to 91. Additionally, females administered
1000 mg/kg/day were noted with decreased body weight gain (52 g), compared with
controls (78 g), over the course of the 13-week study primarily driven by nonsignificant
decreased weight gain from Days 29 to 57 and significant decreased in weight gain from
Days 57 to 91.
Description (incidence and severity):
Decreased food consumption (14.3%) was confined to Days 1 to 8 for males administered
1000 mg/kg/day, this likely contributed to low mean body weight gain, particularly for
the three animals that gained 7 g of body weight or less from Days 1 to 8. Females
administered 500 or 1000 mg/kg/day were noted with higher food consumption
intermittently that did not correlate with significant changes in body weight. Statistically
significant but not biologically relevant changes in food consumption in females was not
VEHA-related.
Description (incidence and severity):
No remarkable ophthalmic observations were noted.
Description (incidence and severity):
Vinyl 2-ethylhexanoate (VEHA)-related effects in hematology test results consisted of
minimally lower red cell mass (i.e., lower red blood cell count, hemoglobin
concentration, and hematocrit) - (males only), minimally lower total white blood cell
count (due to lower absolute neutrophil and/or lymphocyte counts), and minimally lower
platelet count noted for both sexes administered 1000 mg/kg/day. A clear mechanism for
these effects was not identified; however, may have been related to lower body weight
gain and potentially decreased anabolic processes.
No VEHA-related effects were identified in coagulation test results.
Description (incidence and severity):
Vinyl 2-ethylhexanoate (VEHA)-related effects in clinical chemistry test results were
minimal to mild in magnitude, dose-dependent, and noted for animals administered
≥250 mg/kg/day, but more pronounced in animals administered 1000 mg/kg/day. Serum
protein effects consisted of minimally to mildly decreased total protein concentration due
to decreased albumin and globulin concentrations noted for animals administered
1000 mg/kg/day. Lipid effects consisted of minimally to mildly higher cholesterol, high
density lipoprotein, and triglyceride concentrations noted for both sexes administered
≥500 mg/kg/day; mildly higher glucose concentration noted for females administered
≥500 mg/kg/day; and minimally lower low density lipoprotein concentration noted for
males administered ≥250 mg/kg/day. Clear mechanisms for these effects were not
identified; however, may have been related to lower body weight gain and potentially
decreased anabolic processes and/or altered lipid metabolism.
No VEHA-related effects were identified in thyroid hormone results.
Description (incidence and severity):
No VEHA-related effects were identified in urinalysis test results.
Description (incidence and severity):
No VEHA-related elicited or arena observations were noted for either sex. Average
forelimb grip strength was higher for males administered 250 or 500 mg/kg/day,
compared with the control group; however, this was only observed for 1 of 2 trials. The
lack of consistency across trials and because the observations were not dose responsive
suggested the higher forelimb grip strength was not VEHA related.
Vinyl 2-ethylhexanoate-related neurobehavioral observations included an increased
incidence of mildly decreased activity for males administered 1000 mg/kg/day, compared
with controls, which was consistent with locomotor activity finding of decreased activity.
Higher auditory startle response was noted for three females (Animals R0703, R0704,
and R0708) administered 1000 mg/kg/day, compared with controls, on Day 81; higher
auditory startle response was also noted for one female (Animal R0706) administered
1000 mg/kg/day prior to dosing, but this was not one of the animals noted with this
finding on Day 81. Two females (Animals R0505 and R0509) administered
250 mg/kg/day were also noted with increased auditory startle response, and one animal
from each group administered 250, 500, or 1000 mg/kg/day was observed with no
response. Therefore, a lack of a clear dose response or pattern of increased or no response
was not observed for VEHA-treated females, and this finding was likely not
VEHA related.
Other neurobehavioral observations included abnormal (increased or no) approach
response, abnormal (increased or no) auditory startle response for males, increased
extensor thrust, abnormal (increased or decreased) pain response, absent pinna response,
increased reactivity to handling, and mild vocalization to handling. These were noted
infrequently, were transient, or were noted at comparable incidences as for the controls;
therefore, they were considered not VEHA related.

Locomotor Activity
The statistical analysis of motor activity data is presented in the Locomotor Activity
Statistical Analysis Report.
VEHA-related changes in locomotor activity were noted in animals administered
1000 mg/kg/day and included decreased activity for all four measured activities (x + y
ambulations, fine movements, basic movements, and rearing). Decreased activity was
independent of sex and was noted as an overall effect and as a treatment by interval
interaction. Animals administered 500 mg/kg/day were also noted with decreased activity
for fine and basic movements, compared with the controls.
Animals administered 500 or 1000 mg/kg/day were observed with statistically significant
decreases in at least one of the following when assessed for the overall interval
(1 through 60 minutes), compared with the control: x + y ambulation (animals
administered 1000 mg/kg/day only), fine movements, basic movements, and rearing
(animals administered 1000 mg/kg/day only) (see Text Table 4.1). Sex differences or
interactions with treatment were not observed; all further analyses are based on combined
sex data.
Treatment by interval interactions include increased x + y ambulations (7 to 12%),
compared with the control group; in the first interval (1 to 5 minutes), this finding was
noted in all VEHA administered animals but statistically significant for animals
administered 250 or 1000 mg/kg/day. After the first interval, activity was decreased in
multiple measurements. Decreased x + y ambulations (32 to 85%) were noted in animals
administered 1000 mg/kg/day compared to controls. Treatment by interval interactions
noted decreases in fine movements, basic movements, and rearing activities at various
intervals for animals administered 500 mg/kg/day and 1000 mg/kg/day, with higher
magnitude and increased frequency at 1000 mg/kg/day. Animals administered
500 mg/kg/day were noted with decreases between 18 and 27% (fine movements), 14 and
18% (basic movements), and 18 and 30% (rearing activities) at various intervals. Animals
administered 1000 mg/kg/day were noted with decreased fine movements, basic
movements, and rearing activities at all intervals (except at 6 of 12 intervals for rearing
activity), with a magnitude of 28 to 84% (fine movements), 14 to 84% (basic
movements), and 54 to 100% (rearing activities).
Because of the dose-responsive decrease in activity, the consistency across all
measurements, and the magnitude of change (31 to 63% for animals administered
1000 mg/kg/day), the decreased activity was deemed a VEHA-related effect. Decreased
activity in the neurological examination part of the FOB evaluation was also noted for
individual males administered 1000 mg/kg/day. Due to the magnitude of the decreased
activity in animals administered 500 mg/kg/day (4 to 15%) and 1000 mg/kg/day (31 to
63%) decreased activity was deemed adverse only in animals administered
1000 mg/kg/day.
Description (incidence and severity):
Liver weights were increased in males administered ≥500 mg/kg/day and females
administered 1000 mg/kg/day. In males, the increased liver weights correlated with the
microscopic finding of centrilobular hepatocyte hypertrophy.
Kidney weights were increased in animals administered ≥500 mg/kg/day without a
microscopic correlate.
Thymus weights were decreased in females administered 1000 mg/kg/day and correlated
with the microscopic finding of decreased lymphocytes.
Epididymis, prostate, and prostate/seminal vesicle weights were decreased in males
administered 1000 mg/kg/day. These organ weight decreases correlated with tubular
degeneration/atrophy in the testes and cellular debris in the lumen of the epididymis.
The statistically significant increase in thymus weight parameters for females
administered 500 mg/kg/day was considered spurious and not VEHA related because no
microscopic correlates were observed and the individual organ weights were generally
within the range of individual control values.
Description (incidence and severity):
No VEHA-related macroscopic observations were observed.
Description (incidence and severity):
Minimal or slight centrilobular hepatocyte hypertrophy was noted for males administered
≥500 mg/kg/day, and minimal to moderate tubular degeneration/atrophy of the testes and
luminal cell debris of the left epididymis was observed in males administered
≥500 mg/kg/day. Hypertrophy of centrilobular hepatocytes was associated with increased
35
Labcorp Study Number 8461250
liver weights in males administered ≥500 mg/kg/day. Tubular degeneration/atrophy
within the testes and luminal cell debris within the left epididymis correlated with
decreased epididymis, prostate, and prostate/seminal vesicle organ weights in males
administered 1000 mg/kg/day.
Decreased lymphocytes were noted in females administered 1000 mg/kg/day and
occurred at slight or moderate severity in the thymus and moderate severity in the
mesenteric lymph node. Decreased lymphocytes in the thymus correlated with decreased
thymus weights in females administered 1000 mg/kg/day.
Kidneys were evaluated from all animals, and kidneys from males were examined with
immunohistochemistry for Alpha 2u-globulin. Although kidney weights were increased
in animals administered ≥500 mg/kg/day, no microscopic correlate was noted following
hematoxylin and eosin staining or Alpha 2u-globulin immunohistochemistry analysis.
Description (incidence and severity):
Estrous Cycle Determination
Vaginal smears were collected from individual females for 4 consecutive days. The
presence of a full estrous cycle could only be determined from one female administered
500 mg/kg/day (Animal R0604) and one female administered 1000 mg/kg/day
(Animal R0710). Thus, no VEHA-related effect on the female estrous cycle could be
determined. The number of animals at each stage of estrous on the day prior to being
sacrificed has been summarized below. No test item treated females were in preoestrus on
Day 91. On Day 91, VEHA treated females generally had comparable estrous stage
patterns to controls.

Sperm Analysis
A VEHA-related significant decrease in sperm motility and total sperm count was noted
for males administered 1000 mg/kg/day, compared with controls. Males administered
1000 mg/kg/day had decreased mean sperm concentration (density) compared to controls,
however, this was not statistically significant.
During the dosing phase, 3 of 10 males administered 1000 mg/kg/day had 0% motile
sperm, 5 of 10 males had ≤10% motile sperm, and 2 of 10 males had ≥42% motile sperm.
Changes in sperm parameters correlated with minimal to moderate tubular
degeneration/atrophy of the testes and luminal cell debris of the left epididymis was
observed in males administered ≥500 mg/kg/day and decreased epididymis, prostate, and
prostate/seminal vesicle organ weights in males administered 1000 mg/kg/day.
No VEHA-related effects on morphology were noted at any dose level.
Details on results:
Protocol Deviations
Procedure Protocol Deviations
Test Item and Vehicle Control Items
Vehicle Control Item
Formulation
On Day 6 (males)/5 (females) of the dosing phase, vehicle control item
formulations prepared for Group 1 were stored at room temperature
rather than in a refrigerator, set to maintain 2 to 8C.

Inlife Procedures
Dose Administration
On Day 6 (males)/5 (females) of the dosing phase, the formulation for
Group 1 males and several females in Group 1 was stirred for less than
the specified 30 minutes prior to dosing.
On Day 56 of the dosing phase, it could not be confirmed whether
Animal R0405 (Group 1 female) was administered its intended dose due
to a lack of documentation.
On Day 72 of the dosing phase, Animal R0009 (Group 1 male) was not
administered its full intended dose.

Clinical Observations
On Day 37 (males)/36 (females) of the dosing phase, an observation of
no remarkable observations was recorded for the a.m. daily observation,
although only abnormal observations were required to be recorded.
On Day 50 of the dosing phase, the 2-hour postdose observation for
Group 1 males was performed 2 hours 18 minutes postdose.
On multiple occasions, an observation of no remarkable observations
was recorded for the 2-hour postdose observation, although only
abnormal observations were required to be recorded. Individual instances
are recorded in the raw data.
On multiple occasions, the 2-hour postdose observations were not
documented. Individual instances are maintained in the data.

Estrous Cycle
Estrous cycle determination continued from Days 88 through 91 of the
dosing phase, rather than the specified range of Days 89 through 92 of the
dosing phase. Therefore, Day 92 (day of necropsy) was not included.

Thyroid Hormone and Serum Bile Acids Analyses
Compliance Statement
In addition to EPS Titles 40, Part 792, the Compliance Statement from
Principle Investigator for Thyroid Hormone and Clinical Pathology
(Serum Bile Acids) also claims compliance to FDA GLPs Title 21, Part
58, although not required.
Potential Hormone Analyses
Sample Collection and Handling
On 92 of the dosing phase, blood samples (approximately 1.0 mL) were
collected from fasted animals via vena cava for females rather than
specified abdominal aorta due to volume limitations.

Terminal Procedures
Necropsy, Organ Weights,
and Macroscopic
Observations
The head tissues from Animal R0404 (Group 1 female) and
Animal R0310 (Group 4 male) could not be distinguished due to a
mistake during trimming. Tissues were held in neutral-buffered formalin
until otherwise directed. Other tissues including brain, pituitary, eyes,
optic nerves were taken out prior to this happening.
On Day 92 of the dosing phase, the necropsy evaluation was performed
for Animal R0006 (Group 1 male) but was not documented due to an
equipment error.
On Day 92 of the dosing phase, the motility evaluation performed at the
time of necropsy for Animal R0006 (Group 1 male) was not able to
documented due to an equipment error. Necropsy day was moved from
Day 92 of the dosing phase to Day 93 of the dosing phase for males due
to unforeseen equipment errors. Therefore, males were fasted for
~6 hours on Day 93 of the dosing phase prior to necropsy.
On Day 93 of the dosing phase, it could not be confirmed whether the
eye/Harderian gland/optic nerve/testis of Animals R0002 and R0004 (Group 1
males), R0207 (Group 3 male), and R0302 (Group 4 male) were collected/stored
in modified Davidson’s fixative due to lack of documentations.
Only kidney tissues were required to transfer to 70% ethanol following
preservation in neutral-buffered formalin. On Day 93 of the dosing phase, due to
a documentation error, it could not be confirmed whether all tissues from males in
Groups 1 through 3, and Animals R0501 through R0505 (Group 2 females) were
also transferred to 70% ethanol rather than only kidney as required. However,
tissues were taken to slide and read by the pathologist with no concerns.

Histology
Kidney slides from females were inadvertently taken and marked to be
processed for immunohistochemistry, although not required.
Animals R0001and R0003 (Group 1 males) only had one slide of sperm were
stained with hematoxylin and eosin stain rather than two slides of
sperm/animal. Animal R0002 (Group 1 male) had no slides documented
as stained.

These study deviations neither affected the overall interpretation of study findings nor compromised the
integrity of the study.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
sperm measures
Critical effects observed:
not specified
Conclusions:
Male and female rats were administered vehicle control item or 250, 500, or
1000 mg/kg/day VEHA via oral gavage once daily. Vinyl 2-ethylhexanoate-related
observations in animals administered 1000 mg/kg/day included: nonadverse increased
incidence of ataxia in the first week; nonadverse decreased body weight gain; adverse
reduction in activity at Week 12; pronounced alterations in clinical chemistry and
hematology endpoints of minimally lower total white blood cell count, minimally lower
platelet count, decreased total protein concentration, higher cholesterol, higher highdensity
lipoprotein, and higher triglyceride; microscopic findings of centrilobular
hepatocyte hypertrophy (males), decreased lymphocytes (females), tubular
degeneration/atrophy of testes and luminal cell debris of the epididymis (males) with
correlated decreased organ weights; and a significant decrease in sperm motility (males).
For these reasons 1000 mg/kg/day was considered adverse when administered for
13 weeks. Vinyl 2-ethylhexanoate-related observations in animals administered
500 mg/kg/day included: nonadverse reduction in activity at Week 12, alterations in
clinical chemistry and hematology endpoints seen at 1000 mg/kg/day but at a lower
magnitude, centrilobular hepatocyte hypertrophy in males, tubular degeneration/atrophy
of the testes and luminal cell debris of the epididymis with no correlating organ weight
changes. Due to the mild severity of findings and the lack of an impact on the health and
wellbeing of animals administered 500 mg/kg/day, effects for this dose were considered
nonadverse when administered for 13 weeks. Thus, the no observed adverse effect level
(NOAEL) is 500 mg/kg/day.
Executive summary:

The purpose of this study was to evaluate the toxicity of the Test Item, vinyl
2-ethylhexanoate (VEHA), when administered daily via oral gavage to rats for at least
13 weeks.
Male and female Sprague Dawley (Hsd:Sprague Dawley SD) rats were assigned to
four groups, and doses were administered as indicated in the following table. Animals
were dosed via oral gavage once daily for 13 weeks at a volume of 5 mL/kg. The vehicle
control item was corn oil.



Dose Levelb Dose Concentrationb Number of Animals
Groupa (mg/kg/day) (mg/mL) Males Females
1 (Control) 0 0 10 10
2 (Low) 250 50 10 10
3 (Intermediate) 500 100 10 10
4 (High) 1000 200 10 10
a Group 1 was administered vehicle control item only
b Animals were dosed at a volume of 5 mL/kg



Assessment of toxicity was based on mortality, clinical observations, body weights, body
weight changes, food consumption, ophthalmic observations, functional observational
battery (FOB), locomotor activity, estrous cycle determinations, and clinical and
anatomic pathology. Blood samples were collected for thyroid hormone and serum bile
acids analyses.
All animals survived to their scheduled sacrifice.
No VEHA-related ophthalmic observations were noted.
Vinyl 2-ethylhexanoate-related ataxia was noted for animals administered
1000 mg/kg/day; ataxia was confined to one male on Days 6 and 8, and three females
(one on Days 3 and 4, one on Days 3-5, and one on Day 4. Ataxia was not observed after
Day 8 and was deemed nonadverse. Lower, but not significant, mean body weight (9%)
in males and decreased body weight gain in males and females was noted for animals
administered 1000 mg/kg/day, compared with controls. Mean body weight gain over the
13-week dosing phase were lower than controls by 24 and 33% for males and females
administered 1000 mg/kg, respectively. Although decreased body weight gain was
significantly lower, the transient nature, lack of additional correlating observations (such
as decreased body condition), and lack of a need for intervention decreased body weight
gain was considered nonadverse.
A VEHA-related neurobehavioral observation and locomotor activity finding of
decreased activity was noted. An increased incidence of mildly decreased activity for
males administered 1000 mg/kg/day, compared with controls was reported during the
FOB testing on Day 81. Animals administered 1000 mg/kg/day were noted with
decreased activity for all four locomotor measured activities (x + y ambulations,
fine movements, basic movements, and rearing). Decreased activity was independent of
sex and was noted as an overall effect and as a treatment by interval interaction. Animals
administered 500 mg/kg/day were also noted with decreased activity in fine and basic
movements, compared with controls. Due to the magnitude of the decreased activity
(31 to 63%) in animals administered 1000 mg/kg/day, it was considered adverse.
Vinyl 2-ethylhexanoate-related effects in clinical pathology test results were minimal to
mild in magnitude, dose-dependent, and noted for animals administered ≥250 mg/kg/day,
but more pronounced in animals administered 1000 mg/kg/day. Clear mechanisms for
these effects were not identified; however, may have been related to lower body weight
gain and potentially decreased anabolic processes and/or altered lipid metabolism.
Hematology effects consisted of minimally lower red cell mass (i.e., lower red blood cell
count, hemoglobin concentration, and hematocrit) - (males only), minimally lower total
white blood cell count (due to lower absolute neutrophil and/or lymphocyte counts), and
minimally lower platelet count noted for animals administered 1000 mg/kg/day. Clinical
chemistry effects were minimal to mild in magnitude and consisted of decreased total
protein concentration, due to decreased albumin and globulin concentrations noted for
both sexes administered 1000 mg/kg/day; higher cholesterol, high density lipoprotein,
and triglyceride, and glucose (females only) concentrations noted for both sexes
administered ≥500 mg/kg/day; and lower low density lipoprotein concentration noted for
males administered ≥250 mg/kg/day. No VEHA-related effects were identified in
coagulation, thyroid hormone, or urinalysis test results.
Vinyl 2-ethylhexanoate-related microscopic findings were noted in the liver, thymus,
mesenteric lymph node, testes, and epididymis, and these microscopic findings differed
by sex.
Minimal or slight centrilobular hepatocyte hypertrophy (correlated with increased liver
weights) was noted for males administered ≥500 mg/kg/day, and minimal to moderate
tubular degeneration/atrophy of the testes and luminal cell debris of the left epididymis
were observed in males administered ≥500 mg/kg/day. Tubular degeneration/atrophy in
the testes and luminal cell debris in the left epididymis correlated with decreased
epididymis, prostate, and prostate/seminal vesicle organ weights for males administered
1000 mg/kg/day.
Decreased lymphocytes were noted in females administered 1000 mg/kg/day and
occurred at a slight or moderate severity in the thymus (correlated with decreased thymus
weight parameters for females administered 1000 mg/kg/day) and at a moderate severity
in the mesenteric lymph node.
Vinyl 2-ethylhexanoate-related, increased kidney weights were noted for animals
administered ≥500 mg/kg/day but were noted without a microscopic correlate. Liver
weights were also increased for females administered 1000 mg/kg/day without a
microscopic correlate.
VEHA-related decrease in sperm motility, total sperm count, and sperm concentration
(density) was noted for males administered 1000 mg/kg/day. No VEHA-related effects on
morphology were noted at any dose level.
Male and female rats were administered vehicle control item or 250, 500, or
1000 mg/kg/day VEHA via oral gavage once daily. Vinyl 2-ethylhexanoate-related
observations in animals administered 1000 mg/kg/day included: nonadverse increased
incidence of ataxia in the first week; nonadverse decreased body weight gain; adverse
reduction in activity at Week 12; pronounced alterations in clinical chemistry and
hematology endpoints of minimally lower total white blood cell count, minimally lower
platelet count, decreased total protein concentration, higher cholesterol, higher highdensity
lipoprotein, and higher triglyceride; microscopic findings of centrilobular
hepatocyte hypertrophy (males), decreased lymphocytes (females), tubular
degeneration/atrophy of testes and luminal cell debris of the epididymis (males) with
correlated decreased organ weights; and a significant decrease in sperm motility, sperm
count, and nonsignificant decreases in sperm density (males). For these reasons
1000 mg/kg/day was considered adverse when administered for 13 weeks. Vinyl
2-ethylhexanoate-related observations in animals administered 500 mg/kg/day included:
nonadverse reduction in activity at Week 12, alterations in clinical chemistry and
hematology endpoints seen at 1000 mg/kg/day but at a lower magnitude, centrilobular
hepatocyte hypertrophy in males, tubular degeneration/atrophy of the testes and luminal
cell debris of the epididymis with no correlating organ weight changes. Due to the mild
severity of findings and the lack of an impact on the health and wellbeing of animals
administered 500 mg/kg/day, effects for this dose were considered nonadverse when
administered for 13 weeks. Thus, the no observed adverse effect level (NOAEL) is
500 mg/kg/day.

Endpoint conclusion
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Dose descriptor:
NOAEC
500 mg/m³

Additional information

Justification for classification or non-classification

Criteria of EU Directive 67/548/EEC and the CLP (Directive 1272/2008) for systemic target organ Classification and Labeling were not met by Vinyl 2 -ethylhexanoate.