Amines, (2-ethylhexyl)(hydrogenated tallow alkyl)methyl

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 27, 2016 to May 25, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

1078712-76-1

Molecular formula:

C23-27H49-59N (Substance is a UVCB)

Test material form:

liquid

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Crl:CD(SD) rats were used as the test system for this study. This species and strain of animal is
recognized as appropriate for repeated dose toxicity studies. The Sprague Dawley rat was
selected because it is a widely used strain for which significant historical control data are
available. The number of animals used is the minimum necessary to yield scientifically
meaningful results.
Crl:CD(SD) rats (65 males and 65 females) were received in good health from Charles River
Laboratories, Inc., Raleigh, NC, on 28 Jul 2016. The animals were approximately 37 days old at
receipt. Each animal was examined by a qualified technician on the day of receipt and weighed
on the following day. Each animal was uniquely identified by a Monel® metal ear tag displaying
the permanent identification number or with a subcutaneous microchip (BMDS system)
implanted in the dorsoscapular area. All animals were housed for a 14-day acclimation. During
acclimation, each animal was observed twice daily for mortality and changes in general
appearance or behavior.
Data collection during acclimation began on 29 Jul 2016. Individual body weights and cage food
weights were recorded and detailed physical examinations were performed periodically during
acclimation. Ophthalmic examination data were also recorded for animals during acclimation.
All animals were housed throughout acclimation and during the study in an environmentally
controlled room. The room temperature and relative humidity controls were set to maintain
environmental conditions of 73°F ± 5°F (23°C ± 3°C) and 50% ± 20%, respectively. Room
temperature and relative humidity data were monitored continuously and were scheduled for
automatic collection on an hourly basis. Actual
mean daily temperature ranged from 71.8°F to 73.6°F (22.1°C to 23.1°C) and mean daily relative
humidity ranged from 36.5% to 55.0% during the study. Fluorescent lighting provided
illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. The light
status (on or off) was recorded once every 15 minutes. The 12-hour light/12-hour dark
photoperiod was interrupted as necessary to allow for the performance of protocol-specified
activities. Air handling units were set to provide a minimum of 10 fresh air changes per hour.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity over the concentration range of 20.0 to 200 mg/mL for at least
10 days at room temperature (18°C to 24°C) was previously established. However, the
validated method was extended in this study for the analysis of formulations at concentrations as
low as 1.00 mg/mL in order to verify dose formulation concentrations in this study.
Analyses to determine homogeneity and stability of the dosing formulations were conducted
prior to the initiation of dosing. Test substance formulations of sufficient volume for dosing a
group of animals for at least 8 days were prepared. Four 1.0 mL samples were collected from the
top, middle, and bottom stratum of the 2 and 200 mg/mL dosing formulations and from the
middle stratum of the control and 8 and 40 mg/mL dosing formulations. Two samples from each
strata were analyzed using a method validated for concentration in each stratum to assess the
homogeneity of the test substance in the mixtures and used as time-zero data for comparison to
results obtained from later stability samples; the remaining samples were stored at room
temperature. After sampling of the bulk formulations for homogeneity and/or stability, a single
aliquot from the 2 and 200 mg/mL dosing formulations, sufficient in volume for dosing a group
of animals for 1 day, were stored under appropriate conditions. Following at least 6 and 10 days
of storage, and after remixing for a minimum of 30 minutes using a magnetic stirrer, four 1.0 mL
samples were collected from the top and bottom stratum of the formulations. Two samples from
each stratum were analyzed to assess the homogeneity of the test substance in the mixtures after
storage and resuspension; the remaining samples were stored at room temperature as backup
samples. Stability was evaluated by comparing the inter-strata means to the overall
concentrations from the bulk homogeneity data (time-zero).
Concentration of the test substance in the dosing formulations, including the vehicle, was
assessed on the first, fourth, eighth, and last dosing formulations prepared. The 2 and
200 mg/mL dosing formulation samples collected for homogeneity from the middle stratum of
the first dosing formulation preparation were used for concentration verification for Study
Week 0. Four 1.0 mL samples were collected from the middle stratum of the control and each
test substance formulation. Two samples from each stratum were analyzed to assess the
concentration of the test substance in the mixtures; the remaining samples were stored at room
temperature as backup samples.
All analyses were conducted by the Charles River Analytical Chemistry Department using a
validated high performance liquid chromatography method using charged aerosol detection
(CAD)

Duration of treatment / exposure:

90 or 91 consecutive days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

vehicle control

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

40 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

200 mg/kg bw/day (nominal)

Doses / concentrations 5

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10; 15 for vehicle control and 1000 mg/kg/day dose group (5 each/sex for recovery group).

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

Clinical examinations were performed at the time of dose administration and 1 to 2 hours
following dose administration. During the recovery period, the animals were observed once
daily.
Individual body weights were recorded 1 week (± 2 days) prior to randomization, on the day of
randomization, on Study Day 0 (prior to dosing), weekly (± 2 days) during the study period, and
on the day prior to the first day of the scheduled necropsies (nonfasted).

Sacrifice and pathology:

A complete necropsy was conducted on all animals. Animals were euthanized by carbon dioxide
inhalation followed by exsanguination. The necropsies included, but were not limited to,
examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic
cavities, including viscera.
Blood and urine samples for clinical pathology evaluations (hematology, coagulation, serum
chemistry, and urinalysis) were collected from all animals assigned to the scheduled necropsies. The animals were fasted overnight prior to blood collection
while in metabolism cages for urine collection. Blood was collected for hematology and serum
chemistry evaluation from a jugular vein and for coagulation parameters at the time of euthanasia
via the vena cava of animals euthanized by inhalation of carbon dioxide. Blood was collected
into tubes containing potassium K2EDTA (hematology), sodium citrate (coagulation), or no
anticoagulant (serum chemistry).

Other examinations:

FOB assessments were recorded for all animals prior to dose administration during Study
Week 12.
Motor activity was assessed for all animals during Study Week 12.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related clinical observations were limited to the 1000 mg/kg/day group and
included clear, red, and yellow material on various body surfaces (anogenital area, eye[s], mouth,
nose, ventral trunk, and/or urogenital area), hair loss (facial area, dorsal head, forelimb[s],
hindlimb[s], dorsal neck, ventral neck, dorsal trunk, ventral trunk, and/or urogenital area), and
reddened and/or swollen facial area. These observation were generally noted near the end of the
dosing period. Additional test substance-related clinical observations of thin body condition and
unkempt appearance were noted for a select 1000 mg/kg/day group females near the end of the
dosing period and partial closure of right eye was noted for Female No. 5961 (1000 mg/kg/day
group) on Study Day 82.
All other clinical observations in the test substance-treated groups were noted with similar
incidence in the control group, were limited to single animals, were not noted in a dose-related
manner, and/or were common findings for laboratory rats of this age and strain.

Mortality:

mortality observed, treatment-related

Description (incidence):

Seven test substance administered rats were euthanized in extremis or were found dead prior to
scheduled euthanasia.
Of these, 3 deaths, which included 1 male euthanized due to deteriorated clinical
condition/moribundity in the 1000 mg/kg/day group and 2 females found dead or euthanized
in extremis in the 200 mg/kg/day group, were considered test substance-related due to test
substance-related erosion/ulceration of the nonglandular stomach or degeneration/regeneration in
the jejunum.
Two rats had inflammation in the thoracic cavity, which was considered the cause of
moribundity/death, and 1 of these had a lesion of the esophagus consistent with esophageal
perforation which likely lead to thoracic cavity inflammation.1 These 2 deaths were considered
procedural.
Two of the deaths and the relationship to test substance administration were undetermined.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related lower mean cumulative body weight gains (statistically significant
compared to the control group) were noted beginning as early as Study Week 0 to 2 in the
1000 mg/kg/day group males, which continued until the end of the dosing period. At the end of
the dosing period (Study Week 13), the mean body weight in the 1000 mg/kg/day group males
was 9.8% lower than the mean control group body weight. During the recovery period,
cumulative body weights in the 1000 mg/kg/day group males were comparable to the control
group.
In addition, Female No. 5971 (200 mg/kg/day group) and Male No. 5914 and Female No. 5924
(1000 mg/kg/day group) were noted with a significant amount of body weight loss (at least 20%)
and were euthanized in extremis. Female No. 5929 (1000 mg/kg/day group) also lost a
substantial amount of weight from Study Weeks 2 to 4 and was found dead during Study
Week 4.

Food efficiency:

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ophthalmic lesions indicative of toxicity were observed in any of the test substance-treated
groups

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related changes in hematology parameters consisted of a dose dependent increase
in mean absolute reticulocyte counts in the 200 and 1000 mg/kg/day group males and in the 10,
40, 200, and 1000 mg/kg/day group females (maximum increase of 26% and 101%,
respectively). The effect was more pronounced in females. White blood cells were statistically
significantly elevated in females in the 1000 mg/kg/day group by 49%. Neutrophils were
elevated in males and females in the 1000 mg/kg/day group by 61% and 53%, respectively. In
addition, lymphocytes, monocytes, and platelets (minimal increase) were elevated in females in
the 1000 mg/kg/day group by 48%, 59%, and 13%, respectively.
Mean reticulocyte counts in the 1000 mg/kg/day group males and females were similar to the
control group at the end of the recovery period. No other test substance-related changes in
hematology or coagulation parameters were observed compared to the control group for primary
or recovery groups.
Non-test substance-related statistically significant differences were noted, including minimal
decreases in hemoglobin in the 1000 mg/kg/day group males. In addition, there were minimal
decreases in mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration, and
a small increase in basophils in the 1000 mg/kg/day group females. These differences were not
considered test substance-related due to the very small change observed compared to control
group and were attributed to animal variability.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related changes in serum chemistry parameters included minimal statistically
significant reductions in total protein, albumin, globulin, cholesterol, triglycerides (not
statistically significant), and calcium in the 1000 mg/kg/day group males and females. In
addition, alanine aminotransferase and aspartate aminotransferase were slightly higher compared
to the control group in the 1000 mg/kg/day group females. There were no other test substancerelated
changes in serum chemistry parameters.
At the end of the recovery period, albumin remained lower in females (-12%) in the
1000 mg/kg/day group compared to the control group. This resulted in a statistically significant
decrease in the albumin/globulin ratio for this group. There were no other test substance-related
changes observed at the end of the recovery period.
Non-test substance-related statistically significant differences compared to the control group
were noted, including a reduction in serum glucose in the 1000 mg/kg/day group males at the
primary necropsy, higher alkaline phosphatase in the 1000 mg/kg/day group males at the
recovery necropsy, and lower bilirubin in the 1000 mg/kg/day group females at the recovery
necropsy. These changes were small in magnitude and only seen in a few animals in each
respective group and therefore attributed to individual animal variability and not to the test
substance.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis parameters were unaffected by test substance administration

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The following observations were unaffected: handling, open field, sensory, neuromuscular, physiological. Motor activity patterns were unaffected by test substance.

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Organ weight changes considered test substance-related included higher spleen, adrenal gland,
liver, and kidney weights in 1000 mg/kg/day primary necropsy group females.
Test substance-related higher mean absolute and relative (to final body and brain weights) spleen
weights were noted in the 1000 mg/kg/day primary necropsy group females. The weights were
statistically significantly higher, above the Charles River Ashland historical control ranges, and
compatible with statistically significantly higher mean absolute reticulocyte counts in the
1000 mg/kg/day primary necropsy group females.
Mean absolute and relative spleen weights of the 200 and 1000 mg/kg/day primary necropsy
group males also tended to be higher, but most values were not statistically significantly
different, and all values remained within the Charles River Ashland historical control ranges.
Statistically significantly higher mean absolute reticulocyte counts were noted in the hematology
parameters of the 1000 mg/kg/day primary necropsy group males which were compatible with
higher spleen weights in this group. Because spleen weights were higher in the corresponding
1000 mg/kg/day primary necropsy group females, and both 1000 mg/kg/day group males and
females had high reticulocyte counts, but male spleen weight values were within the historical
control ranges and most were not statistically significant, the relationship of the trend in higher
spleen weights of the 200 and 1000 mg/kg/day group males to the test substance was considered
equivocal.
There were no corresponding microscopic alterations in the spleen of males or females, and
spleen weights of males and females (and corresponding reticulocyte counts) at the recovery
necropsy were restored.
Test substance-related statistically significantly higher mean absolute and relative (to final body
and brain weights) adrenal gland weights were noted in the 1000 mg/kg/day primary necropsy
group females. The values were above the Charles River Ashland historical control ranges. The
higher weights were compatible with cytoplasmic vacuolation or hypertrophy/hyperplasia of the
adrenal cortex noted in some 1000 mg/kg/day group females. These findings were considered
likely results of stress associated with test substance-related lesions in the stomach and/or lung.
Absolute and relative mean adrenal gland weights of the 1000 mg/kg/day group females
remained higher at the recovery necropsy, but the mean percent difference values at the recovery
necropsy for the 1000 mg/kg/day group females were all decreased compared to corresponding
percent differences at the primary necropsy, indicating some recovery following the nondosing
period.
Test substance-related higher mean absolute and relative liver and kidney weights (most
statistically significantly higher) were observed in the 1000 mg/kg/day primary necropsy group
females. The liver weight values exceeded the Charles River Ashland historical control ranges,
whereas the kidney weight values remained within the ranges. There were no corresponding
microscopic changes in either organ. The higher liver weights were compatible with the slightly
but statistically significantly higher alanine aminotransferase and aspartate aminotransferase
values of the 1000 mg/kg/day primary necropsy group females. At the Study Week 16/17
(recovery) necropsy, the mean absolute weight values and organ to brain weight ratio values for
the kidneys and liver of the 1000 mg/kg/day group females were all resolved. The mean organ to
final body weight values of the liver and kidneys were still slightly high, but considered to be the
result of a test substance-related effect on final body weight, which had not yet fully recovered.
The percent differences for the mean values of liver and kidney weights at the recovery necropsy
for the 1000 mg/kg/day group females were all decreased compared to corresponding percent
differences at the primary necropsy, which also supported organ weight recovery following the
nondosing period.
Some organ weight differences were statistically significant when compared to the control
groups, but were considered a result of a test substance-related effect on final body weights.
These included the Study Week 12/13 higher mean brain to final body weight ratio and higher
mean kidney to final body weight ratio values of the 1000 mg/kg/day group males, and a higher
mean ovaries to final body weight ratio value of the 1000 mg/kg/day group females. At the
recovery necropsy, a higher mean brain to final body weight ratio value of the 1000 mg/kg/day
recovery necropsy group males was also considered the result of a test substance-related effect
on final body weight.
There were no other test substance-related effects on organ weights. However, some statistically
significant differences were observed when the control and test substance treated groups were
compared. At the recovery necropsy, lower mean absolute pituitary gland weight and lower
mean pituitary to brain weight ratio values of the 1000 mg/kg/day recovery necropsy group
males were attributed to biological variation because these values were not altered in the primary
necropsy groups, and values were within the Charles River Ashland historical control ranges.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related findings of edematous stomach were noted in 1 male in each of the
200 and 1000 mg/kg/day primary necropsy groups, and in two 1000 mg/kg/day primary necropsy
group females.
Test substance-related enlarged adrenal glands were noted in two 1000 mg/kg/day primary
necropsy group females and were consistent with higher mean adrenal gland weights and
microscopic cytoplasmic vacuolation or hypertrophy/hyperplasia in the adrenal cortices in this
group.
There were no gross necropsy observations at the recovery necropsy considered test
substance-related.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related microscopic findings were noted in the nonglandular stomach, jejunum,
mesenteric lymph node, and lungs of various test substance-treated primary necropsy group
males and females, and in the adrenal cortex of 1000 mg/kg/day primary necropsy group
females. At the recovery necropsy, microscopic changes in the nonglandular stomach were
resolved, but persisted in the jejunum, mesenteric lymph node, and lungs of males and females,
and the adrenal cortex of females.

Histopathological findings: neoplastic:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

other: observations in the stomach that were due to direct, site-of-contact injury (erosion and ulcerations of non-glandular stomach).

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

200 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Applicant's summary and conclusion

Conclusions:

The notified substance is not expected to cause target organ toxicity based on an 90-day oral gavage OECD 408 study. The NOAEL assigned of 40 mg/kg/day is based on observations in the stomach that were due to direct, site-of-contact injury (erosion and ulcerations of non-glandular stomach). The two deaths in females that occurred at the 200 mg/kg/day dose group were also attributed to this severe irritation in the stomach. The results from the skin irritation study support that this material may cause severe irritation in the gastro-intestinal system over time.

Additional test-item related findings were typically concluded as being non-adverse by the study director and primarily occurred in the 1000 mg/kg/day group. These findings included:

• Decreased body weight and gain.
• Increased reticulocyte counts that were associated with spleen weight. No microscopic alterations occurred in the spleen and both spleen weight and reticulocyte counts were similar to controls after the recovery period.
• Elevated white blood cells attributed to an inflammatory response.
• Increased liver weight that was likely associated with elevated alanine aminotransferase and aspartate aminotransferase; no changes from control were observed in these parameters after the recovery period and this was likely an adaptive response.
• Increased adrenal gland weight, which was likely associated with microscopic changes (cytoplasmic vacuolation or hypertrophy/hyperplasia). These effects were reduced after the recovery period but still not considered to have returned to normal. These effects were attributed to stress by the study director.
• Increased kidney weight but with no microscopic changes. Kidney weights returned to normal after the recovery period.
• Jejunum: microscopic changes characteristic of degeneration but not considered adverse by the study director, primarily as function was not impacted.
• Microscopic changes in mesenteric lymph nodes; considered non-adverse by the study director.
• Histologic changes in the lungs associated with dosing and/or reflux of an irritating material.

Executive summary:

The objectives of this study were to evaluate the potential toxicity of Amines (2-ethylhexyl)(hydrogenated tallow alkyl) methyl when administered daily by oral gavage to Sprague Dawley rats for 90 consecutive days, as well as to evaluate the recovery, persistence, or progression of any effects following a minimum of a 28-day recovery period.

Three test substance-related deaths occurred prior to the scheduled necropsy. Two 200 mg/kg/day group females were found dead or euthanized in extremis and considered to have a probable cause of death from stomach erosion/ulceration and one 1000 mg/kg/day group male was euthanized in extremis and found to have jejunum degeneration/regeneration. Test substance-related clinical observations were consistent with deteriorating physical condition in the 1000 mg/kg/day group and included clear, red, and yellow material on various body surfaces, hair loss, and reddened and/or swollen facial area. In addition, 1000 mg/kg/day group females were noted with thin body condition and unkempt appearance as well as partial closure of the eyes. Body weight gains were negatively impacted by the test substance in the 1000 mg/kg/day group males during the study period with recovery following the end of dosing. Test substancerelated effects on neurobehavioral assessments were limited to observations of soiled fur and red deposits around the nose and mouth for females in the 1000 mg/kg/day group.

There were test substance-related changes in hematology including increases in reticulocytes in the 200 and 1000 mg/kg/day group males and 10, 40, 200 and 1000 mg/kg/day group females that corresponded with higher spleen weights in the 1000 mg/kg/day group males and females at the primary necropsy. In addition, there were elevated neutrophils (males and females in 1000 mg/kg/day group) and lymphocytes, monocytes, and platelets in females in the

1000 mg/kg/day group. The pattern of elevated white blood cells in the high dose females was consistent with an inflammatory response. Test substance-related changes in serum chemistry parameters included minimal statistically significant reductions in total protein, albumin, globulin, cholesterol, triglycerides (not statistically significant), and calcium in the 1000 mg/kg/day group males and females. In addition, ALT and AST were slightly increased

compared to the control group in the 1000 mg/kg/day group females which corresponded with higher liver weights in this group at the primary necropsy.

Test substance-related changes in organ weights included higher spleen, adrenal gland, kidney, and liver weights in the 1000 mg/kg/day primary necropsy group females. Test substance-related histopathology changes included nonadverse edema of the nonglandular stomach in the 200 and 1000 mg/kg/day group males and 10, 200, and 1000 mg/kg/day group females that corresponded to macroscopic findings of edematous stomach during the primary necropsy. Test substance-related adverse erosion/ulceration of the nonglandular stomach was observed in the 200 and 1000 mg/kg/day group males and females (including the females that were found dead or euthanized in extremis prior to the scheduled necropsy); adverse degeneration of the jejunum (1000 mg/kg/day group male euthanized in extremis); nonadverse cytoplasmic vacuolation of mononuclear cells in the jejunum (1000 mg/kg/day group males and

females) and mesenteric lymph node (10, 40, 200, and 1000 mg/kg/day group males and females); and nonadverse cytoplasmic vacuolation of the adrenal cortex in the 1000 mg/kg/day primary necropsy group females. In addition, adverse granulomatous/pyogranulomatous inflammation was observed in the lungs of various test substance-treated primary necropsy group males and females and was considered related to test substance administration for all primary necropsy groups. Since the observed lung inflammation was likely a result of reflux of test substance into the lungs during oral gavage dosing, this finding was not to be used for determination of the no-observed-adverse-effect level (NOAEL).

Relationships were suspected between gross necropsy, organ weight, clinical pathology, and histopathology observations. These proposed relationships were based on subjective interpretation rather than a statistical analysis of correlation. Enlarged adrenal glands observed at necropsy corresponded with increased adrenal gland weight and histopathologic findings of cytoplasmic vacuolation or hypertrophy/hyperplasia in the adrenal cortex. This finding was determined to be secondary to test substance induced stress. Edematous nonglandular stomach was observed at necropsy and correlated with histopathology findings of test substance attributed edema in the nonglandular stomach. Increased spleen weights in the 1000 mg/kg/day group males and females correlated with increased reticulocyte counts and increased liver weight in the 1000 mg/kg/day group females corresponded with minimally increased ALT and AST levels in this group. Additionally, changes in the jejunum may impact nutrient absorption and correlate with reductions seen in total protein, albumin, globulin, cholesterol and triglycerides in the 1000 mg/kg/day group males and females.