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

Endpoint summary

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

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.

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

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

vehicle control

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

40 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

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

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.

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

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).

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

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.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

August 8, 2016 to May 31, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Sexually mature male and virgin female Sprague Dawley [Crl:CD(SD)] rats were used as the test
system on this study. The animal model, the Crl:CD(SD) rat, is recognized as appropriate for
reproductive toxicity studies and has been proven to be susceptible to the effects of reproductive
toxicants.

Route of administration:

oral: gavage

Vehicle:

corn oil

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test item homogeneity and stability following at least 10 days of room temperature storage at
concentrations of 20 to 200 mg/mL were previously established. An extension validation of test
item homogeneity and stability at a concentration of 2.00 mg/mL was conducted in a concurrent
study to cover the lowest concentration used in the current study.
Prior to the initiation of dose administration, samples for homogeneity and/or concentration
determination were collected from the top, middle, and bottom strata of the first 2 and
200 mg/mL formulations and from the middle stratum of the control, 8, and 40 mg/mL
formulations. In addition, samples for resuspension homogeneity and stability determinations
were collected from the top and bottom strata of an aliquot taken from the 2 and 200 mg/mL
suspensions following room temperature storage for 5 and 11 days; aliquots were mixed for a minimum of 30 minutes prior to sample
collection. Samples for concentration analyses were also collected from the middle stratum of
the middle and last formulations (including the control group) prepared during the study. One
set of samples from each collection was subjected to the appropriate analyses. All remaining
samples were stored at room temperature as back-up. All analyses were conducted by the
Charles River Analytical Chemistry Department using a validated high performance liquid
chromatography method using charged aerosol detection.

Duration of treatment / exposure:

The males were dosed during Study Days 0 through 27 or 28 (14 days prior to pairing
through 1 day prior to scheduled euthanasia) for a total of 28–29 doses. The females were dosed
from Study Day 0 through the day prior to euthanasia (14 days prior to pairing through Lactation
Day 13) for a total of 49–53 doses. Females that failed to deliver were dosed through the day
prior to euthanasia (Post-Mating Day 25) for a total of 39–41 doses.

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

vehicle control

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

40 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

Clinical observation and survival - twice daily and 1-2 hours post-dosing
Body weights - weekly and more frequently for females during gestation
Food consumption - weekly
Estrous cycles

Sacrifice and pathology:

Clinical pathology (hematology, coagulation, serum chemistry) collected from 5 animals/sex/group at scheduled necropsies.
Complete necropsy condcuted on all parental animals

Other examinations:

FOB assessments for 5 animals/sex/group following 28 days of dose administration
Motor activity for 5 animals/sex/group following 28 days of dose administration
Thyroid hormone analysis

Clinical signs:

no effects observed

Mortality:

mortality observed, treatment-related

Description (incidence):

3 females in the 1000 mg/kg/day were euthanized in extremis

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related, significantly (p < 0.05) higher mean absolute neutrophil counts were noted in
the 1000 mg/kg/day group F0 males and correlated with histopathologic findings in the lungs and
nasal cavity.
Test item-related, significantly (p < 0.05 or p < 0.01) higher mean absolute neutrophil counts
were also noted in the 10, 40, and 200 mg/kg/day group F0 females. There were no
histopathologic correlates and, thus, the cause of the increase in neutrophil counts was uncertain
in the females.
There were no other test item-related effects on hematology or coagulation parameters.
However, some statistically significant differences were observed when the control and test
item-treated groups were compared.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related, significantly (p < 0.05 or p < 0.01) lower mean serum albumin values were
noted in the 200 and 1000 mg/kg/day group F0 males. Test item-related, significantly (p < 0.05
or p < 0.01) lower mean serum protein values were also noted in the 200 and 1000 mg/kg/day
group F0 males and were considered secondary to the lower mean albumin values.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Test item-related, significantly (p < 0.01) lower mean heart weights (absolute and relative to
final body weight) were noted in the 200 mg/kg/day group F0 females. There were no
corresponding histopathologic correlates or dose-response pattern. This finding was not
considered adverse.

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were noted in the non-glandular stomach, lungs, and nasal
cavity of the 1000 mg/kg/day group F0 males and females and in the glandular stomach, thymus,
spleen, Peyer’s Patches, mesenteric lymph node, bone marrow (sternum), and adrenal cortex of
the 1000 mg/kg/day group F0 females.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

mortality

other: adverse microscopic findings (hyperplasia, ulceration, necrosis, and neutrophil inflammation in the glandular and non-glandular stomach and decreased lymphoid cellularity in the thymus and spleen)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: absence of systemic toxicity (highest dose tested)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Test item-related effects occurred on the physical condition and survival of F0 females in the

1000 mg/kg/day group. Three females in the 1000 mg/kg/day group were euthanized in extremis

during the breeding period (Study Day 21) or during gestation (Gestation Days 10 and 21)

following adverse clinical signs (thin, pale, and/or cool body) and/or severe body weight losses

prior to euthanasia. At necropsy, a cause of death could not be determined for these females, but

associated test item-related microscopic findings included the following, some of which are

considered to be adverse: hyperplasia in nonglandular stomach, nasal degeneration, neutrophil

inflammation in the nasal cavity, decreased lymphoid cellularity in the thymus and/or

mandibular lymph node, decreased cellularity in the bone marrow; vacuolation and hypertrophy

of the zona fasciculate in adrenal gland cortex; and mixed cell inflammation and type II

pneumocyte hyperplasia in the lungs. One female in the 1000 mg/kg/day group was euthanized

early soon after parturition due to a total litter loss on Postnatal Day (PND) 0. This female was

noted with pale body and cool extremities and microscopic findings, including ulceration,

necrosis and neutrophil inflammation in glandular and non-glandular stomach and intestine;

presence of bacterial and fungal organisms, indicating that the animal was immunosuppressed,

and findings in lymph node, bone marrow, and adrenal cortex attributed to stress response. The

6 remaining females in this group failed to deliver and were necropsied on Post-Mating Day 25;

2 were found to have been gravid. The microscopic findings for these females are summarized

in the text table below. Because the moribundity and unscheduled deaths noted at

1000 mg/kg/day occurred only in females at the highest dosage level and were associated with

clinical and microscopic findings, they were considered test item-related.

All F0 females in the control, 10, 40, and 200 mg/kg/day groups and all F0 males in the control,

10, 40, 200, and 1000 mg/kg/day groups survived to the scheduled necropsies; no test

item-related clinical observations were observed in these same groups at the daily examinations,

weekly detailed examinations, or approximately 1–2 hours following dose administration at any

dosage level.

Mean body weights, body weight gains, and food consumption for F0 females were unaffected by

test item administration during the pre-mating period (Study Days 0–13). In the 200 and

1000 mg/kg/day group F0 females, mean body weight gains were similar to the control group

during Gestation Days 0-7 followed by lower mean body weight gains during the remainder of

gestation (Gestation Days 7-11 and 17-20 at 200 mg/kg/day and Gestation Days 7-20 at

1000 mg/kg/day). The decrements in mean body weight gain during the latter half of gestation

resulted in lower mean body weight gains at 200 and 1000 mg/kg/day when the entire gestation

treatment period (Gestation Days 0–20) was evaluated compared to the control group. In

addition, mean body weights in the 200 and 1000 mg/kg/day groups were 6.9% and 17.3%

lower, respectively, than the control group on Gestation Day 20. The aforementioned body

weight differences at 1000 mg/kg/day correlated with 3 females being euthanized in extremis

during breeding or gestation, the failure of 6 females to deliver (due to totally resorbed litters or

being nongravid), and a total litter loss for 1 female on the same day parturition was complete.

The test item-related effects on mean female body weight at 200 mg/kg/day during gestation

were transient and of small magnitude (≤ 6.9%; not statistically significant), and therefore not

considered adverse. Mean food consumption in the 1000 mg/kg/day group was slightly lower

than the control group during Gestation Days 14-20; mean food consumption was similar to the

control group during Gestation Days 0–14 at 1000 mg/kg/day and throughout gestation at

200 mg/kg/day. All females in the 1000 mg/kg/day group were euthanized in extremis during

the breeding period or gestation, failed to deliver either due to resorbed litters or being

nongravid, or had a total litter loss.

In the 1000 mg/kg/day group, there were no F0 females available for the FOB or motor activity

assessments on Lactation Day 13; therefore, evaluation of these parameters was precluded for

females in this group. No test item-related effects were noted during the FOB or motor activity

evaluations at any dosage level for males and females evaluated in the F0 generation.

Test item-related histopathological findings of squamous cell hyperplasia and hyperkeratosis

(1000 mg/kg/day F0 males only) in the non-glandular stomach were noted in F0 males at 200 and

1000 mg/kg/day and in F0 females at 1000 mg/kg/day. Epithelial degeneration accompanied by

neutrophil inflammation was also noted in the non-glandular stomach of F0 males at

200 mg/kg/day, while mixed inflammation with type II pneumocyte hyperplasia in the lungs and

epithelial degeneration with neutrophil inflammation in the nasal cavity were noted in F0 males

and females at 1000 mg/kg/day. The aforementioned findings were considered due to local

irritation and correlated with increased neutrophil counts in the 1000 mg/kg/day group F0 males.

Test item-related macrophage hyperplasia in the medulla of the mesenteric lymph node was also

noted in the 1000 mg/kg/day group F0 males and females. All of the aforementioned findings

were not considered adverse.

Indirect, likely stress-related, test item-related findings of decreased lymphoid cellularity

(thymus, spleen, Peyer’s patches, mesenteric lymph node); decreased bone marrow cellularity;

increased vacuolation and hyperplasia of the adrenal gland; and ulceration, necrosis, and

neutrophil inflammation of the glandular and non-glandular stomach were noted in 1 F0 female at

1000 mg/kg/day that had a total litter loss. The findings in the glandular and non-glandular

stomach, thymus, and spleen were considered adverse, while findings in the Peyer’s patches,

mesenteric lymph node, bone marrow, and adrenal cortex were not considered adverse.

Additional test item-related clinical pathology findings in the F0 generation included lower mean

serum albumin and total protein values in the 200 and 1000 mg/kg/day group males, lower mean

T4 values in the 40, 200, and 1000 mg/kg/day group males, and higher mean neutrophil counts in

the 10, 40, and 200 mg/kg/day group females. Test item-related lower mean heart weights were

noted in the 200 mg/kg/day group females. Alterations in the serum albumin and total protein

values and heart weights were not considered adverse. The adversity regarding the alterations in

T4 values and neutrophil counts could not be ascertained without additional information;

however, there were no correlating histopathologic findings or alterations in organ weights.

Conclusions:

Based on adverse clinical findings, severe body weight deficits
during gestation, and adverse microscopic findings (hyperplasia, ulceration, necrosis, and
neutrophil inflammation in the glandular and non-glandular stomach and decreased lymphoid
cellularity in the thymus and spleen) , the no-observed-adverse-effect
level (NOAEL) for F0 female systemic toxicity was considered to be 200 mg/kg/day. While test
item-related effects on F0 female body weight and food consumption were noted at
200 mg/kg/day, the differences were small, transient, and not considered to be adverse. Based
on the absence of systemic toxicity for the F0 males at any dosage level, the NOAEL for male
systemic toxicity was considered to be 1000 mg/kg/day, the highest dosage level tested. While
histopathological findings in the non-glandular stomach were noted in F0 males at 200 and
1000 mg/kg/day, these findings were attributed to local irritation of the test item and not
considered adverse. Clinical pathology findings in the F0 generation included lower mean serum
albumin and total protein values in the 200 and 1000 mg/kg/day group males, lower mean T4
values in the 40, 200, and 1000 mg/kg/day group males, and higher mean neutrophil counts in
the 10, 40, and 200 mg/kg/day group females. Lower mean heart weights were also noted in the
200 mg/kg/day group females. Alterations in the serum albumin and total protein values and
heart weights were not considered adverse. The adversity regarding the alterations in T4 values
and neutrophil counts could not be ascertained without additional information; however, there
were no correlating histopathologic findings or alterations in organ weights.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Justification for classification or non-classification

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.