Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Alcohols, C11-14-iso-, C13-rich
EC Number:
271-235-6
EC Name:
Alcohols, C11-14-iso-, C13-rich
Cas Number:
68526-86-3
Molecular formula:
CnH2n+1OH (n = 11 to 14)
IUPAC Name:
Branched alcohols, C11-14, C13 rich
Details on test material:
- Name of test material (as cited in study report): Exxal 13
- Physical state: clear colorless liquid
- Stability under test conditions: stable at room temperature
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The Crl:CD(SD) rat was chosen as the animal model for this study as it is an accepted rodent species for preclinical toxicity testing by regulatory agencies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Crl:CD(SD) rats were received from Charles River Laboratories, Inc., Raleigh, NC. The animals were 7 weeks old and weighed 158 g to 281 g at the initiation of dosing.

- Housing: Animals were group housed (2 to 4 animals of the same sex) until randomization. Following randomization, animals were group housed (2 animals of the same sex and same dosing group together) in solid-bottom cages containing appropriate bedding
- Diet: PMI Nutrition International Certified Rodent LabDiet® 5002 (meal) was provided ad libitum
- Water: Municipal tap water after treatment by reverse osmosis was freely available to each animal via an automatic watering system
- Acclimation period:

ENVIRONMENTAL CONDITIONS:
- Temperature: 68°F to 78°F (20°C to 26°C)
- Humidity (%): 30-70%
- Air changes: 10 or greater air changes per hour with 100% fresh air (no air recirculation)
- Photoperiod (hrs dark/hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
The test substance and vehicle formulations were administered once daily for at least 90 consecutive days orally by gavage.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test substance formulations were previously shown to be stable and homogenous over the range of concentrations used on this study for at least 4 days at room temperature. Therefore, stability and homogeneity assessments were not conducted as part of this study.

Dose formulation samples were collected for concentration analysis as follows: all concentration groups were analyzed on the first, sixth, thirteenth, and last preparations. Analyses were performed by gas chromatography with flame ionization detection using a validated analytical procedure.

Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% of theoretical concentration.
Duration of treatment / exposure:
90 consecutive days
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
10mg/mL concentration, 10mL/kg dose volume
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
30mg/mL concentration, 10mL/kg dose volume
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
100mg/mL concentration, 10mL/kg dose volume
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Based on evidence from structurally related substances and acute toxicity data on the test substance, the low dose was expected to be a no-observed-effect-level (NOEL). A single dose acute toxicity test by the Sponsor concluded that the LD50 was greater than 2000 mg/kg and a 7 day repeat dose study concluded that the LD50 was greater than 1000 mg/kg for this test material. In a 2-week oral study, 5 male rats were dosed orally (gavage) with 144 mg/kg/day (1 mmol/kg/day) of isononanol following a 1 week acclimation. Animals were sacrificed after 14 days and blood was analyzed for plasma cholesterol and triglycerides. The liver was removed for histopathological analysis, analysis of catalase, and CN-insensitive palmitoyl CoA oxidation. Testicular weight was also determined. The rats did not develop testicular atrophy, liver enlargement, hepatic peroxisome induction, or hyperlipidemia. The no-observed-adverse-effect level (NOAEL) for isotridecanol was set at the limit dose of 144 mg/kg/day. Data from a 7 day oral repeat dose study with 2 ethylhexanol reported that animals treated with 1000 mg/kg bw/day had a decreased body weight, increased weight of liver, stomach, kidneys. However, animals at 330 and 100 mg/kg bw/day showed no changes. In a 14-day toxicity study with once daily oral administration of isotridecanol to male and female rats at dosage levels of 150, 400, and 1000 mg/kg/day, adverse findings of tubule degeneration, necrosis, granular casts, and hyaline droplet accumulation in the kidneys of male rats at 400 and 1000 mg/kg/day. The high dose of 1000 mg/kg/day was selected based on the results of these studies and toxicokinetic data, and was expected to produce some effects.

Examinations

Observations and examinations performed and frequency:
Throughout the study, animals were observed for general health/mortality and moribundity twice daily.

Animals were weighed individually within 4 days of receipt, on the day of randomization, on Day 1 (prior to dosing), weekly (± 2 days) during the study period, and on the day prior to the first day of the scheduled necropsy. A fasted weight was recorded on the day of necropsy.

Food consumption:
Food consumption was quantitatively measured once weekly (± 2 days) following randomization and continuing throughout the dosing period.
Sacrifice and pathology:
Opthalmic Examinations:
Ocular examinations were conducted for all animals during the pretreatment period (Day -11) and for all animals in Groups 1 and 4 near the end of the dosing period (Day 85).

Clinical Pathology:
Hematology parameters assessed are as follows: total leukocyte count, erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, reticulocyte count, percent reticulocytes, absolute reticulocytes, differential leukocyte count (percent and absolute of neutrophil, lymphocyte, monocyte, eosinophil, basophil, large unstained cell), red cell distribution width, platelet estimate, red cell morphology.

Coagulation parameters assessed in plasma included activated partial thromboplastin time and prothrombin time.

Serum chemistry parameters assessed included albumin, total protein, globulin [by calculation], albumin/globulin ratio (A/G Ratio) [by calculation], total bilirubin, urea nitrogen, creatinine, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma glutamyltransferase, glucose, total cholesterol, calcium, chloride, phosphorus,potassium, sodium, sorbitol dehydrogenase, triglycerides, and appearance (degree of hemolysis, lipemia, and icterus.

Urinalysis parameters included specific gravity, pH, total volume, color, clarity, protein, glucose, ketones, bilirubin, occult blood.

Terminal Procedures:
All animals underwent necropsy, tissue collection, and organ weight recording on day 91/92. Controls and the high dose group underwent full tissue histology and histopathology assessment, with the low and middle dose groups subject to gross lesion and target tissue analysis. Necropsy examination included evaluation of all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.

Organ Weights:
The organs identified as follows were weighed at necropsy for all animals. Paired organs were weighed together. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated. Organs weighed at necropsy: adrenal glands, brain, epididymides (total and cauda), heart, kidneys, liver, ovaries with oviducts, pituitary, prostate with seminal vesicles, spleen, testes, thymus, thyroid with parathyroids, and uterus.

Tissue Collection: The following tissues were collected and preserved for further analysis: adrenal glands, aorta, bone with marrow, femur, sternum, bone marrow smear (from sternum), brain, cervix, epididymides, eyes with optic nerves, gastrointestinal tract, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, heart, kidneys, larynx, liver, lung, lymph nodes (axillary, mandibular, mesenteric), ovaries with oviducts, pancreas, peripheral nerve, Peyer's patches, pharynx, pituitary, prostate, mandibular salivary glands, skeletal muscle, skin with mammary gland, spinal cord (cervical, lumbar, thoracic), spleen, testes, thymus, thyroid with parathyroids, tongue, trachea, uterus, urinary bladder, vagina.
Other examinations:
Spermatogenic Evaluations:
Sperm was analyzed for determination of sperm motility. Motile and nonmotile spermatozoa per animal in all groups was analzyed. The motility score (percent) for motile (showing motion only) was reported.

The right epididymis was then analyzed for sperm morphology via evaluation by light microscope. Abnormal forms of sperm (double heads, double tails, microcephalic, or megacephalic, etc.) from a differential count of 200 spermatozoa per animal, if possible, were recorded.

The left testis and cauda epididymis from all males were weighed, stored frozen, homogenized, and analyzed for determination of homogenization resistant spermatid count and calculation of sperm production rate.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and will be reported at the 1% and 5% levels.

Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics (number, mean and standard deviation (or S.E. when deemed appropriate)) were reported whenever possible. Values may also be expressed as a percentage of predose or control values when deemed appropriate. Inferential statistics were performed for body weight, food consumption, organ weights, body weight gain, organ weight relative to terminal body weight, clinical pathology data, and spermatogenic parameters when possible, but excluded semi-quantitative data and any group with less than 3 observations. Each dosed group was compared pairwise to the control group.

Clinical pathology data, sperm production rate, and epididymal and testicular sperm numbers were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the control group. Percentages of motile sperm and percentages of sperm with normal morphology were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the nonparametric ANOVA revealed significant (p < 0.05) intergroup variance, Dunn’s test was used to compare the test substance-treated groups to the control group.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Wet fur was observed in 9/10 1000 mg/kg/day treated males and 10/10 1000 mg/kg/day treated females.

All other clinical observations were not considered test substance-related due to a lack of a dose-response, similarity to control groups, or were commonly observed in Sprague Dawley rats.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related lower mean body weight gains were noted in the 1000 mg/kg/day group males throughout the study period (statistically significantly lower on Days 15 to 22 and Days 29 to 36) compared to the control group. The mean body weights were significantly lower in the 1000 mg/kg/day group males on Days 43 and 50, and dropped as low as 9.9% below the concurrent control group on Day 78.

There were no other test substance-related effects on body weight. A statistically significant higher mean body weight gain was noted in the 1000 mg/kg/day group females from Day 1 to 8 when compared to the concurrent control group; however, this difference was likely due to biological variability and was not considered related to test substance administration due to lack of a dose response trend.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption was unaffected by test substance administration. However, some statistically significant differences were observed when the control and test substance-treated groups were compared during the study period. Statistically significant lower mean food consumption was noted from Day 8 to 15 and Day 22 to 29 in the 100 and 1000 mg/kg/day group males, from Day 29 to 36 and Day 36 to 43 in the 100 mg/kg/day group males. Statistically significant higher mean food consumption was noted from Day 8 to 57 and Day 64 to 90 in the 1000 mg/kg/day group females, from Day 29 to 50 and Day 85 to 90 in the 300 mg/kg/day group females, and from Day 43 to 50 in the 100 mg/kg/day group females when compared to the concurrent control group. These differences were likely due to biological variability and were not considered related to test substance administration.
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant lower RBCs, HCT, and HGB were measured in 1000mg/kg/d females compared to concurrent controls. PT was significantly higher in 1000mg/kg/d males, but was significantly lower in 300 and 1000mg/kg/d females compared to concurrent controls. RDW was significantly higher in both 300 and 1000mg/kg/d males and 1000mg/kg/d females compared to concurrent controls.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related slightly lower mean red blood cell mass (mean erythrocyte count, mean hemoglobin, and mean hematocrit) was noted in the 1000 mg/kg/day group males and females. The lowered indicators of red blood cell mass were not considered adverse due to the slight magnitude of change. Additionally, the mean values were within the Charles River Ashland historical control range. Statistically significant higher red cell distribution widths were noted in the 300 mg/kg/day group males and 1000 mg/kg/day group males and females; however, these differences were within the Charles River Ashland historical control range and were not considered related to test substance administration.

Coagulation parameters were unaffected by test substance administration. However, some statistically significant differences were observed when the control and test substance-treated groups were compared. A higher mean prothrombin time was noted in the 1000 mg/kg/day group males and a lower mean prothrombin time was noted in the 300 and 1000 mg/kg/day group females when compared to the concurrent control group at Week 13. These differences were within the Charles River Ashland historical control range (version 3.6) and were not considered related to test substance administration.

Test substance-related effects on serum chemistry parameters included higher mean albumin, total protein, and globulin values in the 1000 mg/kg/day group males and females, lower mean chloride values in the 1000 mg/kg/day group males and 300 and 1000 mg/kg/day group females, adverse higher mean urea nitrogen values in the 1000 mg/kg/day males and nonadverse slightly higher mean urea nitrogen values in the 100, 300, and 1000 mg/kg/day group females, higher mean creatinine and alkaline phosphatase values in the 1000 mg/kg/day group males, and higher mean cholesterol values in the 1000 mg/kg/day group females.

Other statistically significant differences were observed when the control and test substance treated groups were compared, however, these differences were not considered related to test substance administration and were attributed to biologic variation because they were of a magnitude of change commonly observed in rats of this age and strain under similar study conditions.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Urinalysis parameters were unaffected by test substance administration. However, some statistically significant differences were observed when the control and test substance-treated groups were compared. Higher mean specific gravity was noted in the 100 mg/kg/day group males, lower mean pH was noted in the 100 and 300 mg/kg/day group males, and higher mean total volume was noted in the 1000 mg/kg/day group males and females. These differences were not considered related to test substance administration and were attributed to biologic variation because they were of a magnitude of change commonly observed in rats of this age and strain under similar study conditions.
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related organ weight changes were noted in the kidneys (males and females), liver (males and females), thyroid/parathyroid glands (males and females), adrenal glands (females), heart (females), and pituitary gland (females) in the 100, 300, and/or 1000 mg/kg/day groups.

Statistically significant, dose-dependent, higher kidney weights (absolute and relative to terminal body and brain weights) were identified in the 300 and 1000 mg/kg/day group males and females, with multiple weights out of the normal range for the Charles River Ashland historical control database. Higher kidney weights correlated histologically with tubular degeneration/necrosis, regeneration, granular casts, and the accumulation of hyaline droplets within the proximal tubules of the 300 and 1000 mg/kg/day group males.

Statistically significant, dose-dependent, higher liver weights (absolute and relative to terminal body and brain weights) were identified in the 300 and 1000 mg/kg/day group males and females, with numerous weights out of the normal range for the Charles River Ashland historical control database. Higher liver weights correlated histologically with hepatocellular hypertrophy in the 300 and 1000 mg/kg/day group males and females.

Test substance-related higher thyroid/parathyroid gland weights were noted in the 100, 300, and 1000 mg/kg/day group males and females, with multiple weights in all dose groups out of the normal range for the Charles River Ashland historical control database. Higher thyroid/parathyroid gland weights (absolute and relative to brain weight) were statistically significant in the 300 mg/kg/day group females. Higher thyroid/parathyroid gland weights correlated histologically with minimal follicular cell hypertrophy/hyperplasia in the thyroid gland in the 100 (females only), 300, and 1000 mg/kg/day group males and females.

Dose-dependent, higher adrenal weights (absolute and relative to terminal body and brain weights) were identified in the 300 and 1000 mg/kg/day group females. Higher adrenal weights were statistically significant in the 300 (absolute and relative to brain weight) and 1000 (absolute and relative to terminal body and brain weights) mg/kg/day group females. There were no histologic correlates.

Statistically significant, dose-dependent, higher heart weights (absolute, total body, and brain weights) were identified in the 1000 mg/kg/day group females. There were no test substance related histologic correlates.

Higher pituitary gland weights were identified in the 1000 mg/kg/day group females, with 4 of 10 pituitary gland weights out of the normal range for the Charles River Ashland historical control database. Higher pituitary weights were not statistically significant, but were dose dependent. There were no histologic correlates.

Finally, some organ weight differences were statistically significant when compared to the control group, but were considered to be the result of a test substance-related effect on final body weight. Higher brain weight, right epididymis weight, and prostate/seminal vesicle weight (relative to terminal body weight) were statistically significant in the 1000 mg/kg/day group males, but are likely the result of a statistically-significant lower terminal body weight, as all values were within the normal range for the Charles River Ashland historical control database and there were no histologic correlates.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related dark red discoloration was noted in the kidneys of 2 males in the 1000 mg/kg/day group. These changes correlated histologically to mild renal tubular degeneration and necrosis; granular casts and tubular regeneration were also observed histologically in the kidneys of these animals.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related microscopic findings were noted in the liver, kidneys, thyroid glands, and lungs of the 100, 300, and/or 1000 mg/kg/day group males and/or females. These findings were considered to be related to non-human relevant pathways or were nonadverse in nature.

Other effects:
no effects observed
Description (incidence and severity):
Spermatogenic evaluations:

No test substance-related changes in spermatogenesis or sperm morphology were detected on this study. Lower progressive sperm motility was statistically significant in the 1000 mg/kg/day group males at the terminal necropsy, but was within the Charles River Ashland historical control reference range (IVOS version 14). The lowered value was not considered test substance-related because it was attributed to a single animal (No. 4006), and lacked correlating pathology findings; further, there were no statistically significant differences on any other measured sperm parameters.

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Final Body Weight

 

          Males

          Females
  Dosage (mg/kg/d)  0  100  300  1000  0  100  300  1000
 Mean Bodyweight (g) at end of study 632.0  597.9 617.4 563.4* 304.4   298.2 310.1 313.4
 %Diff  N/A -5.39 -2.31 -10.6   N/A -2.04 1.87 2.96 

Final body weight data was subjected to a one-way analysis of variance (ANOVA). If a statistically significant difference (p<0.05) is present in the ANOVA, a comparison of the control group to each treated group by Dunnett's test followed. All analyses were two-tailed for significance levels of 5% and 1%.

Statistical Test: Anova & Dunnett's Test

Group Factor Test: Analysis of Variance p < 0.05

Test: Dunnett 2 Sided p < 0.05

N/A = NOT APPLIC

Anova & Dunnett: * = p ≤ 0.05

Haematological Parameters

            Males           Females
 Dosage (mg/kg/d)  0  100  300  1000  0  100  300  1000
Number of Animals Assessed  10  9  10  9  10  10  10  10
White Blood Cells (thousands/uL) - Mean  9.94 8.99 9.13 11.56  7.13 7.34 7.00 6.58 
  %Diff  N/A -9.6 -8.1   16.3   N/A 2.9 -1.8 -7.7
Red Blood Cells (millions/uL) - Mean 9.31 9.18   9.25 9.02  8.48 8.64 8.30  7.90**
  %Diff   N/A -1.4  -0.6 -3.1    N/A 1.9 -2.1 -6.8
Hemoglobin (g/dL) - Mean 16.4 16.0 16.0 15.5 15.5 15.4 14.9 14.1** 
  %Diff   N/A -2.4 -2.4 -5.5   N/A -0.6 -3.9 -9.0
Hematocrit (%) - Mean 50.6 49.5 49.6 48.4 47.4 46.9 45.7 42.9**
  %Diff   N/A -2.2 -2.0  -4.3   N/A -1.1 -3.6 -9.5
Mean Corpuscular Volume (femtoliters) - Mean 54.4 53.9 53.7 53.6 55.9 54.3 55.1 54.3
  %Diff   N/A -0.9 -1.3 -1.5    N/A -2.9 -1.4  -2.9
Mean Corpuscular Hemoglobin (picograms) - Mean  17.6 17.4 17.3 17.2  18.3 17.8 18.0 17.8
  %Diff   N/A  -1.1 -1.7  -2.3   N/A -2.7 -1.6 -2.7
Mean Corpuscular Hemoglobin Concentration (g/dL) - Mean  32.3   32.4 32.2 32.1   32.8 32.8 32.6 32.8
  %Diff   N/A  0.3 -0.3   -0.6   N/A  0.0  -0.6 0.0
Platelet Count (thousands/uL) - Mean 1033 1012  937  972 990 1004 990   1006
  %Diff   N/A  -2.0 -9.3 -5.9   N/A 1.4  0.0  1.6
Prothrombin Time (seconds) - Mean   16.3 17.2a 17.0 17.8**a  16.7  16.3 15.7** 14.7** 
  %Diff   N/A 5.5 4.3 9.2   N/A -2.4 -6.0 -12.0
Activated Partial Thromboplastin Time (seconds) - Mean 15.2 15.7a 15.1  15.4a  15.5 15.4  14.8  13.5
  %Diff   N/A 3.3 -0.7 1.3  N/A  -0.6 -4.5 -12.9
Reticulocyte Count (%) - Mean  2.2 2.4  2.3 2.3 2.4 2.2 2.1 2.0
  %Diff   N/A 9.1 4.5 4.5   N/A -8.3 -12.5 -16.7
Reticulocyte Absolute (thousands/uL) - Mean 202.7 215.8 211.4 211.1 200.5   186.1 175.8 155.8 
  %Diff   N/A 6.5 4.3 4.1   N/A -7.2 -12.3 -22.3 
Neutrophil Count (%) - Mean 23.5 16.5 22.7  21.4 14.4 16.4 16.7 16.7
  %Diff   N/A -29.8 -3.4 -8.9   N/A 13.9  16.0 16.0
Lymphocyte Count (%) - Mean  71.5 80.1 72.8  75.2 82.1 79.6  79.7  78.7
  %Diff   N/A 12.0 1.8 5.2   N/A  -3.0 -2.9 -4.1 
Monocyte Count (%) - Mean 3.0  1.9** 2.6  2.2  2.1 2.3 1.9 2.8
  %Diff   N/A -36.7  -13.3  -26.7   N/A 9.5 -9.5 33.3 
Eosinophil Count (%) - Mean  1.6 1.0 1.5 0.8 1.0 1.2 1.2 1.5
  %Diff   N/A -37.5  -6.3 -50.0   N/A 20.0  20.0 50.0
Basophil Count (%) - Mean  0.2  0.2  0.2 0.1   0.1 0.1 0.1 0.1
  %Diff   N/A 0.0  0.0  -50.0   N/A 0.0 0.0    0.0
Large Unstained Cell Count (%) - Mean 0.3  0.3 0.3  0.3 0.3 0.4 0.4  0.4 
  %Diff   N/A 0.0  0.0 0.0    N/A 33.3 33.3  33.3
Neutrophil Absolute (thousands/uL) - Mean 2.55 1.45 2.05 2.47 1.11  1.17 1.12  1.07
  %Diff   N/A -43.1  -19.6 -3.1    N/A 5.4 0.9 -3.6 
Lymphocyte Absolute (thousands/uL) - Mean 6.90 7.24  6.66 8.69 5.77 5.88 5.63 5.21 
  %Diff   N/A 4.9  -3.5  25.9   N/A 1.9 -2.4  -9.7
Monocyte Absolute (thousands/uL) - Mean  0.31 0.18 0.24 0.26  0.15 0.15  0.14 0.18
  %Diff   N/A -41.9 -22.6  -16.1   N/A 0.0 -6.7 20.0
Eosinophil Absolute (thousands/uL) - Mean 0.15 0.08 0.14 0.09  0.07 0.09 0.08 0.09
  %Diff    N/A -46.7 -6.7   -40.0   N/A 28.6 14.3 28.6
Basophil Absolute (thousands/uL) - Mean 0.02 0.02 0.02 0.01 0.1 0.1 0.1 0.01 
  %Diff   N/A 0.0 0.0 -50.0   N/A 0.0  0.0   0.0
Large Unstained Cell Absolute (thousands/uL) - Mean 0.02  0.03 0.03 0.03 0.02  0.03  0.03 0.03 
  %Diff   N/A 50.0 50.0   50.0   N/A 50.0 50.0 50.0 
Red Cell Distribution Width (%) - Mean  12.1 12.1 12.7* 12.9**  11.0 10.9 11.3 11.5**
  %Diff   N/A 0.0 5.0 6.0    N/A -0.9 2.7 4.5

Haematology data was subjected to a one-way analysis of variance (ANOVA). If a statistically significant difference (p<0.05) is present in the ANOVA, a comparison of the control group to each treated group by Dunnett's test followed. All analyses were two-tailed for significance levels of 5% and 1%.

Statistical Test: Anova & Dunnett's Test

Group Factor Test: Analysis of Variance p < 0.05

Test: Dunnett 2 Sided p < 0.05

* = Significantly different from the control group at 0.05 using Dunnett's test

** = Significantly different from the control group at 0.01 using Dunnett's test

a = data from 10 animals

N/A = NOT APPLICABLE

Clinical Chemistry Parameters

            Male           Female
 Dosage (mg/kg/d)  100  300  1000  0  100  300  1000
 Number of Animals Assessed  10  10  10  10  10  10  10  10
Albumin (g/dL) - Mean  4.1  4.0 4.2 4.5**  4.8 4.7 5.1  5.4*
  %Diff N/A  -2.4 2.4 9.8  N/A  -2.1 6.3  12.5
Total Protein (g/dL) - Mean  6.9  6.8 7.0 7.4* 7.8 7.6 8.0 8.7**
  %Diff  N/A  -1.4 1.4 7.2  N/A  -2.6 2.6  11.5
Globulin (g/dL) - Mean  2.7 2.7  2.8 3.0 2.9 2.9 3.0 3.3** 
  %Diff  N/A  0.0  3.7 11.1  N/A  0.0 3.4 13.8
Albumin/Globulin Ratio - Mean  1.51  1.49 1.48 1.51 1.64 1.64 1.72 1.65
  %Diff  N/A  -1.3  -2.0 0.0  N/A  0.0 4.9 0.6 
Total Bilirubin (mg/dL) - Mean  0.0 0.0 0.0 0.0 0.03 0.0 0.01 0.00
  %Diff  N/A  N/A N/A N/A  N/A  -100.0 -66.7  -100.0
Urea Nitrogen (mg/dL) - Mean 8.5 9.0 9.8 11.7** 9.8 11.4 11.3  11.6 
  %Diff  N/A   5.9 15.3 37.6  N/A 16.3 15.3 18.4 
Creatinine (mg/dL) - Mean  0.36 0.36 0.40 0.50** 0.44 0.41 0.44 0.44
  %Diff   N/A 0.0 11.1 38.9  N/A -6.8 0.0 0.0
Alkaline Phosphatase (International Unit/L) - Mean  163 153 160 213  104 75 74 58
  %Diff  N/A  -6.1 -1.8 30.7  N/A  -27.9 -28.8 -44.2
Alanine Aminotransferase (International Unit/L) - Mean   37 36 38 43 61 28 28 43
  %Diff  N/A  -2.7 2.7 16.2  N/A  -54.1 -54.1 -29.5
Gamma Glutamyltransferase (International Unit/L) - Mean    0.0 0.0 0.0 0.0  0.0   0.0 0.0 0.0 
  %Diff  N/A    N/A   N/A  N/A   N/A    N/A  N/A   N/A 
Glucose (mg/dL) - Mean 106 108 109 108 107 108 115 101
  %Diff   N/A 1.9 2.8 1.9  N/A  0.9 7.5  -5.6
Cholesterol (mg/dL) - Mean  62 49** 58 64 75 70 82 121**
  %Diff  N/A  -21.0 -6.5 3.2  N/A  -6.7 9.3 61.3
Calcium (mg/dL) - Mean 10.6 10.4  10.5 10.7  11.2  10.9 11.1 11.5 
  %Diff

N/A

-1.9 -0.9 0.9   N/A -2.7 -0.9 2.7
Chloride (mg/dL) - Mean  106  105 104*  103** 106 106 104* 101**
  %Diff
 N/A
-0.9 -1.9 -2.8   N/A  0.0 -1.9  -4.7
Phosphorus (mg/dL) - Mean 7.5 7.7 7.6  8.3* 6.6 6.5  6.5  6.0
  %Diff N/A 2.7 1.3 10.7  N/A  -1.5 -1.5 -9.1
Potassium (Millequivalents/L) - Mean 5.92 6.24  5.85 6.00  5.49 5.37 5.39 5.00 
  %Diff  N/A  5.4 -1.2 1.4  N/A  -2.2 -1.8 -8.9 
Sodium (Millequivalents/L) - Mean 146 146 146  146 145 145 144 144
  %Diff  N/A  0.0 0.0 0.0   N/A  0.0 -0.7 -0.7
Sorbitol Dehydrogenase (International Unit/L) - Mean  9 3  7 20  6 7 13
  %Diff  N/A  -66.7 -22.2 -11.1  N/A  -70.0 -65.0 -35.0 
Triglyceride (mg/dL) - Mean 70 69 89 77 58 36 75 89 
  %Diff  N/A  -1.4 27.1 10.0  N/A  -37.9 29.3 53.4 
 Aspartate Aminotransferase (International Unit/L) - Mean 110 103 100 101 137 85 85 92
  %Diff   N/A -6.4 -9.1 -8.2 N/A -38.0 -38.0  -32.8

Clinical chemistry data was subjected to a one-way analysis of variance (ANOVA). If a statistically significant difference (p<0.05) is present in the ANOVA, a comparison of the control group to each treated group by Dunnett's test followed. All analyses were two-tailed for significance levels of 5% and 1%.

Statistical Test: Anova & Dunnett's Test

Group Factor Test: Analysis of Variance p < 0.05

Test: Dunnett 2 Sided p < 0.05

* = Significantly different from the control group at 0.05 using Dunnett's test

** = Significantly different from the control group at 0.01 using Dunnett's test

N/A = NOT APPLICABLE

Organ Weight Changes

Organ Weight Changes

(Absolute =g,

RBW & RBrW =%)

 Male              Female           
Dosage (mg/kg/d)  0  100  300  1000  0  100  300  1000

Brain (Number

Animals Assessed)

10   10  10  10 10   10  10  10

Absolute 

(%Diff)

2.2236

2.2982

(3.35)

2.2200

(-0.16)

2.2701

(2.09)

2.0111

1.9982

(-0.64)

1.9924

(-0.93)

1.9906 

(-1.02)

  RBW

(%Diff)

0.35426

0.38591

(8.93)

0.36099

(1.90)

0.40626**

(14.68)

0.66551

0.67203

(0.98)

0.64714

(-2.76)

0.63866 

(-4.03)

  RBrW  

(%Diff)

 N/A  N/A   N/A   N/A   N/A   N/A   N/A   N/A 

Adrenal

Gland

 (Number

Animals Assessed)

 10  10  10  10 10   10  10  10

  Absolute 

(%Diff)

0.07205

0.06527

(-9.41)

0.06352

(-11.84)

0.06368

(-11.62)

0.07077

0.07742

(9.40)


0.08285*

(17.07)

 0.08912**

(25.93)

  RBW 

(%Diff)

0.01156

0.01098

(-5.01)

0.01033

(-10.6)

0.01142

(-1.22)

0.02352

0.02605

(10.73)

0.02668

(13.44)

0.02844**

(20.93)

  RBrW 

(%Diff)

3.23911

2.84576

(-12.14)

2.86650

(-11.5)

2.81418

(-13.11)

3.52328

3.89126

(10.44)

4.15966*

(18.06)

4.47257**

(26.94) 

Pituitary

Gland  

(Number

Animals Assessed)

10   10  10  10 10   10  10  10

  Absolute 

(%Diff)

0.01752

0.01635

(-6.68)

0.01643

(-6.22)

0.01527

(-12.84)

0.02117

0.02244

(6.0)

0.02433

(14.93)

0.02586

(22.15) 

  RBW 

(%Diff)

0.00279

0.00274

(-1.86)

0.00267

(-4.21)

0.00273

(-2.28)

0.00703

0.00759

(7.9)

0.00783

(11.41)

0.00826

(17.48)

  RBrW 

(%Diff)

0.78588

0.71139

(-9.47)

0.74156

(-5.63)

0.67455

(-14.17)

1.05576

1.12145

(6.22)

1.22266

(15.81)

1.30659

(23.75)

Prostate and

Seminal Vesicle

 (Number

Animals Assessed)

 10  10  10  10  0  0  0

  Absolute 

(%Diff)

3.4766

3.8590

(10.99)

3.4903

(0.39)

3.7064 

(6.61)

N/A  N/A   N/A  N/A

  RBW 

(%Diff)

0.55316

0.65084 *

(17.66)

0.56822

(2.72)


0.66062**

(19.43)

 N/A   N/A   N/A   N/A 

  RBrW 

(%Diff)

156.78668 

168.43561

(7.42)

157.51726

(0.47)

163.26884

(4.13)

N/A  N/A   N/A   N/A 

Thyroid and

Parathyroid 

(Number

Animals Assessed)

 10  10  10  10 10   10  10  10

  Absolute 

(%Diff)

0.0287

0.0325

(13.24)

0.0330

(14.98)

0.0335

(16.72)

0.0196

0.0250

(27.55)

0.0285*

(45.41)

0.0253 

(29.08)

  RBW

 (%Diff)

0.00452

0.00545

(20.48)

0.00532

(17.57)

0.00595

(31.58)

0.00649

0.00849

(30.91)

0.00913

(40.65)

0.00810

(24.83)

  RBrW

 (%Diff)

1.28870

1.41050

(9.45)

1.48616

(15.32)

1.47401

(15.38)

0.97422

1.25212

(28.52)

1.42643*

(46.42)

1.27812

(31.19)

Heart 

(Number

Animals Assessed)

10   10  10  10 10   10  10  10

  Absolute  

(%Diff)

1.9681

 1.9491

(-0.97)

 2.0486

(4.09)

1.9758

(0.39)

1.0643

1.0679

(0.34)

1.1676

(9.71)

 1.2361**

(16.14)

  RBW

 (%Diff)

0.31394

0.32694

(4.14)

0.33232

(5.85)

0.35147

(11.95)

0.35190

0.35969

(2.21)

0.37735

(7.23)

0.39436 *

(12.07)

  RBrW 

(%Diff)

88.50759

84.96344

(-4.00)

92.47024

(4.48)

87.39278

(-1.26)

53.00385

53.45991

(0.86)

58.62830

(10.61)

62.13871**

(17.2)

Kidney  

(Number

Animals Assessed)

10   10  10  10 10  10  10  10

  Absolute 

(%Diff)

3.8786

4.3631

(12.49)

4.6355**

(19.51)

5.6232 **

(44.98)

1.9646

2.0619

(4.96)


2.2791**

(16.01)


2.5723 ** 

(30.93)

  RBW 

(%Diff)

0.61379

0.73365 **

(19.57)


0.75184 **

(22.49)

1.00224 **

(63.28)

0.64794

0.69245

(6.87)

0.73641**

(13.65)

0.82507**

(27.34)

  RBrW

 (%Diff)

174.362

190.064

(9.00)

208.911**

(19.8)

247.600**

(42.00)

97.820

103.303

(5.6)

114.438**

(16.99)

129.413**

(32.30)

Liver  

(Number

Animals Assessed)

 10  10  10  10 10  10  10  10

  Absolute

 (%Diff)

19.2268

19.3775

(0.78)

23.9217**

(24.41)

33.0867 **

(72.09)

9.6231

10.6543

(10.72)


13.3312**

(38.53)

20.8864**

(117.04)

  RBW 

(%Diff)

3.04029

3.24829

(6.84)

3.86936**

(27.27)

5.86001**

(92.75)

3.16223

3.57302

(12.99)

4.31054**

(36.31)

6.62629**

(109.55)

  RBrW 

(%Diff)

864.329

843.314

(-2.43)

1078.002*

(24.72)

1454.926**

(68.33)

479.400

532.330

(11.04)

668.710**

(39.49)

1048.128**

(118.63)

Ovary

& Oviduct  

(Number Animals

Assessed)

 0  0  0  0 10   10  10  10

  Absolute 

(%Diff)

 N/A  N/A    N/A  N/A 0.1189 

0.1453

(22.20) 

0.1367 

(14.97)

0.1160

(-2.44)

  RBW

 (%Diff)

 N/A  N/A    N/A  N/A  0.03943

0.04901

(24.30)

0.04441

(12.64)

0.03721

(-5.62)

  RBrW 

(%Diff)

 N/A  N/A    N/A  N/A  5.89873

7.27375*

(23.31)

6.83842

(15.93)

5.81503

(-1.41)

Spleen

 (Number

Animals Assessed)

 10  10  10  10 10   10  10  10
  Absolute
 (%Diff)
 0.8893

0.8271

(-6.99)

0.7853

 (-11.69)

0.7992 

(-10.13)

0.5214 

0.5697

(9.26)

0.5502

(5.52)

0.5408 

(3.72)

  RBW 

(%Diff)

0.14059

0.13970

(-0.63)

0.12704

(-9.64)

0.14220

(1.15)

0.17210

0.19221

(11.68)

0.17826

(3.58)

0.17246

(0.21)

  RBrW 

(%Diff)

39.87752 

36.04878

(-9.60)

35.38476

(-11.27)

35.20814

(-11.71)

25.96571 

28.67102

(10.42)

27.61524

(6.35)

27.09098

(4.33)

Thymus  

(Number

Animals Assessed)

10   10  10  10 10   10  10  10

  Absolute

(%Diff)

0.2964 

0.2565

(-13.46)

0.2516

(-15.11)

0.2722

(-8.16)

 0.2157


0.2901* 

(34.49)

0.2936*

(36.11)

0.2585 

(19.84)

  RBW 

(%Diff)

0.04688

0.04291

(-8.46)

0.04085

(-12.86)

0.04839

(3.23)

0.07111

0.09861*

(38.68)

0.09534*

(34.08)

0.08243

(15.92)

  RBrW 

(%Diff)

13.30782

11.14347

(-16.27)

11.35036

(-14.71)

12.02158

(-9.66)

10.74031

14.64683*

(36.37)

14.75972*

(37.42)

12.95199

(20.59)

Testis (Left)  

(Number

Animals Assessed)

10   10  10  10  0  0  0

  Absolute  

(%Diff)

1.8662

1.9312

(3.48)

1.8679

(0.09)

1.7697

(-5.17)

 N/A   N/A   N/A   N/A 

  RBW 

(%Diff)

0.29669

0.32453

(9.38)

0.30363

(2.34)

0.31674

(6.76)

 N/A   N/A   N/A   N/A 

  RBrW 

(%Diff)

83.89486

84.19161

(0.35)

84.31183

(0.50)

78.00178

(-7.02)

 N/A   N/A   N/A   N/A 

Testis (Right)  

(Number

Animals Assessed)

10   10  10  10  0  0  0

  Absolute

 (%Diff)

1.8818

1.9087

(1.43)

1.8448

(-1.96)

1.7974

 (-4.49)

 N/A   N/A   N/A   N/A 

  RBW

 (%Diff)

0.29932

0.32039

(7.03)

0.29992

(0.20)

0.32166

(7.46)

 N/A   N/A    N/A

N/A

RBrW 

(%Diff)

84.60778

83.17584

(-1.69)

83.26390

(-1.59)

79.21517

(-6.37)

 N/A   N/A    N/A  N/A 

Uterus  

(Number

Animals Assessed)

 0  0  0 10   10  10  10

  Absolute 

(%Diff)

 N/A   N/A   N/A   N/A  0.8058

0.6459

(-19.8)

0.7714

(-4.27)

0.7899 

(-1.97)

  RBW 

(%Diff)

N/A  N/A  N/A N/A 0.26917

0.21764

(-19.41)

0.25102

(-6.74)

0.25285

 (-6.06)

  RBrW 

(%Diff)

 N/A   N/A   N/A   N/A  40.17810

32.25662

(-19.71)

38.77816

(-3.48)

39.59528

(-1.45)

Left epididymis

(Number

Animals Assessed)

10   10  10  10  0  0  0

Absolute 

(%Diff)

0.7089 

 0.7541

(6.37)

 0.7323

(3.30)

0.7098

(0.13) 

 N/A   N/A    N/A    N/A 

RBW 

(%Diff)

 0.11285 

0.12644

(12.03) 

0.11898 

(5.42)

0.12671 

(12.28)

  N/A    N/A    N/A    N/A 

RBrW 

(%Diff)

31.92298 

32.80353 

(2.76)

33.03223 

(3.48)

31.31221 

(-1.91)

  N/A    N/A    N/A    N/A 

Right epididymis

(Number

Animals Assessed)

 10  10  10  10  0  0  0  0

Absolute 

(%Diff)

0.7080 

0.7450 

(5.23)

0.7280

(2.82) 

0.7330 

(3.53)

  N/A  N/A    N/A   N/A

RBW

(%Diff)

0.11277

 0.12552

(11.30)

0.11826 

(4.86)

0.13109* 

(16.24)

  N/A   N/A   N/A   N/A

RBrW 

(%Diff)

31.84442 

32.48383 

(2.01)

32.83681 

(3.12)

32.33187

(1.53) 

  N/A   N/A   N/A   N/A

Epididymis,

Cauda, Left

(Number

Animals Assessed)

10   10  10  10  0  0  0  0

Absolute 

(%Diff)

0.31335 

 0.34926

(11.46)

0.34004

(8.52)  

0.33079

(5.57)  

  N/A    N/A    N/A    N/A 

RBW

(%Diff)

0.05016 

 0.05860

(16.83)

0.05523

(10.10) 

 0.05924

(18.09)

 N/A   N/A   N/A   N/A

RBrW

(%Diff)

14.11727 

15.18402

(7.56)

15.34381

(8.69)

14.58295

(3.30) 

  N/A    N/A    N/A    N/A 

Epididymis,

Cauda, Right

(Number

Animals Assessed)

 10  10  10  10  0 0  0  0

Absolute 

(%Diff)

0.30806 

0.34356*

(11.52) 

0.34248*

(11.17) 

0.34203

(11.03) 

  N/A    N/A    N/A    N/A 

RBW

(%Diff)

0.04931 

0.05792*

(17.47) 

0.05571

(12.99) 

 0.06125**

(24.22)

  N/A    N/A    N/A    N/A 

RBrW

(%Diff)

13.87633 

14.98335

(7.98) 

15.45874

(11.40) 

15.06905

(8.59)

   N/A   N/A    N/A    N/A 

RBW = Relative to Body Weight

RBrW = Relative to Brain Weight

Values derived from the control group animals at all time points evaluated were considered as concurrent control values for purposes of constructing a ‘normal’ range for the present study. Organ weights (absolute and relative) were subjected to a parametric one-way analysis of variance (ANOVA).6 If the results of the ANOVA were significant (p < 0.05), Dunnett’s test was applied to the data to compare the test substance-treated groups to the control group. The ‘normal’ historical control range for comparison with individual animal values was represented by values within the Charles River Ashland historical control low and high observed value range for organ weights. The group mean values were compared to the Charles River Ashland historical control database mean ± 2 SD (standard deviations) for organ weights.

Statistical Test: Anova & Dunnett's Test

Group Factor Test: Analysis of Variance p < 0.05

Test: Dunnett 2 Sided p < 0.05

* = Significantly different from the control group at 0.05 using Dunnett's test

** = Significantly different from the control group at 0.01 using Dunnett's test

N/A = NOT APPLICABLE

Histopathological Findings

            Males           Females  
Dosage (mg/kg/d)  0  100  300  1000  0  100  300  1000
Liver   (number of animals assessed)  10

 10

 10  10  10  10  10  10
Hypertrophy, centrilobular (minimal)  0  0  3  10  0  0  8  10
 Minimal

 -

 3  0  -  -  4  0
 Mild  -  -  0  3  -  - 4  0
 Moderate  -  -  0  7  -  -  0  10
Necrosis, regionally extensive 0  0  0  0  0 0 0

 1

Severe

 -

 -

 -

 -

 -

-

 -

 1

 Kidney  10  10 10   10  10  10  10  10
Degeneration/necrosis; proximal convoluted tubule  1  4  10  0  0  0  1
  Minimal  1  4  2  -  -  -  1
  Mild  0  0  8  -  -  -  0
Regeneration; proximal convoluted tubule   2  6  10  0  0  0  0
  Minimal  1  4  2  -  -  -  1
  Mild  1  2  6  -  -  -  -
  Moderate  0  0  2  -  -  -  -
Cast; granular  1  1  9  0  0  0  0
  Minimal  1  4  2  -  -  -  -
  Mild  1  2  6  -  -  -  -
  Moderate  0  0  1  -  -  -  -
Accumulation; hyaline droplets   3  6  10  0  0  0  1
  Minimal  3  6  10  -  -  -  1
Thyroid Gland  10  10  10  10  10  10  10  10
Hypertrophy/hyperplasia; follicular cell  1  1  5  1  2  7  7
 Minimal  1  3  5  1  2  7  7
Lungs 10   10  10  10  10  10  10  10
Macrophage aggregation  4  2  4  7  1  2  2  7
Minimal  2  4  6  1  2  2  6
Mild  0  0  1  0  0  0  1
Inflammation, mononuclear cell  3  2  5  0  2  3  4
  Minimal  3  2  5  -  2  2  4
  Mild  0  0  0  -  0  1  0

- = No noteworthy findings

Test substance-related histologic changes within the liver were present in the 300 and 1000 mg/kg/day group males and females and consisted of dose-dependent, minimal to moderate hepatocellular hypertrophy, often in centrilobular regions, with increased eosinophilic cytoplasm. Additionally, severe hepatocellular necrosis was observed in the caudate lobe from a single female in the 1000 mg/kg/day group, and was characterized as coagulation necrosis of centrilobular and midzonal hepatocytes with associated hemorrhage, inflammatory cells, and islands of few remaining periportal hepatocytes. Given the absence of hepatocellular necrosis (with the exception of a single 1000 mg/kg/day group female) and liver enzymes that were comparable to the concurrent control group, liver findings were considered non-adverse.

Test substance-related histologic changes within the kidneys were present in the 100, 300, and 1000 mg/kg/group males. Histologic changes consisted of tubular degeneration/necrosis, regeneration, the presence of granular casts, and hyaline droplet accumulation. Minimal to mild tubular degeneration/necrosis was characterized by individual or grouped proximal convoluted tubules containing epithelial cells with vacuolated cytoplasm and/or pyknotic nuclei, with occasional sloughing of cells into the lumen. Minimal to moderate regeneration within the proximal convoluted tubules was characterized as slightly enlarged tubular epithelial cells with basophilic cytoplasm. Minimal to moderate granular casts consisted of dilated tubules at the corticomedullary junction lined by flattened epithelial cells and containing granular, eosinophilic debris. Finally, minimal hyaline droplet accumulation was observed within tubular epithelial cells and was characterized as eosinophilic, refractile, finely granular to globular intracytoplasmic material. These renal changes were not observed in female rats, with the exception of a single female in the 1000 mg/kg/day group with minimal tubular degeneration/necrosis, hyaline cast formation, and a ‘very minimal’ accumulation of intracytoplasmic hyaline droplets, in which a test substance-related effect could not be excluded. The nature/composition of the hyaline droplets and related histopathological changes was not determined but they likely represent α-2u-globulin mediated kidney effects in rats and it is well established that these are not relevant to humans and hence are not adverse effects for humans.

Test substance-related changes within the thyroid glands were present in the 100 (females only), 300, and 1000 mg/kg/day group males and females and consisted of an increased prevalence of thyroid follicular cell hypertrophy/hyperplasia. Thyroid gland changes were considered to be non-adverse, and may represent an adaptive change secondary to isotridecanol-related hepatic microsomal enzyme induction and associated increased thyroxine metabolism. Pituitary gland hypertrophy (pars distalis) secondary to xenobiotic-induced hepatocellular microsomal enzyme induction has also been reported concurrently with thyroid follicular hypertrophy in rodents; pituitary gland changes were not observed histologically in the current study, however higher pituitary gland weights were present in the 1000 mg/kg/day group females.

Finally, test substance-related histologic changes were present in the lungs of the 1000 mg/kg/day group males and females. An increased prevalence of macrophage aggregation, characterized by increased numbers of foamy macrophages within the alveolar spaces that occasionally contained golden, brown granular material (consistent with hemosiderin) was observed in the 1000 mg/kg/day group males and females. Additionally, an increased incidence of mononuclear inflammation was observed in the 1000 mg/kg/day group females. These findings were considered non-adverse.

Minimal to mild mononuclear cell infiltrates and fibrosis were observed in the hearts of multiple control and 1000 mg/kg/day group rats, and was attributed to rodent progressive cardiomyopathy, a common spontaneous change in Sprague Dawley rats (particularly males).

Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Spermatogenesis Parameters

            Male
 Dosage (mg/kg/d)  100  300  1000
Number Animals Assessed 10   10  10  10

MOTILITY - Mean

92 93 93 87
  %Diff  N/A  1.1 1.1  -5.4 
CAUDA EPID, LT WEIGHT (GRAMS) - Mean  0.3134 0.3493 0.3400  0.3308
  %Diff  N/A  11.5  8.5   5.6
CAUDA EPID, LT CONCENTRATION (MILLIONS/GRAM) - Mean 819.0 871.2  865.1 858.0
  %Diff  N/A   6.4 5.6 4.8 
TESTIS, LEFT WEIGHT (GRAMS) - Mean 1.87 1.93   1.87 1.77
  %Diff N/A  3.2  0.0 -5.3
TESTIS, LEFT CONCENTRATION (MILLIONS/GRAM) - Mean 136.7  143.9 134.1 134.1
  %Diff  N/A  5.3  -1.9  -1.9
SPERM PRODUCTION RATE (MILLIONS/GRAM/DAY) - Mean 22.4 23.6 22.0 22.0
  %Diff N/A  5.4  -1.8 -1.8
NORMAL MORPHOLOGY - Mean %

99.5

99.7

99.7

99.7 

  %Diff

 N/A 

N/A 

 N/A 

N/A 

NORMALLY SHAPED HEAD SEPARATED FROM FLAGELLUM - Mean %

0.3

0.2

0.1

0.2

  %Diff

  N/A

 N/A 

 N/A 

 N/A 

HEAD ABSENT WITH NORMAL FLAGELLUM - Mean %

0.3

0.1

0.3

0.2

  %Diff

  N/A

 N/A 

 N/A

 N/A 

ABNORMAL HEAD - Mean %

0.0

0.0

0.0

0.0

  %Diff

 N/A

  N/A

  N/A

  N/A

ABNORMAL FLAGELLUM - Mean %

 0.0

0.0

0.0

0.0

  %Diff

  N/A

  N/A

  N/A

  N/A

OTHER - Mean %

0.0

0.0

0.0 

0.0

  %Diff

 N/A

  N/A

  N/A

 N/A

PROGRESSIVE MOTILITY - Mean %   82.0 81.0  77.0   67.0** 
 %Diff  N/A -1.2   -6.1 -18.3 

Spermatogenesis data was subjected to a one-way analysis of variance (ANOVA). If a statistically significant difference (p<0.05) is present in the ANOVA, a comparison of the control group to each treated group by Dunnett's test followed. All analyses were two-tailed for significance levels of 5% and 1%.

The percentage of motile spermatozoa and percentage of sperm with normal morphology were subjected to the Kruskal-Wallis nonparametric ANOVA test followed by the Dunn’s Test. Epididymal and testicular sperm numbers and sperm production rates were subjected to a parametric ANOVA test and Dunnett’s test.

** = Significantly different from the control group at 0.01 (non-parametric analysis)

Applicant's summary and conclusion

Conclusions:
Isotridecanol does not cause toxicity relevant to humans under the conditions of this subchronic study at dosage levels up to 1000mg/kg/d; the NOAEL for isotridecanol in rats is 300mg/kg/d.
Executive summary:

A 90 -day subchronic study was conducted in rats to assess the toxicity of isotridecanol. The test substance was administered by oral gavage at a dose of 0, 100, 300, and 1000mg/kg/d for 90 consecutive days. The control animals received a carrier (corn oil) dose. Observations were made as to the nature, onset, severity, and duration of toxicological signs. Based on the results of this study, oral administration of isotridecanol to rats at dosage levels of 100, 300, and 1000 mg/kg/day for a minimum of 90 days resulted in microscopic changes in the liver, kidneys, thyroid glands, and lungs of the 100, 300, and/or 1000 mg/kg/day group males and/or females. Liver histologic changes correlated with dose-dependent, statistically significant higher liver weights in the 300 and 1000 mg/kg/day group males and females; liver findings were considered non-adverse, as were lung and thryoid findings. Renal histologic changes were present in the 100, 300, and 1000 mg/kg/day group males. Histologic changes correlated with dose-dependent higher kidney weights in the 300 and 1000 mg/kg/day group males. Similar kidney weight changes were observed in the 300 and 1000 mg/kg/day group females, but no histologic correlates were identified, with the exception of a single female in the 1000 mg/kg/day group. The histopathological changes in kidney likely represent α-2u-globulin mediated kidney effects in rats and it is well established that these are not relevant to humans and hence are not adverse effects for humans. No test substance-related organ weight changes outside the historical control range were observed across all reproductive tissues assessed in males and females (epididymides, ovaries with oviducts, prostate with seminal vesicles, testes, uterus); similarly, no test substance-related histological findings were observed for any reproductive tissues assessed in males or females (cervix, epididymides, ovaries with oviducts, prostate, seminal vesicles, testes, uterus, vagina). Additionally, no test substance-related effects were observed on spermatogenesis endpoints (mean testicular and cauda epididymal sperm numbers and sperm production rate, motility, and morphology) in males at any dosage level.