Alcohols, C9-11-branched

Repeated dose toxicity: oral

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 1

Reference Type:

study report

Title:

Unnamed

Year:

2020

Reference 2

Reference Type:

publication

Title:

Characterization of Xenobiotic-Induced Hepatocellular Enzyme Induction in Rats: Anticipated Thyroid Effects and Unique Pituitary Gland Findings

Author:

Zabka TS, Fielden MR, Garrido R, Tao J, Fretland AJ, Fretland JL, Albassam MA, Singer T, and Kolaja KL.

Year:

2011

Bibliographic source:

Toxicologic Pathology, 39: 664-677.

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

Test material information

Constituent 1

Reference substance name:

Alcohols, C9-11-branched

EC Number:

271-360-6

EC Name:

Alcohols, C9-11-branched

Cas Number:

68551-08-6

Molecular formula:

CnH2n+1OH (n = 10 to 12)

IUPAC Name:

Branched alcohols, C10-12, C11 rich

Test material form:

liquid

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 8 weeks old and weighed 169 g to 307 g at the initiation of dosing.

- Housing: Animals were group housed (3 animals of the same sex) until randomization. Following randomization, animals were group housed (2 to 3 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; animals were fasted overnight prior to blood collection for clinical pathology evaluations and prior to necropsy

- Water: Municipal tap water after treatment by reverse osmosis and UV irradiation was freely available to each animal via an automatic watering system

ENVIRONMENTAL CONDITIONS:
- Temperature: 66°F to 77°F (19°C to 25°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 (including the comparator test substance, isotridecanol) formulations have been previously shown to be stable and homogeneous over the range of concentrations used on this study for at least 11 days at room temperature.Therefore, stability and homogeneity of test substance formulations were not assessed on this study.

Test substance and comparator test substance (isotridecanol) dosing formulations were prepared at appropriate concentrations to meet dose level requirements. The dosing
formulations were prepared approximately weekly.

Dose formulation samples were collected for concentration analysis as follows: all concentration groups were analyzed 3 days before the first dose, and on Days 21, 49, and 84. 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.

The analyzed dosing formulations contained 90.8% to 101% of the test substance, which was within the protocol-specified range of target concentrations for suspensions (85%-115%). The test substance was not detected in the analyzed vehicle formulation administered to the control group.

Duration of treatment / exposure:

90 consecutive days

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

10mg/mL concentration, 10mL/kg dose volume

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

30mg/mL concentration, 10mL/kg dose volume

Doses / concentrations 3

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 dosage level was expected to be a no-observed-effect level (NOEL). A single-dose acute toxicity test 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 substance. 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) was set at the dose limit 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/day had a decreased body weight and increased liver, stomach, and kidney weights. However, animals at 100 and 330 mg/kg/day showed no changes. In a 90-day subchronic study in rats exposed to 1000 mg/kg/day of isotridecanol, the animals had increased liver weights and showed evidence of thyroid hypertrophy/ hyperplasia hypothesized to be a secondary effect of liver metabolism. A high dosage level of 1000 mg/kg/day for the test substance and the comparator substance, isotridecanol, was selected based on the results of the aforementioned studies and toxicokinetic data, and was expected to produce some effects without causing severe toxicity.

Based on the previously reported 90-day repeat dose study on isotridecanol, which yielded a test substance-related secondary, adaptive finding in the thyroid (follicular hyperplasia and hypertrophy, increased thyroid weights) at study termination, three additions were made to this study design. The first was to add a 1000mg/kg/d dose group of isotridecanol, to fully examine the nature of the thyroid effect previously observed. The second was an additional interim necropsy scheduled for half-way through the study (Day 47/48), with five additional animals of each sex at the control and high dose groups (1000mg/kg/d isoundecanol, and 1000mg/kg/d isotridecanol). This was to examine whether a temporal relationship existed between the nature and severity of findings in the thyroid. The third additional element was to take additional thyroid hormone sampling times throughout the study design of 90 days, to get a temporally-sensitive dataset to compare quantitative hormone data with any qualitatively-based histopathological findings. To ensure that there wasn't any negative impact on the animals due to these additional blood samples being taken throughout the study, additional hematological parameters were monitored throughout the study as well.

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, 1 week (± 2 days) prior to randomization, on the day of randomization, on Day 1 (prior to dosing), weekly (± 2 days) during the study period, on the day prior to the first day of the scheduled necropsy, and on the day of the scheduled necropsies. A fasted weight was recorded on the day of necropsy.

Food consumption was quantitatively measured once weekly (± 2 days) starting on Day 1 and continuing throughout the dosing period.

Sacrifice and pathology:

Opthalmic Examinations:
Ocular examinations were conducted for all animals during the pretreatment period (Day -6) and near the end of the dosing period (Day 89).

Clinical Pathology:
Hematology parameters assessed are as follows: differential leukocyte count, erythrocyte count, total hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean platelet volume, platelet count, reticulocyte count, red cell distribution width, and total leukocyte count.

Coagulation parameters assessed in plasma included activated partial thromboplastin time, fibrinogen, prothrombin time, and sample quality parameters (degree of hemolysis, lipemia, and icterus).

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

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

Thyroid Hormone Assessment: Blood samples were processed for serum, and serum was analyzed for triiodothyronine (Total T3), thyroxine (Total T4), and thyroid stimulating hormone (TSH).

Determination of estrous cycles: Vaginal lavages were performed for all females on the day of the terminal necropsy. Giemsa stained slides were prepared from all animals, and the slides were microscopically examined to determine the stage of the estrous cycle.

Terminal Procedures:
Ten males and ten females from each dose group 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.

Five males and five females from the controls and high dose test substance and comparator groups underwent necropsy, gross lesion and target tissue analysis (consisting of liver and thyroid), as well as organ weight recording on day 47/48.

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 and coagulating glands, 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 (with joint), sternum, bone marrow smear (from femur), brain, cervix, epididymides, eyes with optic nerves, gastrointestinal tract, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, harderian glands, heart, kidneys, larynx, liver (sections of two lobes), lung (including bronchi), lymph nodes (axillary, mandibular, mesenteric), ovaries with oviducts, pancreas, peripheral nerve (sciatic), Peyer's patches, pharynx, pituitary, prostate with coagulating glands, mandibular salivary glands, seminal vesicles, skeletal muscle (quadriceps), skin with mammary gland, spinal cord (cervical, lumbar, thoracic), spleen, testes, thymus, thyroid with parathyroids, tongue, trachea, uterus, urinary bladder, vagina.

Other examinations:

SPERMATOGENIC EVALUATION:
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) and progressively motile (showing net forward motion) sperm 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 were 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. For parametric/non-parametric statistics (Levene's test for homogeneity of group variances), with the groups being compared using an overall one-way ANOVA F-test (if Levene's test was not significant) or the Kruskal-Wallis test (if it was significant). If the oveerall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett's or Dunn's test, respectively. This was performed for body weight, body weight gain,food consumption, hematology, coagulation, serum chemistry, urinalysis, organ weights, organ weight relative to terminal body weight. Each dosed group was compared pairwise to the control group.

Macroscopic and microscopic findings were recorded in terms of incidence (Fisher's exact test to conduct pairwise group comparisons).

Thyroid hormone variables and spermatogenesis parameters were assessed using parametric statistical methods as described above.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related clinical observations were noted in the 1000 mg/kg/day group males and females. Test substance- and isotridecanol-related clinical observations noted in males and females in both 1000 mg/kg/day groups included the following: slight salivation and wet fur noted on the cranium, dorsal cervical, dorsal thoracic, mouth, muzzle, and ventral cervical areas. In addition, test substance- and isotridecanol-related ploughing was noted in both 1000 mg/kg/day groups. Please see attached tables for more information.

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. These observations were considered nonadverse since the animals remained in overall good health (no effects on mortality and/or body weights).

Mortality:

no mortality observed

Description (incidence):

All animals survived until scheduled necropsy.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights were unaffected by test substance administration. However, some statistically significant differences were observed when the control and test substance-treated groups were compared; however, these differences were not considered test substance-related because the differences were considered due to biological variability and/or lacked a dose-response trend. The mean body weight in the 1000 mg/kg/day isotridecanol group females at the end of dosing on Day 90 was slightly lower compared to the control group females, due to a single animal with significant body weight loss from Days 85 to 90. Therefore, this change was not considered isotridecanol-related.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Test substance- and isotridecanol-related higher mean food consumption (often times statistically significant) was noted in the 1000 mg/kg/day group males and females throughout the dosing period compared to the control groups. Higher mean food consumption in these groups began in the males during Day 22-29 and in the females during Day 8-15, and were intermittently higher each week thereafter. With the exception of the 1000 mg/kg/day group males in the test substance group (not statistically significant, but considered higher as part of a trend), the mean food consumption during Day 1-90 was statistically significantly higher. These changes were considered nonadverse since the animals remained in overall good health (no effects on mortality and/or body weights).

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. All findings observed were typical in prevalence and appearance for laboratory rats of this age and strain.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Instances of transient, ultimately nonadverse, changes in hematological outcomes were noted. Test substance- and isotridecanol-related lower mean red blood cell counts, hemoglobin, and hematocrit were noted in the 1000 mg/kg/day group females on Days 16 and 30 when compared to the control group. Test substance- and isotridecanol-related lower mean hemoglobin and hematocrit were noted in the 1000 mg/kg/day group males on Days 30 and 58 when compared to the control group. Isotridecanol-related lower mean hemoglobin and hematocrit were noted in the 1000 mg/kg/day group females on Day 58 when compared to the control group. Test substanceand isotridecanol-related lower mean absolute reticulocyte counts were noted in the 1000 mg/kg/day group females on Day 58 when compared to the control group. All of these changes were considered to be nonadverse due to their slight magnitude of change (see table below).

Isotridecanol-related lower mean absolute reticulocyte counts were noted in the 1000 mg/kg/day isotridecanol-treated group females on Day 91/92 when compared to the control group, which was considered nonadverse. Some other statistically significant differences were observed when the control and test substance- or isotridecanol-treated groups were compared, but differences were not considered test substance-related 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.

All of these changes were considered to be nonadverse due to their slight magnitude of change and since they were in a direction that was not biologically significant. Please see attached tables for more information.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related lower mean bile acid value were noted in the 300, 1000, 1000 mg/kg/day group males and females on Day 91/92 when compared to the control group. Higher mean cholesterol, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) levels were noted in the test substance and isotridecanol 1000 mg/kg/day male groups and isotridecanol 1000 mg/kg/day female group on Day 91/92 when compared to the control group. Mean cholesterol and HDL levels were higher in the test substance 1000 mg/kg/day female group on Day 91/92 when compared to the control group. Mean chloride was lower in the test substance and isotridecanol 1000 mg/kg/day male and female groups on Day 91/92 when compared to the control group. Mean alkaline phosphatase (ALP) was slightly higher in the test substance and isotridecanol 1000 mg/kg/day male groups on Day 91/92 when compared to the control group. Mean urea nitrogen (UREAN) was higher in the test substance and isotridecanol 1000 mg/kg/day male groups on Day 91/92 when compared to the control group. Mean urea nitrogen (UREAN) was higher in the test substance and isotridecanol 1000mg/kg/d male groups on Day 91/92 when compared to the control group. Since the higher urea nitrogen was considered to correlate to the adverse microscopic findings noted in the kidneys of the 1000 mg/kg/day male groups, these changes were considered to be adverse. All other changes were considered to be nonadverse due to their slight magnitude of change (see table below).

Some other statistically significant differences were observed when the control and test substance- or isotridecanol-treated groups were compared, but differences were not considered test substance-related 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. Please see attached tables for more information.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Test substance- and isotridecanol-related higher urine mean volume and lower mean pH were noted in the 1000 mg/kg/day male groups and lower mean pH was noted in the 1000 mg/kg/day female groups when compared to the control group. Test substance-related lower mean pH was also noted in the 100 (not statistically significant, but part of a dose-responsive trend) and 300 mg/kg/day group males. Since the higher urine mean volume was considered to correlate to the adverse microscopic findings noted in the kidneys of the 1000 mg/kg/day male groups, these changes were considered to be adverse. Please see attached tables for more information.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related higher liver, kidney, and thyroid/parathyroid weights were noted in 1 or both sexes at doses of 1000mg/kg/day at the scheduled interim euthanasia, and noted in 100, 300, and 1000 mg/kg/day dose groups at terminal euthanasia. Test substance-related higher mean adrenal gland weights were noted in the 300 and 1000mg/kg/day females (see table below).

Interim Euthanasia: Test substance-related higher mean liver and kidney weights (absolute and relative to terminal body and brain weights) and trends of higher mean thyroid/parathyroid gland weights (absolute and relative to terminal body and brain weights) were noted in the 1000 mg/kg/day group males and females. In the interim euthanasia group animals, similar organ weight changes in the liver, kidney, and thyroid/parathyroid glands were seen between the test substance and comparator 1000 mg/kg/day male and female groups. There were no other test substance-related or statistically significant effects on organ weights.

Terminal euthanasia: Test substance-related higher mean liver and thyroid/parathyroid gland weights (absolute and relative to terminal body and brain weights) were noted in the 100, 300, and 1000 mg/kg/day group males and females; test substance-related higher mean kidney weights (absolute and relative to terminal body and brain weights) were noted in the 100, 300, and 1000 mg/kg/day group males and 300 and 1000 mg/kg/day group females; and test substance-related higher mean adrenal gland weights were noted in the 300 and 1000 mg/kg/day group females. In the terminal euthanasia group animals, similar organ weight changes in the liver, thyroid/parathyroid gland, and kidney weights were seen between the test substance and 1000 mg/kg/day isotridecanol group males and females.

There were no other test substance-related effects on organ weights. However, some statistically significant differences were observed in the thymus at the interim euthanasia and the heart at the terminal euthanasia between the control and test substance-treated groups. These values were within the range of Charles River Ashland historical control values and were not considered test substance-related.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In the interim euthanasia group animals, macroscopic findings of liver enlargement was noted in both the test substance and isotridecanol 1000 mg/kg/day male groups at a similar incidence (1/5 males in 1000mg/kg/d test substance group; 1/4 males in 1000mg/kg/d isotridecanol group); but macroscopic findings in the kidneys were only in the test substance 1000 mg/kg/day group males (2/5 males).

Overall, in the terminal euthanasia group, test substance-related macroscopic findings of enlargement of the liver, kidney, and thyroid gland were observed; small and/or abnormal consistency of the testes; and small epididymides were noted in the 1000 mg/kg/day group males.

Terminal Euthanasia Incidence:
THYROID: Enlargement (1/10 of the 1000 mg/kg/day group males)
KIDNEY: Enlargement of the kidney was noted in the 100mg/kg/d group males (2/10) and 1000 mg/kg/day group males (2/10; 1/10 males with dilatation of the kidney)
LIVER: Enlargement (3/10 males in 1000mg/kg/d group; 1/10 males in 1000mg/kg/d isotridecanol group); adhesion (1/10 males in 1000mg/kg/d isotridecanol group); mass (2/10 males in 1000mg/kg/d isotridecanol group); pale focus (1/10 females @100mg/kg/d; 2/10 males and 1/10 females @300mg/kg/d; 1/10 males and 2/10 females @1000mg/kg/d; 2/10 males and females @1000mg/kg/d isotridecanol group); pale discoloration (1/10 males@300mg/kg/d); abnormal appearance (1/10 control group males)
EPIDIDYMES: Small epididymides and testes were only noted in the 100 and 1000 mg/kg/day group males (small epididymis in 2/10 animals @100 mg/kg/day; small epididymis in 1/10 animals @ 1000mg/kg/day
TESTES: Small right and left testes in 2/10 animals @ 100mg/kg/day; small right and left testes in 1/10 animals @ 1000mg/kg/day), and abnormal consistency of the left and/or right testes was noted in the 100 and 300 mg/kg/day group males (2/10 animals and 1/10 animals, respectively).

The findings of small testes and epididymis were noted in one test substance 1000 mg/kg/day group male that correlated with degeneration (of the seminiferous tubules within the testes) and decreased intraluminal contents (of spermatids) within the epididymides. Given that this can be a background findings in rats, these findings were considered incidental. The macroscopic finding of liver enlargement was noted in both the test substance and isotridecanol 1000 mg/kg/day male groups (3/10 and 1/10, respectively). There were no other macroscopic findings considered to be test substance-related.

Findings that were not test-substance related:
JEJUNUM: 1/10 males @1000mg/kg/d with diverticulum
SPLEEN: 1/10 females @300mg/kg/d with abnormal appearance; "small" spleen in 1/10 females @1000mg/kg/d isotridecanol
THYMUS: 1/10 males @1000mg/kd/ with "dark focus"
UTERUS: 1/10 control females and 3/10 females @100mg/kg/d with "material accumulation"

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related microscopic findings were noted in the liver and thyroid glands in the males and females at 1000mg/kg/d; kidney findings were recorded in males only at 1000mg/kg/day in the interim scheduled euthanasia.

At the interim euthanasia:
Test substance-related findings of hepatocellular hypertrophy within the liver and hypertrophy/hyperplasia within the thyroid glands were noted in the 1000 mg/kg/day group males and females and degeneration/necrosis and regeneration of the proximal convoluted tubules, granular casts, and hyaline droplet accumulation within the kidney were noted in the 1000 mg/kg/day group males. Similar findings at a similar incidence and/or severity were present in the isotridecanol 1000 mg/kg/day groups (see tables below).

At the terminal euthanasia:
In the liver, histological changes of minimal to moderate hepatocellular hypertrophy in the test substance 100, 300, and 1000 mg/kg/day group males and females correlated with liver enlargement and higher mean liver weights (see tables below). Given the absence of changed in liver enzymes suggestive of hepatocellular damage (i.e., aspartate aminotransferase and alanine aminotransferase), the liver findings were considered nonadverse.

Renal histological changes were present in the test substance 1000 mg/kg/day group males and consisted of minimal to moderate degeneration/necrosis and regeneration of the proximal convoluted tubules, minimal to moderate presence of granular casts, and minimal accumulation of hyaline droplet accumulation in the test substance 100 and 1000 mg/kg/day group males (see tables below). The spectrum of these findings was consistent with alpha-2u globulin nephropathy. Hyaline droplet accumulation composed of alph-2u globulin is common in young control group male rats. Alpha-2u globulin accumulation is a male rat-specific effect, not occurring in female rats or other species. Microscopically, xenobiotic-accentuated alpha-2u globulin accumulation is characterized by increased numbers of variably-sized round to irregular shaped droplets primarily within the P2 segment of proximal convoluted tubules. Microscopic findings frequently noted in association with alpha-2u globulin accumulation include tubular epithelial cell necrosis, granular casts, and higher incidence/severity of chronic progressive nephropathy, and are collectively termed alph-2u globulin nephropathy. While considered a male rat-specific phenomenon, the finding in this study was considered adverse due to the presence of individual cell necrosis and granular casts, as well as higher urea nitrogen and creatinine. Higher mean kidney weights were also noted primarily in the test substance 1000 mg/kg/day group males and females which were considered largely adaptive via the mechanism of microsomal enzyme induction. However, regeneration within the proximal convoluted tubules was considered to also have contributed and correlated with enlargement and increased mean kidney weights in males. Higher urea and urea/creatinine noted in males correlated with gross observations, organ weights, and microscopic findings in the test substance 1000 mg/kg/day group males and were suggestive of renal dysfunction and hyaline droplet accumulation. Given the microscopic findings within the kidney and associated elevations in urea nitrogen and creatinine in the test substance 1000 mg/kg/day group males, the findings within the kidneys were considered adverse in the test substance 1000 mg/kg/day group males, but ultimately not human-relevant.

In the thyroid, histological changes were present in the test substance 100, 300, and/or 1000 mg/kg/day group males and females and consisted of hypertrophy/hyperplasia of thyroid follicular cells. These findings correlated with higher mean thyroid/parathyroid gland weights and lower T3 and T4 and higher TSH in both males and females and were considered to have contributed to higher TSH and lower T3 and T4 levels. Thyroid gland changes (inclusive of serum thyroid hormones) were considered to be nonadverse and were considered to represent an adaptive change secondary to test substance-related hepatic microsomal enzyme induction and associated increased thyroxin metabolism where higher TSH and/or lower T3 and T4 were seen (see tables below).

Adrenal gland histological changes were present in the test substance 100, 300, and 1000 mg/kg/day group males and consisted of cytoplasmic vacuolation of the adrenal cortical cells (see tables below). There were no histological correlates for the increased mean adrenal gland weights in the test substance 300 and 1000 mg/kg/day group females. Given the absence of any other changes in the adrenal gland (e.g., organ weight changes in the females), findings within the adrenal gland were considered nonadverse.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

SPERMATOGENIC EVALUATIONS:

There were no test substance- or isotridecanol-related changes in spermatogenesis or sperm morphology detected at either the interim or terminal euthanasia on this study. Lower mean testis concentration and sperm production rate were statistically significant in the test substance-treated 100 mg/kg/day group males at the terminal necropsy when compared to the control group, but was within the Charles River Ashland reproductive historical control reference range (version 2016.03). The lower value was not considered test substance-related because it lacked a dose response, was attributed to 2 animals, and lacked correlating pathology findings; further, there were no statistically significant differences on any other measured sperm parameters. See tables below for data.

ESTROUS CYCLE EVALUATIONS:

There were no test substance- or isotridecanol-related changes in estrous cycle detected at the terminal euthanasia on this study, given the variability in the data and that there was no microscopic evidence of any overt pathology in the reproductive tissues to support an effect on estrous.

THYROID HORMONE EVALUATIONS:

On Day 16: test substance- and isotridecanol-related lower mean T3 (not statistically significant, but dose-responsive) was noted in the 1000 mg/kg/day group males, test substance- and isotridecanol-related lower mean T3 was noted in the 300 and 1000 mg/kg/day group females and test substance- and isotridecanol-related lower mean T4 and higher mean TSH was noted in the 1000 mg/kg/day group females (not statistically significant for the 1000 mg/kg/day isotridecanol-treated females, but dose-responsive), when compared to the control group. See tables below for data.

On Day 30, test substance- and isotridecanol-related lower mean T3 (not statistically significant, but dose-responsive) was noted in the 300 and 1000 mg/kg/day group males, test substance- and isotridecanol-related lower mean T4 and higher mean TSH (TSH not statistically significant, but
dose-responsive) was noted in the 1000 mg/kg/day group males, test substance- and isotridecanol-related lower mean T3 was noted in the 300 and 1000 mg/kg/day group females, test substance- and isotridecanol-related lower mean T4 and higher mean TSH was noted in the 1000 mg/kg/day group females, when compared to the control group. See tables below for data.

On Day 58, test substance-related lower mean T3 (not statistically significant, but dose-responsive) was noted in the 300 and 1000 mg/kg/day group males, test substance- and isotridecanol-related higher mean TSH (not statistically significant, but dose-responsive) was noted in the 1000 mg/kg/day group males, test substance-related lower mean T3 and T4 (not statistically significant, but dose-responsive) was noted in the 1000 mg/kg/day group females, and test substance- and isotridecanol-related higher mean TSH (not statistically significant, but dose-responsive) was noted in the 1000 mg/kg/day group females, when compared to the control group. See tables below for data.

On Day 91/92, test substance-related lower mean T3 (not statistically significant, but generally dose-responsive) was noted in the 100, 300 and 1000 mg/kg/ day group males, isotridecanol-related lower mean T4 was noted in the 1000 mg/kg/day group males, test substance- and isotridecanol-related higher mean TSH was noted in the 1000 mg/kg/day group males (not statistically significant for the 1000 mg/kg/day comparator males, but dose-responsive), test substance-related lower mean T3 was noted in the 300 and 1000 mg/kg/day group females, test substance- and isotridecanol-related lower mean T4 was noted in the 1000 mg/kg/day group females, and test substance-related higher mean TSH (not statistically significant, but dose-responsive) was noted in the 1000 mg/kg/day group females, when compared to the control group. See tables below for data.

These changes in thyroid hormone levels (statistically significant, unless otherwise noted) were considered related to higher mean thyroid/parathyroid gland weights and hypertrophy/hyperplasia of thyroid follicular cells, and part of an adaptive, nonadverse change.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

2

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

urinalysis

Remarks on result:

other: Not human relevant; observed renal changes in male rats only were consistent with alpha-2u globulin accumulation, which is a well-known male rat-specific effect, not occurring in female rats or other species, including humans

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Final Body Weights

Dosage (mg/kg/d)	Male 0	Male 100	Male  300	Male 1000	Male 1000^#	Female  0	Female  100	Female 300	Female 1000	Female 1000^#
Mean body weight (g) at end of study	602.9	591.5	597.0	541.9	578.0	291.4	294.2	295.8	298.0	271.9
%Diff	N/A	-1.9	-0.98	-10.12	-4.13	N/A	1.009	1.015	1.023	-6.692

N/A = NOT APPLICABLE

# = Isotridecanol Group

Summary of Test Substance-Related Hematology Findings

Dosage (mg/kg/d)	Male 0	Male 100	Male 300	Male 1000	Male 1000^#	Female 0	Female 100	Female 300	Female 1000	Female 1000^#
Red blood cells (mil/μL)
Day 16	7.745	7.810	7.728	7.819	7.689	7.919	7.831	7.742	7.575**	7.565**
Day 30	8.209	8.246	8.323	8.036	7.990	8.166	7.984	7.997	7.881*	7.689**
Hemoglobin (g/dL)
Day 16	15.28	15.22	15.10	15.09	14.76	15.31	15.20	14.97	14.49**	14.11**
Day 30	15.46	15.27	15.68	14.82**	14.57**	15.38	15.15	14.95	14.61**	13.90**
Day 58	15.43	15.09	14.93	14.44**	14.29**	14.66	15.16	14.60	14.40	13.71**
Hematocrit (%)
Day 16	49.42	49.22	48.85	49.50	48.17	48.50	47.91	47.14	46.51**	45.69**
Day 30	50.19	49.89	50.84	48.49*	47.35**	49.31	48.17	47.86	47.26**	45.31**
Day 58	49.20	47.79	47.45	46.64**	45.34**	45.93	47.04	45.48	45.35	43.39*
Reticulocytes (10^9/L)
Day 58	239.57	222.76	226.97	199.87	211.56	191.10	215.12	203.23	157.03	157.44
Day 91/92	215.83	222.17	227.37	204.08	206.79	205.14	213.61	191.02	170.65	140.09*

* = Significantly different from the vehicle control group at 0.05 using Anova & Dunnett’s test.

** = Significantly different from the vehicle control group at 0.01 using Anova & Dunnett’s test.

Bold = Test substance-related change.

# = Isotridecanol Group

Test Substance-Related Serum Chemistry Findings on Day 91/92

Dosage (mg/kg/d)	Male 0	Male 100	Male 300	Male 1000	Male 1000^#	Female 0	Female 100	Female 300	Female 1000	Female 1000^#
Bile acid (umol/L)	13.44	14.82	9.65	4.47**	8.54	15.14	10.95	9.81	5.23	5.33
Cholesterol (mg/dL)	46.3	48.9	52.7	61.9***	75.9**	63.2	62.6	75.1	91.5*	121.0*
High density lipoprotein cholesterol (mg/dL)	11.9	12.3	13.8	18.1**	20.6**	19.1	18.9	21.4	27.8*	35.8*
Low density lipoprotein cholesterol (mg/dL)	12.3	13.2	14.0	16.7	23.1***	12.6	11.8	14.4	12.8	20.7*
Chloride (mEq/L)	105.3	103.9	103.8	102.3*	101.5*	105.4	105.4	104.0	100.8*	97.3*
Alkaline phosphatase (U/L)	87.5	86.3	90.3	126.4*	118.0*	45.0	41.5	48.3	42.0	39.7
Urea Nitrogen (mg/dL)	8.1	9.4	10.0	12.2**	11.8**	9.4	10.7	10.9	11.2	11.8

* =Significantly different from the vehicle control group at 0.01 using Anova & Dunnett’s test.

**=   Significantly different from the vehicle control group at 0.01 using Kruskal-Wallis & Dunn’s test.

*** = Significantly different from the vehicle control group at 0.05 using Kruskal-Wallis & Dunn’s test.

Bold = Test substance-related change.

# = Isotridecanol Group

Test Substance-Related Organ Weight Changes (Absolute and Relative) at Interim Euthanasia (Day 47/48)

Organ Weight Changes (Absolute =g, RBW & RBrW =%)

Dosage (mg/kg/d)	Male 0	Male 100	Male 300	Male 1000	Male 1000^#	Female 0	Female 100	Female 300	Female 1000	Female 1000^#
Thyroid and Parathyroid  (Number Animals Assessed)	5	-	-	5	5	5	-	-	5	5
Absolute	0.018	-	-	0.023 (24.29)	0.023 (25.38)	0.019	-	-	0.023 (24.41)	0.025 (34.88)
RBW  (%Diff)	0.004	-	-	0.005 (24.72)	0.005 (35.93)	0.007	-	-	0.008 (16.32)	0.009 (31.42)
RBrW  (%Diff)	0.83	-	-	1.08 (29.76)	1.05 (26.90)	0.96	-	-	1.19 (24.34)	1.24 (29.55)
Kidney   (Number Animals Assessed)	5	-	-	5	5	5	-	-	5	5
Absolute	3.61	-	-	4.61 (27.64)	4.37 (21.08)	1.92	-	-	2.34* (22.19)	2.37* (23.75)
RBW  (%Diff)	0.71	-	-	0.91** (29.34)	0.94** (33.28)	0.72	-	-	0.84* (16.93)	0.87** (23.75)
RBrW  (%Diff)	164.81	-	-	220.79* (33.97)	202.03 (22.58)	98.47	-	-	120.46* (22.33)	118.34* (20.18)
Liver   (Number Animals Assessed)	5	-	-	5	5	5	-	-	5	5
Absolute	18.76	-	-	30.15** (60.68)	28.99** (54.53)	9.95	-	-	18.00** (80.86)	18.70** (87.94)
RBW  (%Diff)	3.65	-	-	5.97** (63.39)	6.24**  (70.61)	3.72	-	-	6.43*  (73.11)	6.88** (85.22)
RBrW  (%Diff)	858.18	-	-	1444.17** (68.28)	1340.08** (56.15)	511.20	-	-	926.89* (81.31)	933.93* (82.69)

RBW = Relative to body weight

RBrW = Relative to brain

% change is % change from control.

- = not applicable.

* = Statistically significantly different from the control group at p ≤ 0.05 using Dunnett’s test.

** = Statistically significantly different from the control group at p≤ 0.01 using Dunnett’s test.

Bold= Values considered to be test substance-related.

# = Isotridecanol Group

Test Substance-Related Organ Weight Changes (Absolute and Relative) at Terminal Euthanasia (Day 91/92)

Organ Weight Changes (Absolute =g, RBW & RBrW =%)

Dosage (mg/kg/d)	Male 0	Male 100	Male 300	Male 1000	Male 1000^#	Female 0	Female 100	Female 300	Female 1000	Female 1000^#
Adrenal Gland  (Number Animals Assessed)	10	10	10	10	10	10	10	10	10	10
Absolute	0.065	0.0652 (0.493)	0.067 (3.018)	0.06491 (-0.046)	0.06338 (-2.40222)	0.069	0.073 (6.54)	0.080 (17.01)	0.084** (22.41)	0.091** (32.84)
RBW  (%Diff)	0.011	0.011 (3.480)	0.011 (4.798)	0.012 (11.518)	0.01107 (3.03812)	0.024	0.025 (4.944)	0.027 (15.06)	0.028 (19.54)	0.034** (43.79)
RBrW  (%Diff)	2.964	2.991 (0.931)	3.09208 (4.332)	3.057 (3.142)	2.90557  (-1.96103)	3.453	3.587 (3.88)	3.874 (12.19)	4.190** (21.35)	4.562** (32.13)
Thyroid and Parathyroid  (Number Animals Assessed)	10	10	10	10	10	10	10	10	10	10
Absolute	0.024	0.029 (19.08)	0.028 (15.58)	0.029 (22.32)	0.034** (41.44)	0.018	0.020  (12.92)	0.020 (11.15)	0.029** (57.65)	0.027** (48.09)
RBW (%Diff)	0.004	0.005 (20.65)	0.005 (16.25)	0.005** (36.43)	0.006** (48.19)	0.006	0.007 (11.34)	0.007  (8.66)	0.010** (55.03)	0.010** (57.27)
RBrW (%Diff)	1.101	1.309 (18.93)	1.282 (16.47)	1.384* (25.73)	1.559** (41.58)	0.91	1.00 (9.85)	1.00 (6.62)	1.43** (57.18)	1.35** (48.17)
Kidney   (Number Animals Assessed)	10	10	10	10	10	10	10	10	10	10
Absolute	3.60	4.22 (17.15)	4.17 (15.60)	5.00** (38.55)	5.04** (39.83)	1.87	1.98  (6.85)	2.16* (16.44)	2.37** (27.76)	2.44** (31.20)
RBW  (%Diff)	0.60	0.72** (19.27)	0.70** (15.86)	0.92** (53.50)	0.87** (44.97)	0.64	0.67 (6.01)	0.73 (15.46)	0.80** (25.35)	0.90** (41.87)
RBrW  (%Diff)	165.00	193.24* (17.12)	192.22 (16.50)	235.01** (42.43)	231.33** (40.20)	93.28	96.84 (3.82)	104.16 (11.67)	118.55** (27.09)	121.81** (30.59)
Liver   (Number Animals Assessed)	10	10	10	10	10	10	10	10	10	10
Absolute	17.62	19.80 (12.35)	22.40 (27.15)	28.76** (63.22)	32.68** (85.45)	9.23	10.40 (12.69)	12.84* (39.22)	17.80** (92.99)	19.72** (113.80)
RBW  (%Diff)	2.93	3.35 (14.19)	3.73** (27.48)	5.29** (80.67)	5.62** (91.70)	3.17	3.54 (11.73)	4.36** (37.65)	5.98** (89.09)	7.26** (129.28)
RBrW  (%Diff)	806.02	905.95 (12.40)	1033.94* (28.28)	1354.34** (68.03)	1498.68** (85.94)	463.55	508.14 (9.62)	618.79* (33.49)	890.90** (92.20)	987.24** (112.97)

RBW = Relative to Body Weight

RBrW = Relative to Brain Weight

% change is % change from control.

- = not applicable.

* = Statistically significantly different from the control group at p ≤ 0.05 using Dunnett’s test.

** = Statistically significantly different from the control group at p ≤ 0.01 using Dunnett’s test.

Bold= Values considered to be test substance-related.

# = Isotridecanol Group

Test Substance-Related Histopathological Findings at Interim Euthanasia (Day 47/48)

Dosage (mg/kg/d)	Male 0	Male 100	Male 300	Male 1000	Male 1000^#	Female 0	Female 100	Female 300	Female 1000	Female 1000^#
Liver  (number of animals assessed)	5	N/A	N/A	5	5	5	N/A	N/A	5	5
Hypertrophy, hepatocellular	0	N/A	N/A	3	5**	0	N/A	N/A	5**	5**
Minimal	-	N/A	N/A	2	1	-	N/A	N/A	5**	1
Mild	-	N/A	N/A	1	4*	-	N/A	N/A	0	4*
Kidney (number of animals assessed)	0	N/A	N/A	2	1	N/A	N/A	N/A	N/A	N/A
Degeneration/necrosis; proximal convoluted tubule	N/A	N/A	N/A	2	1	N/A	N/A	N/A	N/A	N/A
Minimal	N/A	N/A	N/A	2	1	N/A	N/A	N/A	N/A	N/A
Regeneration; proximal convoluted tubule	N/A	N/A	N/A	2	1	N/A	N/A	N/A	N/A	N/A
Minimal	N/A	N/A	N/A	1	0	N/A	N/A	N/A	N/A	N/A
Mild	N/A	N/A	N/A	1	1	N/A	N/A	N/A	N/A	N/A
Cast; granular	N/A	N/A	N/A	2	1	N/A	N/A	N/A	N/A	N/A
Minimal	N/A	N/A	N/A	2	1	N/A	N/A	N/A	N/A	N/A
Accumulation; hyaline droplets	N/A	N/A	N/A	2	1	N/A	N/A	N/A	N/A	N/A
Minimal	N/A	N/A	N/A	2	1	N/A	N/A	N/A	N/A	N/A
Thyroid gland (number of animals assessed)	5	N/A	N/A	5	1	5	N/A	N/A	5	5
Hypertrophy/hyperplasia; follicular cell	0	N/A	N/A	8**	1	0	N/A	N/A	5**	5**
Minimal	-	N/A	N/A	8**	1	-	N/A	N/A	3	5**
Mild	-	N/A	N/A	0	1	-	N/A	N/A	2	0

- = no noteworthy findings

N/A = not applicable.

* = Statistically significantly different from the control group at ≤ 0.05 using Fisher’s Exact test.

** = Statistically significantly different from the control group at ≤ 0.01 using Fisher’s Exact test.

# = Isotridecanol Group

Test Substance-Related Histopathological Findings at Terminal Euthanasia (Day 91/92)

Dosage (mg/kg/d)	Male 0	Male 100	Male 300	Male 1000	Male 1000^#	Female 0	Female 100	Female 300	Female 1000	Female 1000^#
Liver  (number of animals assessed)	10	10	10	10	10	10	10	10	10	10
Hypertrophy, hepatocellular	0	5*	10**	10**	10**
Minimal	-	5*	8**	0	0
Mild	-	0	2	10**	2
Moderate	-	0	0	0	8**
Kidney (number of animals assessed)	10	10	10	10	10	10	10	10	10	10
Degeneration/necrosis; proximal convoluted tubule	0	1	2	10**	9**	0	0	0	0	0
Minimal	-	0	2	5*	6*	-	-	-	-	-
Mild	-	1	0	4	2	-	-	-	-	-
Moderate	-	0	0	1	1	-	-	-	-	-
Regeneration; proximal convoluted tubule	0	3	3	10**	9**	0	0	0	0	0
Minimal	-	2	2	2	3	-	-	-	-	-
Mild	-	1	1	5*	3	-	-	-	-	-
Moderate	-	0	0	3	3	-	-	-	-	-
Cast; granular	0	1	1	10**	9**	0	0	0	0	0
Minimal	-	0	1	3	5*	-	-	-	-	-
Mild	-	1	0	4	3	-	-	-	-	-
Moderate	-	0	0	3	1	-	-	-	-	-
Accumulation; hyaline droplets	0	1	0	10**	9**	0	0	0	0	0
Minimal	-	1	-	10**	9**	-	-	-	-	-
Adrenal gland (number of animals assessed)	10	10	10	10	10	10	0	0	10	10
Vacuolation; cortical	0	4	2	3	1	0	-	-	0	0
Minimal	-	4	2	2	1	-	-	-	-	-
Mild	-	0	0	1	0	-	-	-	-	-
Thyroid gland (number of animals assessed)	10	10	10	10	10	9	10	10	10	10
Hypertrophy/hyperplasia; follicular cell	0	4	5*	8**	9**	0	0	2	8**	10**
Minimal	-	4	5*	8**	7**	-	-	2	8**	10**
Mild	-	0	0	0	2	-	-	0	0	0

- = no noteworthy findings.

* = Statistically significantly different from the control group at ≤ 0.05 using Fisher’s Exact test.

** = Statistically significantly different from the control group at ≤ 0.01 using Fisher’s Exact test.

# = Isotridecanol Group

Spermatogenesis Parameters at Terminal Euthanasia

Dosage (mg/kg/d)	0	100	300	1000	1000^#
Number Animals Assessed	10	10	10	10	10
MOTILITY (%) - Mean	87	90^a	91^b	84	84
S.D.	7.1	5.0	4.4	8.2	6.2
CAUDA EPID, LT WEIGHT (GRAMS) - Mean	0.2759	0.2549	0.3082	0.2946	0.3087
S.D.	0.03399	0.06883	0.02295	0.06525	0.04241
CAUDA EPID, LT CONCENTRATION (MILLIONS/GRAM) - Mean	566.4	519.6	582.4	550.3	626.9
S.D.	138.60	291.79	139.05	159.35	112.88
TESTIS, LEFT WEIGHT (GRAMS) - Mean	1.742	1.609	1.829	1.678	1.849
S.D.	0.1356	0.5901	0.1236	0.4127	0.1120
TESTIS, LEFT CONCENTRATION (MILLIONS/GRAM) - Mean	90.0	67.2*	94.0	87.7	90.0
S.D.	14.37	33.04	12.26	17.45	10.51
SPERM PRODUCTION RATE (MILLIONS/GRAM/DAY) - Mean	14.8	11.0*	15.4	14.4	14.8
S.D.	2.36	5.42	2.01	2.86	1.72
NORMAL MORPHOLOGY - Mean %	99.9	99.6	99.7	97.0	99.5
S.D.	0.34	0.44	0.36	6.76	0.58
NORMALLY SHAPED HEAD SEPARATED FROM FLAGELLUM - Mean %	0.1	0.3	0.2	1.2	0.3
S.D.	0.21	0.37	0.26	1.96	0.42
HEAD ABSENT WITH NORMAL FLAGELLUM - Mean %	0.1	0.1	0.1	1.9	0.2
S.D.	0.16	0.18	0.17	4.98	0.26
ABNORMAL HEAD - Mean %	0.0	0.0	0.0	0.0	0.0
S.D.	0.0	0.0	0.0	0.0	0.0
ABNORMAL FLAGELLUM - Mean %	0.0	0.0	0.0	0.0	0.0
S.D.	0.0	0.0	0.0	0.0	0.0
OTHER - Mean %	0.0	0.0	0.0	0.0	0.0
S.D.	0.0	0.0	0.0	0.0	0.0

# = Isotridecanol Group

Summary of Test Substance-Related Thyroid Hormone Findings

	Males (mg/kg/day)					Females (mg/kg/day)
	0	100	300	1000	1000^#	0	100	300	1000	1000^#
Total T3 (pg/mL)
Day 16	389	381	392	350	351	435	414	319*	353*	309*
Day 30	357	331	307	294	318	378	324	282*	305*	317**
Day 58	489	482	409	418	504	570	581	517	423**	526
Day 91/92	522	451	462	434	518	694	583	459*	476*	571
Total T4 (pg/mL)
Day 16	50,420	52,470	54,720	48,433	47,607	52,467	56,510	47,520	42,173*	41,767*
Day 30	51,133	53,830	47,480	40,400*	43,953**	54,727	58,260	47,550	38,200*	42,627*
Day 58	34,450	42,290	47,380*	37,960	45,900**	43,380	48,230	48,290	33,410	42,250
Day 91/92	39,400	45,580	45,880	35,060	26,290*	43,600	41.740	38,980	32,290*	21,620*
TSH (ng/mL)
Day 16	9.1	6.8	7.5	9.2	10.9	3.5	3.3	3.4	6.2**	5.1
Day 30	9.9	12.6	12.8	16.6	15.7	7.4	6.7	6.6	9.2	8.7
Day 58	13.1	12.6	15.2	19.0	20.5	6.5	5.5	7.8	13.8	11.6
Day 91/92	7.4	10.3	9.9	17.1**	14.4	4.9	4.2	4.3	7.5	5.9

* =Significantly different from the vehicle control group at 0.01 using Dunnett’s test.

**=   Significantly different from the vehicle control group at 0.05 using Dunnett’s test.

Bold = Test substance-related change

# = Isotridecanol Group

Applicant's summary and conclusion

Conclusions:

Isoundecanol does not cause human-relevant toxicity under the conditions of this subchronic study at dosage levels up to 1000mg/kg/d (the highest dose tested); the NOAEL for isoundecanol is 1000mg/kg/d.

Executive summary:

A 90 -day subchronic study was conducted in rats to assess the toxicity of isoundecanol. 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 isoundecanol to rats at dosage levels of 100, 300, and 1000 mg/kg/day for a minimum of 90 days was well tolerated.

There were no test substance-related effects on body weight, estrous cycle, spermatogenesis, or ophthalmic findings.

Test substance-related findings were limited to clinical observations, food consumption, hematological findings, clinical biochemistry findings, urinalysis findings, organ weight changes, gross pathological findings, and histopathological findings; the majority of these findings were non-adverse in nature due to slight magnitude of changes and/or being part of adaptive, non-adverse changes in the animal model. Adverse findings were not human relevant, and were limited to kidney findings in the male rat.

Clinical observations in the high dose group males and females (slight salivation, wet fur on cranium, dorsal cervical, dorsal thoracic, mouth, muzzle, and ventral cervical areas) and ploughing (animal diving into bedding and pushing it with its head and forelimbs) in high dose males only. These observations were considered nonadverse since the animals remained in overall good health (no effects on mortality and/or body weights). Higher mean food consumption was observed in males and females at the high dose throughout the dosing period (these changes were considered nonadverse since the animals remained in overall good health (no effects on mortality and/or body weights). Non-adverse changes in hematological parameters were observed in high dose male and females due to their slight magnitude of change. Clinical biochemistry changes were limited to mean urea nitrogen, which was higher in the test substance 1000 mg/kg/day male group when compared to the control group. Since the higher urea nitrogen was considered to correlate to the adverse microscopic findings noted in the kidneys of the 1000 mg/kg/day male groups, these changes were considered to be adverse. All other clinical biochemistry changes were considered to be nonadverse due to their slight magnitude of change. Since the higher urine mean volume and lower mean pH were considered to correlate to the adverse microscopic findings noted in the kidneys of the 1000 mg/kg/day male groups, these changes were considered to be adverse. Thyroid hormone changes followed a trend of decreased T3 and T4, with increases in TSH as dose and time increased. These changes in thyroid hormone levels were considered related to higher mean thyroid/parathyroid gland weights and hypertrophy/hyperplasia of thyroid follicular cells, and part of an adaptive, non-adverse change. Administration of the test substance for 90 days resulted in microscopic change in the liver and thyroid glands of the test substance 100, 300, and 1000 mg/kg/day group males and females and in the kidneys and adrenal glands of the 100, 300, and 1000 mg/kg/day group males.  Liver histological changes of minimal to moderate hepatocellular hypertrophy in the test substance 100, 300, and 1000 mg/kg/day group males and females correlated with liver enlargement and higher mean liver weights. Given the absence of changes in liver enzymes suggestive of hepatocellular damage (i.e., aspartate aminotransferase and alanine aminotransferase), the liver findings were considered non-adverse. Thyroid histological changes were present in the test substance 100, 300, and/or 1000 mg/kg/day group males and females and consisted of hypertrophy/hyperplasia of thyroid follicular cells. These findings correlated with higher mean thyroid/parathyroid gland weights and lower T3 and T4 and higher TSH in both males and females and were considered to have contributed to higher TSH and lower T3 and T4 levels. Thyroid gland changes were considered to be nonadverse and may represent an adaptive change secondary to test substance-related hepatic microsomal enzyme induction and associated increased thyroxin metabolism where elevations of TSH and decreases in T3 and/or T4 can be seen (Zabka, et al. 2011). For additional information on the human relevance of the thyroid findings, please consult the Appendix in Section 13. Adrenal gland histological changes were present in the test substance 100, 300, and 1000 mg/kg/day group males and consisted of cytoplasmic vacuolation of the adrenal cortical cells. There were no histological correlates for the increased mean adrenal gland weights in the test substance 300 and 1000 mg/kg/day group females. Given the absence of any other changes in the adrenal gland (e.g., organ weight changes in the females), findings within the adrenal gland were considered nonadverse.

Microscopic findings of proximal convoluted tubule degeneration/necrosis, proximal convoluted tubule regeneration, granular casts, and hyaline droplet accumulation in the kidneys along with higher urea nitrogen and creatinine values were considered adverse in the test substance 1000 mg/kg/day group males. Therefore, the no-observed-adverse-effect level (NOAEL) for isoundecanol was considered to be 300 and 1000 mg/kg/day for the males and females, respectively. However, since the renal changes observed in the male rats in this study were in association with alpha-2u globulin accumulation, which is a well-known male rat-specific effect, not occurring in female rats or other species, including humans, and thus are of no human relevance.

Thus, in the absence of the alpha-2u globulin accumulation effect, the NOAEL relevant for humans was considered to be 1000 mg/kg/day, the highest dosage level tested for both sexes of rats in this study.