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Toxicological information

Repeated dose toxicity: oral

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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
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
Limit dose chosen based on toxicokinetic data on test substance
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

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 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.:
700 mg/kg bw/day (actual dose received)
Remarks:
70mg/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 is 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 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) for isooctanol 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. A high dosage level of 700 mg/kg/day was selected based on the results of the aforementioned studies and toxicokinetic data, and was expected to produce some effects without causing severe toxicity.

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) 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 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:
no effects observed
Description (incidence and severity):
There were no test substance-related clinical observations. All clinical observations in the test substance-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain.
Mortality:
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, a statistically significantly lower mean body weight gain was noted in the 100 mg/kg/day group males compared to the control group from Days 64–71; this difference was transient and did not occur in a dose-related manner, and therefore was not considered test substance-related.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was unaffected by test substance administration. However, some statistically significant differences were observed in the 700 mg/kg/day group males and in the 100, 300, and 700 mg/kg/day group females compared to the control group. These differences in food consumption were not of sufficient magnitude to affect mean body weights and body weight gains in these groups, and therefore were not considered test substance-related.
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
A statistically significantly lower mean percentage of eosinophils were noted in the 700 mg/kg/day group males compared to the control group. Statistically significantly lower mean platelet counts were noted in the 300 and 700 mg/kg/day group females compared to the control group. The aforementioned differences were within the range of values in the Charles River Ashland historical control data (version 3.6), and therefore were not considered test substance-related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related higher mean triglyceride values were noted in the 700 mg/kg/day group males and females compared to the control group (difference was statistically significant for females only).

The elevated triglyceride levels were not considered to be adverse due to the mild nature of the change. No other test substance-related effects on serum chemistry parameters were noted for males and females at any dosage level. Other statistically significant differences were not considered test substance-related because they were within the Charles River Ashland historical control reference ranges and/or did not occur in a dose-related manner.
Urinalysis findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related higher liver and kidney weights were noted in 1 or both sexes at doses of 100, 300, and 700 mg/kg/day.

Statistically significantly higher mean absolute and relative (to brain and body weights) liver weights were seen in the 300 and 700 mg/kg/day female groups. Liver weights in the 100 mg/kg/day group females were approximately 8% higher than the control group levels and the group mean absolute liver weight slightly exceeded the Charles River Ashland historical control reference range; therefore, the apparent dose-related response in this group was also sufficient to be considered an effect of test substance administration. In contrast, a dose-related response in liver weight changes was not seen in the males. Higher liver weights (15% to 19%) were seen only in the 700 mg/kg/day group males with statistical significance limited to liver to body weight ratio. However, using absolute liver weights as examples, all mean values for the control, 300, and 700 mg/kg/day groups exceeded the Charles River Ashland historical control reference range. Higher liver weights in the 300 (females only) and 700 mg/kg/day groups were attributed to centrilobular hepatocellular hypertrophy seen microscopically.

Statistically significant higher mean absolute (females only) and relative (to brain and body weights) kidney weights were seen in the 700 mg/kg/day male and female groups. The 19% higher absolute kidney weight seen in the 700 mg/kg/day group males was sufficient to be considered an effect of test substance administration. There was no microscopic correlation in either sex for the higher kidney weights seen at 700 mg/kg/day.

There were no other test substance-related or statistically significant effects on organ weights.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related microscopic findings were noted in the liver and kidney of the 300 and/or 700 mg/kg/day groups.

In the liver, minimal centrilobular hypertrophy was characterized by enlargement of centrilobular hepatocytes by modestly increased amounts of pale eosinophilic cytoplasm. This finding was observed in a small number of males (4/10) and females (3/10) from the 700 mg/kg/day group and a single 300 mg/kg/day group female and was considered the microscopic correlation for the higher liver weights seen in those groups. Higher liver weights in the 100 mg/kg/day group females were likely due to hepatocellular hypertrophy too subtle to be detected with routine microscopic examination. One female from the 700 mg/kg/day group with centrilobular hypertrophy also had focal areas of hepatocellular necrosis. These foci were randomly distributed and a similar finding was seen in a 100 mg/kg/day group male in the absence of centrilobular hypertrophy; therefore its association with test substance administration was considered unlikely. In addition, evaluation of serum chemistry parameters gave no indication of test substance-related liver injury or dysfunction.

In the kidney, minimal hyaline droplet accumulation was seen only in the males at 700 mg/kg/day. It would be best characterized as a ‘very minimal’ change. At these magnifications similar but multifocal to rare globules were also seen in the lower dose and control group males such that the appearance in the male rats from the 700 mg/kg/day group was considered an exacerbation of a normal physiological process; a similar finding was not seen in the females. One male from the 700 mg/kg/day group had fairly uniformly distributed (mild) dilated renal tubules containing granular casts. This indication of prior renal tubular injury/necrosis (granular casts) or active tubular necrosis was not observed in other rats; therefore, its association with test substance administration was unclear. Evaluation of urinalysis and serum chemistry parameters gave no indication of test substance-related kidney injury or dysfunction.

There were no other definitive test substance-related histologic changes. One female from the 700 mg/kg/day group had uniformly distributed (mild) macrophage aggregates within the subcapsular sinus of the mesenteric lymph node. These macrophages were enlarged by an abundant amount of pale foamy cytoplasm and often contained small numbers of variably sized eosinophilic globules/droplets. The distribution of this finding indicated a process that had developed locally elsewhere and was delivered to the lymph node via the lymphatics; however, the site of origin, nature of the foamy contents and globular material, and association with test substance administration were undetermined.

Confounding the organ weight and histologic evaluations of the liver for the males was the presence of centrilobular fatty change in half of the control group males. This change was characterized by 1 or more large (macrovesicular), clear, round, and well-defined intracytoplasmic vacuoles consistent with lipid. The change was graded minimal to moderate and likely contributed to the atypically higher control group liver weights. This microscopic finding also transitioned into or abutted a variable thick zone of microvesicular periportal fatty change (not diagnosed). The cause(s) of the liver fatty change was not determined and a comparable finding in the centrilobular region was not seen in test substance-treated rats or in the control group females. Polycystic liver (biliary) and kidney disease was seen in a control group male as a spontaneous finding (autosomal recessive condition) that also resulted in higher individual animal and group mean liver and kidney weights. The above findings and 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.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Spermatogenic evaluations:

No test substance-related effects were observed on spermatogenesis endpoints (mean testicular and cauda epididymal sperm numbers and sperm production rate, motility, progressive motility, and morphology) in males at any dosage level. Differences from the control group were not statistically significant.

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 700 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Final Body Weights

 

          Males

          Females
Dosage (mg/kg/d)  0  100  300  700  0  100  300  700
 Mean Bodyweight (g) at end of study  628.7  602.9  582.9  603.8 303.4 302.0   294.4 312.7 
 %Diff  N/A -4.1   -7.3 -4.0  N/A  -0.05 -3.0  3.3 

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 APPLICABLE

(No statistically significant findings to report)

Haematological Parameters

            Males           Females
 Dosage (mg/kg/d)  0  100  300  700  0  100  300  700
Number of Animals Assessed  10  10  10  10  10  10  10  10
White Blood Cells (thousands/uL) - Mean  10.23  9.96  9.31  11.56 6.07  5.73  5.32  5.67 
  %Diff  N/A  -2.6  -9.0  13.0   N/A -5.6  -12.4  -6.6 
Red Blood Cells (millions/uL) - Mean  8.61  8.81  8.66  8.50  8.12 8.12  8.10   8.03
  %Diff   N/A  2.3  0.6  -1.3   N/A 0.0  -0.2   -1.1
Hemoglobin (g/dL) - Mean 15.8   15.9  15.7  15.5  15.6 15.4  15.3  15.3 
  %Diff   N/A 0.6  -0.6  -1.9    N/A -1.3  -1.9  -1.9 
Hematocrit (%) - Mean 46.4  46.5   46.3  45.8 44.7  44.2  43.9   43.8
  %Diff   N/A 0.2  -0.2   -1.3   N/A -1.1  -1.8  -2.0 
Mean Corpuscular Volume (femtoliters) - Mean 53.8  52.8  53.6  53.9  55.1  54.4  54.2  54.5 
  %Diff   N/A -1.9  -0.4   0.2   N/A -1.3   -1.6 -1.1 
Mean Corpuscular Hemoglobin (picograms) - Mean   18.4  18.1 18.2  18.2   19.2 19.0  18.8  19.0 
  %Diff   N/A  -1.6  -1.1 -1.1    N/A -1.0  -2.1  -1.0 
Mean Corpuscular Hemoglobin Concentration (g/dL) - Mean   34.2  34.2 34.0   33.9  34.9 34.8  34.7  34.9 
  %Diff   N/A  0.0  -0.6  -0.9   N/A  -0.3  -0.6 0.0 
Platelet Count (thousands/uL) - Mean  929  948 859   861 1046   971  952* 926** 
  %Diff   N/A  2.0 -7.5  -7.3    N/A  -7.2 -9.0   -11.5
Prothrombin Time (seconds) - Mean   16.6 17.8  17.4   16.8 16.5   16.6 16.1   16.0
  %Diff   N/A 7.2  4.8  1.2    N/A 0.6  -2.4   -3.0
Activated Partial Thromboplastin Time (seconds) - Mean 12.6   14.1  14.3 14.1   12.6  12.0  12.6  12.3
  %Diff   N/A  11.9 13.5  11.9   N/A  -4.8  0.0  -2.4 
Reticulocyte Count (%) - Mean  2.2   2.2 2.2  2.1 2.1   2.3 2.1  2.2 
  %Diff   N/A 0.0  0.0  -4.5    N/A 9.5  0.0  4.8 
Reticulocyte Absolute (thousands/uL) - Mean 185.9  192.6  189.4  176.7   170.3  182.8 171.7  171.5 
  %Diff   N/A 3.6   1.9 -4.9    N/A 7.3  0.8   0.7
Neutrophil Count (%) - Mean 19.7   20.4 16.6   18.4  17.7 16.7  23.0   18.8
  %Diff   N/A  3.6 -15.7  -6.6    N/A -5.6   29.9 6.2 
Lymphocyte Count (%) - Mean  75.5  75.1  79.4   77.6 78.1   79.5  73.2 77.8 
  %Diff   N/A -0.5  5.2  2.8    N/A  1.8 -6.3  -0.4 
Monocyte Count (%) - Mean  2.4 2.5   2.3  2.4 2.4  2.0  1.9  2.0 
  %Diff   N/A 4.2   -4.2  0.0   N/A -16.7  -20.8  -16.7 
Eosinophil Count (%) - Mean  1.7 1.4  1.0  0.8**  1.2  1.4   1.4 0.8 
  %Diff   N/A  -17.6 -41.2  -52.9    N/A  16.7  16.7 -33.3 
Basophil Count (%) - Mean   0.2 0.1   0.2  0.2  0.1 0.1  0.1   0.1
  %Diff   N/A -50.0   0.0  0.0   N/A 0.0    0.0   0.0  
Large Unstained Cell Count (%) - Mean 0.5   0.5 0.5   0.6 0.5  0.4   0.4 0.6 
  %Diff   N/A 0.0   0.0  20.0   N/A -20.0   -20.0 20.0 
Neutrophil Absolute (thousands/uL) - Mean  1.99 2.03  1.54  2.11   1.06  0.93  1.25 1.05 
  %Diff   N/A  2.0 -22.6  6.0    N/A -12.3  17.9  -0.9 
Lymphocyte Absolute (thousands/uL) - Mean 7.76  7.47 7.40  8.98  4.76  4.59  3.86  4.43 
  %Diff   N/A -3.7  -4.6  15.7    N/A -3.6  -18.9  -6.9 
Monocyte Absolute (thousands/uL) - Mean   0.25 0.25   0.21  0.27  0.14 0.11  0.11  0.12 
  %Diff   N/A 0.0  -16.0  8.0    N/A -21.4  -21.4  -14.3 
Eosinophil Absolute (thousands/uL) - Mean  0.17 0.14  0.10  0.10  0.07   0.08 0.08  0.04 
  %Diff    N/A -17.6  -41.2  -41.2    N/A 14.3  14.3  -42.9 
Basophil Absolute (thousands/uL) - Mean 0.02  0.01   0.02 0.02  0.00   0.01  0.01 0.01 
  %Diff   N/A -50.0   0.0 0.0    N/A   N/A  N/A   N/A 
Large Unstained Cell Absolute (thousands/uL) - Mean 0.05  0.05   0.04  0.07 0.03   0.02 0.02  0.03 
  %Diff   N/A 0.0   -20.0 40.0    N/A  -33.3  -33.3  0.0 
Red Cell Distribution Width (%) - Mean  12.0 12.3  12.5   12.4 11.1  11.2  11.2  11.5 
  %Diff   N/A 2.5  4.2  3.3    N/A 0.9  0.9   3.6

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

N/A = NOT APPLICABLE

Clinical Chemistry Parameters

            Male           Female
 Dosage (mg/kg/d)  100  300  700  0  100  300  700
 Number of Animals Assessed  10  10  10  10  10  10  10  10
Albumin (g/dL) - Mean   4.0  4.0  3.9  3.8 4.6  4.4   4.6 4.5 
  %Diff N/A  0.0  -2.5  -5.0   N/A  -4.3  0.0  -2.2 
Total Protein (g/dL) - Mean  6.9   6.8  6.8 6.6  7.6  7.4  7.7   7.7
  %Diff  N/A  -1.4  -1.4  -4.3   N/A  -2.6  1.3  1.3 
Globulin (g/dL) - Mean   2.9 2.8   2.9  2.8 3.1  3.0  3.1  3.1 
  %Diff  N/A   -3.4 0.0  -3.4   N/A  -3.2   0.0  0.0
Albumin/Globulin Ratio - Mean   1.40 1.42  1.35  1.37  1.50  1.47   1.48  1.48
  %Diff  N/A  1.4   -3.6 -2.1   N/A  -2.0  -1.3  -1.3 
Total Bilirubin (mg/dL) - Mean  0.01 0.00   0.00  0.00 0.03  0.05  0.03   0.00
  %Diff  N/A  -100.0 -100.0 -100.0  N/A  66.7  0.0   -100.0
Urea Nitrogen (mg/dL) - Mean 9.4  9.6  8.9  10.1  12.1  10.3*   11.3 10.2* 
  %Diff  N/A  2.1  -5.3  7.4   N/A  -14.9  -6.6  -15.7 
Creatinine (mg/dL) - Mean  0.39  0.36  0.35  0.32  0.44   0.41 0.44   0.41
  %Diff   N/A -7.7  -10.3  -17.9   N/A  -6.8  0.0   -6.8
Alkaline Phosphatase (International Unit/L) - Mean  134  146  128  141  66   70  57  72
  %Diff  N/A  9.0  -4.5  5.2   N/A  6.1   -13.6  9.1
Alanine Aminotransferase (International Unit/L) - Mean    38 36  30  45  30   29  30  29
  %Diff  N/A  -5.3  -21.1  18.4   N/A  -3.3  0.0   -3.3
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 120  108   112 104*   105 111   112  115
  %Diff   N/A  -10.0  -6.7 -13.3   N/A  5.7   6.7  9.5
Cholesterol (mg/dL) - Mean  57  53   61  69 66  65  71   90*
  %Diff  N/A  -7.0  7.0  21.1   N/A  -1.5  7.6   36.4
Calcium (mg/dL) - Mean  10.5  10.5  10.5 10.3  10.8  10.6 10.8  10.8 
  %Diff

N/A

 0.0  0.0  -1.9   N/A -1.9   0.0  0.0
Chloride (mg/dL) - Mean   105   104  105   103*  106  105  105  103**
  %Diff
 N/A
-1.0  0.0  -1.9   N/A  -0.9   -0.9 -2.8 
Phosphorus (mg/dL) - Mean 6.7   6.9   6.5 6.8 5.1   5.0  5.0  5.4
  %Diff N/A 3.0  -3.0  1.5   N/A  -2.0  -2.0   5.9
Potassium (Millequivalents/L) - Mean 5.62  5.82   5.75  5.59 5.14  4.99  4.93   5.00
  %Diff  N/A  3.6  2.3  -0.5   N/A   -2.9 -4.1  -2.7 
Sodium (Millequivalents/L) - Mean 145  145  146  145 144  144   144  144
  %Diff  N/A  0.0   0.7  0.0  N/A   0.0  0.0  0.0
Sorbitol Dehydrogenase (International Unit/L) - Mean  8 12   11  10  7  10  11  8
  %Diff  N/A  50.0   37.5  25.0  N/A  42.9  57.1  14.3 
Triglyceride (mg/dL) - Mean 66   60  77  102  40  33 46  68* 
  %Diff  N/A  -9.1   16.7  54.5  N/A  -17.5  15.0  70.0 
 Aspartate Aminotransferase (International Unit/L) - Mean  88  86  66  93 82   74  71  71
  %Diff   N/A  -2.3 -25.0   5.7   N/A -9.8  -13.4  -13.4 

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  700  0  100  300  700

Brain

(Number Animals

Assessed)

10   10  10  10 10   10  10  10

Absolute

(%Diff)

 2.1488

2.1751

(1.22) 

2.1287 

(-1.21)

2.1227 

(-1.21)

1.9743 

1.9962

 (1.11)

2.0046 

(1.53)

1.9916 

(0.88)

  RBW

(%Diff)

0.35052 

0.36683 

(4.66)

0.36847

(5.12)

0.35507 

(1.30)

 0.65633

0.66466 

(1.27)

 0.68751

(4.75)

0.64028

(-2.44)  

  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)

 0.06440

 0.05904

(-8.32)

 0.06130

(-4.81)

0.05777 

(-10.40)

0.06877 

0.06526 

(-5.10)

0.07084

(3.01) 

0.07175 

(4.33)

  RBW 

(%Diff)

0.01043

0.00986 

(-5.53)

0.01064 

(1.93)

0.00971 

(-6.97)

0.02279 

0.02173

(-4.65)  

0.02444 

(7.27)

0.02299 

(0.91)

  RBrW 

(%Diff)

 3.00390

2.71062 

(-9.76)

2.88395 

(-3.99)

2.72839

(-9.17)

3.47799 

3.27360 

(-5.88)

3.54678 

(1.98)

3.60044

(3.52) 

Pituitary Gland  

(Number Animals

Assessed)

10   10  10  10 10   10  10  10

  Absolute  

(%Diff)

0.01440 

 0.01578

(9.58)

0.01533 

(6.46)

0.01429 

(-0.76)

0.02073 

0.01975 

(-4.72)

0.02154 

(3.91)

0.01983 

(-4.34)

  RBW 

(%Diff)

0.00232a

0.00263 

(13.35)

0.00264 

(13.58)

0.00238 

(2.40)

0.00686 

0.00660 

(-3.71)

 0.00751

(9.59)

0.00635 

(-7.46)

  RBrW 

(%Diff)

 0.67035

0.72470 

(8.11)

0.71998 

(7.40)

0.67522 

(0.73)

 1.05116

0.99169 

(-5.66)

 1.08721

(3.42)

0.99664 

(-5.19)

Prostate and Seminal

Vesicle 

(Number Animals

Assessed)

 10  10  10  10  0  0  0

  Absolute  

(%Diff)

3.4057 

3.6635 

(7.57)

3.4464 

(1.2)

3.3892 

(-0.48)

N/A  N/A   N/A  N/A

  RBW 

(%Diff)

 0.54800

0.61569 

(12.35)

0.59973 

(9.44)

0.56810 

(3.67)

 N/A   N/A   N/A   N/A 

  RBrW 

(%Diff)

 158.41334

168.59533 

(6.43)

 162.15135

(2.36)

159.63902 

(0.77)

N/A  N/A   N/A   N/A 

Thyroid and

Parathyroid 

(Number Animals

Assessed)

 9  10  10  10 10   10  10  10

  Absolute  

(%Diff)

0.0308 

 0.0301

(-2.27)

0.0282 

(-8.44)

0.0272 

(-11.7)

0.0235 

0.0210 

(-10.6)

0.0230 

(-2.13)

0.0234 

(-0.43)

  RBW

 (%Diff)

0.00491 

0.00510 

(3.87)

0.00487 

(-0.84)

0.00448 

(-8.71)

 0.00783

0.00702 

(-10.31)

0.00785 

(0.26)

 0.00747

(-4.49)

  RBrW

 (%Diff)

1.42932 

 1.38554

(-3.06)

1.32399 

(-7.37)

1.29473 

(-9.42)

 1.19139

1.05337

(-11.58)

 1.14631

(-3.78)

1.17700 

(-1.21)

Heart 

(Number Animals

Assessed)

10   10  10  10 10   10  10  10

  Absolute 

(%Diff)

1.7268 

1.8577 

(7.58)

1.7025 

(-1.41)

1.8555 

(7.38)

1.0671 

1.0617 

(-0.51)

1.0962 

(2.72)

1.1464 

(7.21)

  RBW 

(%Diff)

0.27847 

0.31376 

(12.67)

0.29351 

(5.40)

0.31069 

(11.57)

0.35207 

0.35177 

(-0.087)

0.37347 

(6.08)

0.36716

(4.28)

  RBrW 

(%Diff)

 80.38262

85.39757 

(6.24)

79.99742 

(-0.48)

87.63876 

(9.03)

 54.11132

53.22837 

(-1.63)

54.60384 

(0.91)

57.70350 

(6.64)

Kidney

 (Number Animals

Assessed)

10   10  10  10 10  10  10  10

  Absolute

 (%Diff)

 3.6315

 3.9072

(7.59)

3.9456 

(8.65)

4.3300 

(19.2)

1.9169a

2.0262 

(10.9)

2.0576 

(7.34)

2.1990bb 

(14.7)

  RBW 

(%Diff)

0.58922a

0.65288 

(10.81)

0.68107 

(15.59)

0.71438a

(21.24)

0.63554a 

0.67426 

(6.09)

0.70445b 

(10.84)

0.70424b 

(10.81)

  RBrW

 (%Diff)

168.754a 

179.468 

(6.35)

 185.179

(9.73)

204.355bb 

(21.10)

97.225aa 

101.563 

(4.46)

102.706 

(5.64)

110.448bb 

 (13.6)

Liver

 (Number Animals

Assessed)

 10  10  10  10 10  10  10  10

  Absolute 

(%Diff)

 19.2799a

17.8857 

(-7.23)

19.0057 

(-1.42)

 22.2437

(15.4)

9.0890a

9.8483 

(8.35)

10.5596bb

(16.18)

12.4913bb

(37.43)

  RBW 

(%Diff)

3.08141aa

2.95992 

(-3.94)

3.25233 

(5.55)

3.66531bb

 (18.95)

 3.01773aa

3.27174 

(8.42)

3.60557bb 

(19.48)

4.00617bb 

(32.75) 

  RBrW 

(%Diff)

899.116a 

821.475 

(-8.64)

891.903 

(-0.80)

1051.393 

(16.94)

461.562aa 

493.642 

(6.95)

527.036bb

 (14.19)

628.372bb

 (36.14)

Ovary &

Oviduct  

(Number Animals

Assessed)

 0  0  0  0 10   10  10  10

  Absolute

 (%Diff)

 N/A  N/A    N/A  N/A 0.1325 

0.1393 

(5.13)

0.1297 

(-2.11)

0.1311

(-1.06) 

  RBW

 (%Diff)

 N/A  N/A    N/A  N/A   0.04361

0.04627 

(6.10)

 0.04417

(1.29)

0.04193 

(-3.84)

  RBrW

 (%Diff)

 N/A  N/A    N/A  N/A  6.68764 

7.00152 

(4.69)

6.43764 

(-3.74)

6.58300 

(-1.56 )

Spleen  

(Number Animals

Assessed)

 10  10  10  10 10   10  10  10

  Absolute 

(%Diff)

0.7703  

0.7260 

(-5.75)

0.7136 

(-7.36)

0.8183 

(6.23)

0.5013 

0.5461 

(9.00)

0.5484 

(9.40)

0.5224 

(4.21)

  RBW 

(%Diff)

0.12339 

 0.12101

(-1.93)

0.12267 

(-0.58)

0.13661 

(10.71)

0.16557 

0.18175 

(9.77)

0.18590 

(12.28)

 0.16704

(0.89)

  RBrW

 (%Diff)

35.92457 

 33.36782

(-7.12)

 33.52540

(-6.68)

38.68547 

(7.69)

25.47595 

 27.41840

(7.62)

27.24243 

(6.93)

26.24529

(3.02)

Thymus

 (Number Animals

Assessed)

10   10  10  10 10   10  10  10

  Absolute

 (%Diff)

0.2902 

0.2660 

(-8.34)

 0.2491

(-14.16)

0.2391 

(-17.55)

0.2419 

0.2376 

(-1.78)

0.2035 

(-15.87)

0.2244 

(-7.23)

  RBW

 (%Diff)

 0.04745

 0.04382

(-7.65)

0.04288 

(-9.63)

0.04011 

(-15.47)

 0.07995

0.07889 

(-1.32)

0.06955 

(-13.01)

 0.07166

(-10.37)

  RBrW 

(%Diff)

 13.52623

 12.21427

(-9.70)

 11.71417

(-13.40)

 11.30696

(-16.41)

 12.32138

 11.97464

(-2.81)

 10.13445

(-17.74)

 11.26475

(-8.58)

Testis (Left)

 (Number Animals

Assessed)

10   10  10  10  0  0  0

  Absolute 

(%Diff)

1.7629 

1.7916 

(1.62)

1.8145

 (2.93)

1.7997 

(2.09)

 N/A   N/A   N/A   N/A 

  RBW 

(%Diff)

  0.28543 

 0.30103 

(5.47) 

 0.31410

(10.05) 

0.30170 

(5.70)

 N/A   N/A   N/A   N/A 

  RBrW 

(%Diff)

82.10306 

 82.33135

(0.28)

85.30869 

(3.90)

 84.93569

(3.45)

 N/A   N/A   N/A   N/A 

Testis (Right)  

(Number Animals

Assessed)

10   10  10  10  0  0  0

  Absolute

 (%Diff)

1.7545 

1.7848

 (1.73)

1.8378 

(4.75)

1.8373 

(4.72)

 N/A   N/A   N/A   N/A 

  RBW

 (%Diff)

0.28387

0.30006 

(5.71)

 0.31843

(12.17)

0.30792 

(8.47)

 N/A   N/A    N/A

N/A

  RBrW

 (%Diff)

81.72414 

82.05858 

(0.41)

 86.43094

(5.76)

86.68989

 (6.08)

 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  0.7695 

0.8329 

(8.24)

 0.7086

(-7.91)

0.6432 

(-16.4)

  RBW

 (%Diff)

N/A  N/A  N/A N/A 0.25648 

0.28132 

(9.69)

0.24445 

(-4.69)

 0.20619

(-19.61)

  RBrW 

(%Diff)

 N/A   N/A   N/A   N/A  38.97070 

41.96597 

(7.69)

35.44759 

(-9.04)

 32.40343

(-16.85)

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

a = Group Factor Test ANOVA p<0.05

aa = Group Factor Test ANOVA p<0.01

b = Dunnett 2 Sided p < 0.05

bb = Dunnett 2 Sided p < 0.01

N/A = NOT APPLICABLE

Histopathological Findings

            Males           Females
Dosage (mg/kg/d)  0  100  300  700  0  100  300  700
Liver   (number of animals assessed)  10

 10

 10  10  10  10  10  10
 Hypertrophy, centrilobular (minimal)  0  0  0  4  0  0  1  3
Fatty change, centrilobular   5  0  0  0  0  0  0  0
 Minimal

 2

 -  -  -  -  -  -
 Mild  2  -  -  -  -  -  -  -
 Moderate  1  -  -  -  -  -  -  -
 Kidney (number of animals assessed)  10  10  10  10  10  0  2  10
 Accumulation, hyaline droplet (minimal)  0  0  0  7  0 -  0  0

Spermatogenesis Parameters

            Male
 Dosage (mg/kg/d)  100  300  700
Number Animals Assessed 10   10  10  10

MOTILITY - Mean

86  85  85  85
  %Diff  N/A  -1.2   -1.2  -1.2
PROGRESSIVE MOTILITY (%) - Mean 75   73  74  72
  %Diff  N/A   -2.7  -1.3 -4.0 
CAUDA EPID, LT WEIGHT (GRAMS) - Mean  0.303 0.309  0.308 0.312 
  %Diff  N/A   2.0  1.7  3.0
CAUDA EPID, LT CONCENTRATION (MILLIONS/GRAM) - Mean 677.2   789.9 568.0  785.2 
  %Diff  N/A   16.6 -16.1  15.9 
TESTIS, LEFT WEIGHT (GRAMS) - Mean 1.763   1.792  1.815 1.800
  %Diff N/A   1.6 2.9   2.1
TESTIS, LEFT CONCENTRATION (MILLIONS/GRAM) - Mean  148.9  135.4 140.8   126.7
  %Diff  N/A   -9.1 -5.4  -14.9 
SPERM PRODUCTION RATE (MILLIONS/GRAM/DAY) - Mean 24.4  22.2  23.1  20.8 
  %Diff N/A   -9.0 -5.3  -14.8 
NORMAL MORPHOLOGY - Mean %  98.9 99.3  99.1  99.3 
  %Diff  N/A  N/A   N/A  N/A 

NORMALLY SHAPED HEAD SEPARATED FROM FLAGELLUM - Mean %

0.4   0.3 0.2  0.4 
  %Diff   N/A  N/A   N/A   N/A 
HEAD ABSENT WITH NORMAL FLAGELLUM - Mean % 0.8   0.4 0.7  0.4 
  %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

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.

(No statistically significant findings to report)

Applicant's summary and conclusion

Conclusions:
Isooctanol does not cause toxicity under the conditions of this subchronic study at dosage levels up to 700mg/kg/d (the highest dose tested); the NOAEL for isooctanol is 700mg/kg/d.
Executive summary:

A 90 -day subchronic study was conducted in rats to assess the toxicity of isooctanol. The test substance was administered by oral gavage at a dose of 0, 100, 300, and 700mg/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 isooctanol to rats at dosage levels of 100, 300, and 700 mg/kg/day for a minimum of 90 days was well tolerated. Test substance-related findings were limited to nonadverse higher triglyceride values in the 700 mg/kg/day group males and females, alterations in organ weights in the 300 and 700 mg/kg/day group males and females, and microscopic findings in the kidney and liver in the 700 mg/kg/day group males and the 300 and 700 mg/kg/day group females. No test substance-related organ weight changes 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, progressive motility, and morphology) in males at any dosage level. Therefore, the no-observed-adverse-effect level (NOAEL) was considered to be 700 mg/kg/day, the highest dosage level tested.