2,6,8-trimethylnonan-4-ol

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 August - 30 September 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to test guidelines and in accordance with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproductive/Developmental Toxicity Screening Test. July, 2000.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,6,8-trimethylnonan-4-ol

EC Number:

204-606-8

EC Name:

2,6,8-trimethylnonan-4-ol

Cas Number:

123-17-1

Molecular formula:

C12H26O

IUPAC Name:

2,6,8-trimethylnonan-4-ol

Details on test material:

- Name of test material (as cited in study report): 2,6,8-Trimethylnonan-4-nonanol
- Molecular weight (if other than submission substance): 186.3
- Physical state: Liquid, transparent pale yellow
- Analytical purity: The purity of the major component Trimethyl nonanol was 96.05 ± 0.055%, water was 0.32 ± 0.002%, and five minor components were present at 0.1% or greater as measured by area percent gas chromatography and coulometric Karl Fisher water determination. The structure was confirmed via infrared (IR) spectroscopy and gas chromatographic mass spectrometry (GC/MS).
- Lot/batch No.: RC1355T3D4

Test animals

Species:

rat

Strain:

other: CRL:CD(SD)Rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

Supplier and Location
Charles River Laboratories Inc. (Raleigh, North Carolina)

Age at Study Start
Approximately eight weeks of age at initiation of treatment

Physical and Acclimation
Each animal was evaluated by a laboratory veterinarian, or a trained animal/toxicology technician under the direct supervision of a laboratory veterinarian, to determine the general health status and acceptability for study purposes upon arrival at the laboratory (fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International - AAALAC International). The animals were housed two-three per cage in stainless steel cages, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle), and acclimated to the laboratory for at least one week prior to the start of the study.

Housing
Animals were housed one per cage in stainless steel cages. The relative humidity was maintained within a range of 40-70%. The average room temperature was maintained at 22 ± 1°C (with a maximum permissible excursion of ± 3°C). A 12-hour light/dark photocycle was maintained for all animal room(s) with lights on at 6:00 a.m. and off at 6:00 p.m. Room air was exchanged approximately 12-15 times/hour. Cages had wire mesh floors and were suspended above absorbent paper. Cages contained a feed crock and a pressure activated lixit valve-type watering system.

Feed and Water
Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form. Feed and municipal water was provided ad libitum. Analyses of the feed were performed by PMI Nutrition International to confirm the diet provides adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source was periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants were conducted at periodic intervals by an independent testing facility. The results indicated no contaminants that would interfere with the conduct of the study or interpretation of the results.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

Oral gavage is the preferred route of exposure according to OECD Guideline 422.

Male rats were dosed daily for 14 days prior to mating and continuing throughout the mating for 33 days. Female rats were dosed once daily for 14 days prior to breeding, and during breeding (two weeks), gestation (three weeks), and lactation through post partum day 4.

Dose Levels and Justification
The high-dose level was based upon data obtained from a preliminary range-finding study and was expected to induce some toxic effects, but not death or obvious suffering. The lower dose levels were selected to provide dose response data for any toxicity that may have been observed among the high-dose group rats and to establish a NOEL.

Dose Preparation
The test material was administered in a corn oil vehicle, such that a dose volume of 4 ml/kg body weight yielded the targeted dose. Dose volumes were adjusted using the most current body weight. Dose formulations were prepared periodically throughout the study period based upon stability.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis
Homogeneity
The low- and high-dose formulations from the first mix of the main study were analyzed to confirm homogeneous distribution of the test material concurrent with the start of the study.

Stability
Stability of the test material in the vehicle was initiated prior to the start of the study at concentrations of 0.25, 2.5 and 250 mg/ml. Dose formulations for the current study were prepared and used within these stability limits.

Concentration Verification
Analysis of all dosing solutions from the first mix of the main study were initiated prior to the start of dosing using gas chromatography with flame ionization and external standards to determine target concentrations.

Duration of treatment / exposure:

Male rats were dosed daily for 14 days prior to mating and continuing throughout the mating for 33 days. Female rats were dosed once daily for 14 days prior to breeding, and during breeding (two weeks), gestation (three weeks), and lactation through post partum day 4.

Frequency of treatment:

Daily throughout the treatment period

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 20, 100 and 500 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

12/sex/dose level

Control animals:

yes, concurrent vehicle

Details on study design:

Experimental Design and Critical Dates
Groups of 12 male and 12 female Crl:CD(SD) rats were administered the test material daily, by gavage, at dose levels of 0 (control), 20, 100, or 500 mg/kg/day. Female rats were dosed once daily for approximately two weeks prior to breeding, during breeding (two weeks), gestation (three weeks), and lactation through postpartum day 4. Male rats were dosed beginning approximately two weeks prior to breeding and continuing through breeding (two weeks) for a minimum exposure period of 33 days. Effects on general toxicity, neurobehavioral activity, clinical chemistry, urine parameters, hematology, gonadal function, mating behavior, conception, development of the conceptus, parturition and early postnatal growth and survival were evaluated. In addition, a gross necropsy of the adults was conducted with histopathologic examination of tissues. The key study parameters and study schedule are presented in Table 1. Gavage dosing for both males and females began on August 10, 2005. The adult males were necropsied on September 12, 2005. The adult females were necropsied from September 19, 2005 to September 30, 2005. In the offspring, litter size, pup survival, sex, body weight, and the presence of gross external abnormalities were assessed. Pups were euthanized on PND 4.

Positive control:

No data

Examinations

Observations and examinations performed and frequency:

Daily In-Life Observations
A cage-side examination was conducted at least twice daily. This examination was typically performed with the animals in their cages and was designed to detect significant clinical abnormalities that were clearly visible upon a limited examination, and to monitor the general health of the animals. The animals were not hand-held for these observations unless deemed necessary. Significant abnormalities that were observed included, but were not limited to: decreased/increased activity, repetitive behavior, vocalization, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor, twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in fecal consistency, and fecal/urinary quantity. In addition, all animals were observed for morbidity, mortality, and the availability of feed and water at least twice daily.

Cage-side examinations were conducted on dams and their litters, at least twice daily. These examinations were described as above.

Clinical Observations
Clinical examinations were conducted on all animals once daily throughout the study. During the exposure period, these examinations were conducted at the time of anticipated peak effects after dosing. Females were observed for signs of parturition beginning on or about GD 20 (see litter data). Clinical observations included a careful, hand-held examination of the animal with an evaluation of abnormalities in the eyes, urine, feces, gastrointestinal tract, extremities, movement, posture, reproductive system, respiration, skin/hair-coat, and mucous membranes, as well as an assessment of general behavior, injuries or palpable mass/swellings.

Detailed Clinical Observations
Detailed clinical observations (DCO) were conducted on all rats pre-exposure and weekly throughout the study. Mated females received DCO examinations on GD 0, 7, 14, and 20, and LD 3. Mated females received DCO examinations on GD 0, 7, 14, and 20, and LD 3. The DCO was conducted at approximately the same time each examination day prior to dosing, according to an established format. The examination included cage-side,
hand-held and open-field observations, which were recorded categorically or using explicitly defined scales (ranks).

Functional Tests
The functional tests (sensory evaluation, rectal temperature, grip performance and motor activity) were conducted pre-exposure and during the last week of the treatment period. For the females, this took place on LD 4.

Sensory Evaluation
The sensory evaluation included a test for nociception (responsiveness to tail pinch) and for startle response (responsiveness to sharp noise). The evaluation was conducted in a small clear plastic box.

Rectal Temperature
Rectal temperature was measured by carefully placing a rectal thermistor (Physitemp, Clifton, New Jersey) approximately 4 cm into the rectum for approximately 10 seconds. Temperature was then recorded. The thermistor was validated at 37°C before and after the study. The instrument was re-calibrated if the temperature recordings differed from the reference thermometer by more than ± 0.5°C.

Grip Performance
Hindlimb grip performance was tested according to the procedure described by Mattsson et al. (1986). Briefly, the observer placed the rat’s forelegs on a plastic bench and the hindfeet were set on a horizontal screen attached to an electronic strain gauge (Chatillon, Greensboro, North Carolina). The observer then smoothly but firmly pulled backward on the tail until the rat’s grip on the screen was broken. An electronic strain gauge was used to record the rat’s resistance to the pull in grams. The average of three trials was used for statistical analysis. Forelimb grip performance was similarly tested. In this application, a bench was not used, and the rats were placed so that the forefeet were on the screen and the hindfeet
were suspended approximately 10 cm above the plastic platform.
Instrument Validation: A standard 500-gram weight attached to a fine-gauge wire was suspended from the load cell and was checked just before and just after testing (a 1% tolerance, i.e., 500 ± 5 grams, was acceptable).

Reference
Motor Activity
An automated system was used for motor activity (MA) data collection. No entry into the MA test room was allowed during the testing period. Each test session consisted of ten 8-minute epochs, totaling 80 minutes of testing per animal per test session. This duration was chosen based on the results of a validation study indicating that performance of control animals approached asymptote in 70-80 minutes in CD rats (Dow Chemical Company report, 2001). Activity counts for each epoch were recorded.
Motor Activity Cage Calibration: Cages used for testing were calibrated prior to testing each day. Calibration was performed with a rod attached to a rotary motor that broke the infrared beam at a constant speed. The duration of each beam break was calculated to ensure equivalence across chambers.
Motor Activity Cage Allocation: Rats were allocated to the motor activity cages in such a way that the counterbalancing of treatment groups and sexes across cages and test times was maximized.

Body Weights/Body Weight Gains
All rats were weighed at least once during the pre-exposure period and on the first day of dosing. Male body weights continued to be recorded weekly throughout the study. Females were weighed weekly during the pre-mating and mating periods. During gestation, females were weighed on GD 0, 7, 14, 17, and 20. Females that delivered litters were weighed on LD 1 and 4. Females that failed to mate or deliver a litter were weighed at least weekly until termination. Body weight analyses were conducted for the following days: GD 0, 7, 14 and 20. Body weight gains were determined for the following intervals: GD 0-7, 7-14, 14-20, 0-20, and LD 1-4.

Feed Consumption
Feed consumption was determined weekly during the two week pre-breeding period for males and females by weighing feed crocks at the start and end of a measurement cycle. Feed consumption was not measured for males or females due to co-housing during breeding. Following breeding, feed consumption was not measured for males. For females during gestation, feed consumption was measured on GD 0, 7, 14, and 20. After parturition, feed consumption was measured on LD 1 and 4. Feed consumption was not recorded for females that failed to mate or deliver a litter. Feed consumption was calculated using the following equation:
Feed consumption (g/day) = (initial weight of crock - final weight of crock)/(# of days in measurement cycle)

Reproductive examinations are explained further in the reproduction/developmental tox section.

Reference
Mattsson, J. L., Johnson, K. A., and Albee, R. R. (1986). Lack of neuropathologic consequences of repeated dermal exposure to 2,4-Dichlorophenoxyacetic acid in rats. Fund. Appl. Toxicol. 6, 175-181.

Sacrifice and pathology:

Clinical Pathology
Animals were fasted overnight prior to blood collection. Blood samples were obtained from the orbital sinus following anesthesia with CO2 at the scheduled necropsy. Blood was not obtained from animals that failed to deliver a litter.

Hematology
Sample Preparation
Blood samples were mixed with ethylenediamine-tetraacetic acid (EDTA), smears will be prepared, stained with Wright-Giemsa stain, cover-slipped and filed for evaluation at the discretion of the pathologist.
Hematologic parameters were assayed using an Advia 120 Hematology Analyzer (Bayer Corporation, Tarrytown, New York).
Assays
Hematocrit (HCT)
Hemoglobin (HGB) concentration
Red blood cell (RBC) count
Total white blood cell (WBC) count
Differential WBC count
Platelet (PLAT) count
Reticulocyte (RET) count
RBC indices:
Mean Corpuscular Hemoglobin (MCH)
Mean Corpuscular Volume (MCV)
Mean Corpuscular Hemoglobin Concentration (MCHC)

Coagulation
Sample Preparation
Blood samples were collected in sodium citrate tubes, centrifuged and plasma collected and assayed using an ACL9000 (Instrumentation Laboratory, Lexington, Massachusetts).
Assay
Prothrombin time (PT)

Clinical Chemistry
Sample Preparation
Blood samples were collected in glass tubes and placed on ice. Sera were separated from cells as soon as possible following blood collection. Each serum sample was aliquoted into vials for use, as warranted for serum T3, T4 and TSH hormone assays. Serum samples were stored at –20°C. The serum samples were shipped on dry ice to Anilytics Incorporated, Gaithersburg, Maryland for analysis. Serum parameters listed below were measured using a Hitachi 912 Clinical Chemistry Analyzer (Roche Diagnostics, Indianapolis, Indiana).
Enzyme Activities of:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Concentrations of:
Albumin (ALB)
Cholesterol (CHOL)
Creatinine (CREA)
Electrolytes (NA, K, PHOS, CL and CA)
Glucose (GLU)
Total bilirubin (TBIL)
Total protein (TP)
Triglycerides (TRIG)
Urea nitrogen (UN)

Urinalysis
Urine samples were obtained from all males the week prior to the scheduled necropsy. Animals were housed in metabolism cages and the urine collected overnight (approximately 16 hours). Feed and water was available during this procedure.
Assays
Color, appearance, specific gravity (refractometer) and urine volume Semiquantitative analyses (Multistix Reagent Strips, Bayer Corporation, Elkhardt, Indiana on the Clinitek 200+) of:
pH
Bilirubin
Glucose
Protein
Ketones
Blood
Urobilinogen
Microscopic Exam:
Urine samples were collected from each male by manual compression of the urinary bladder. The urine samples were pooled from each group, and the microsediment were characterized microscopically.

Anatomic Pathology
Adult Necropsy
Adult males (fasted) were weighed and then submitted for necropsy after at least four weeks (actual: TD 34) of exposure. Adult females (fasted) were weighed and then submitted for necropsy on LD 5, or at least 24 days after the end of the mating period for females not producing a litter. The animals were anesthetized by the inhalation of CO2 and blood was collected from the orbital sinus (all males, all females that littered), their tracheas was exposed and clamped, and the animals were euthanized by decapitation.

A complete necropsy was conducted on all animals by a veterinary pathologist or a technician qualified to recognize lesions, assisted by a team of trained individuals. The necropsy included an examination of the external tissues and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined. The eyes were examined in situ by application of a moistened microscope slide to each cornea. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised. The nasal cavity was flushed via the nasopharyngeal duct and the lungs were distended to an approximately normal inspiratory volume with neutral, phosphate-buffered 10% formalin using a hand-held syringe and blunt needle. The uteri of all females were stained with an aqueous solution of 10% sodium sulfide stain (Kopf et al., 1964) for approximately two minutes and was examined for the presence and number of implantation sites. After evaluation, uteri were gently rinsed with saline and preserved in neutral phosphate-buffered 10% formalin. Weights of the adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes, thymus, and thyroid with parathyroids (weighed after fixation) were recorded, and organ:body weight ratios calculated.

Representative samples of tissues were collected and preserved in neutral, phosphate-buffered 10% formalin, with the exception of the testes and
epididymides that were fixed in Bouin’s or another appropriate fixative. In addition, sections of remaining liver were flash frozen in liquid nitrogen immediately after procurement for later UGT enzyme determination (approximately one gram). The frozen liver samples were stored at –80 ºC until analyzed. Transponders were removed and placed in jars with the tissues.

Microsome Isolation For Total UGT Activity
Frozen stored samples of the liver were thawed on ice, and homogenized using a Potter-Elvehjem apparatus. Microsomes were isolated using the method outlined by Guengerich (1982). A 1:5 ratio of wet weight tissue to homogenization buffer was used for all samples. Briefly, tissues were homogenized in a Tris-buffered KCl solution containing EDTA and an antioxidant (butylated hydroxyanisole). Upon centrifugation at 9,000xg to remove cell debris, blood cells, and large organelles, recovered supernatant were recentrifuged at 100,000xg for 60 minutes to pellet microsomes. The pellets were then resuspended in a pyrophosphate solution to remove nucleic acid polymers and centrifuged again at 100,000xg for 60 minutes. Upon discarding the supernatant, the pelleted microsomes were resuspended in a Trisbuffered 20% glycerol solution containing an antioxidant, frozen on dry ice and stored at -80°C until used. All centrifugation steps were carried out at 0-5°C and total microsomal proteins for individual animal determined using the Pierce BCA™ method (Pierce Chemical Co., Rockford, IL). Microsomal UGT activity was measured as the conjugation of a 1-naphthol substrate using the fluorometric method outlined by MacKenzie and Hanninen (1980) and modified for use in a microplate
fluorometer. Appropriate positive controls were run with enzyme assays.

Offspring Necropsy
All pups surviving to PND 4 were euthanized by intraperitoneal administration of sodium pentobarbital solution, examined for gross external alterations, and then discarded. Any pups found dead or which were euthanized in moribund condition were examined to the extent possible and discarded.

Histopathology
Histologic examination of tissues was conducted on all control and high-dose adult rats. Examination of tissues from the remaining groups was limited to those tissues that demonstrated treatment-related histologic effects at the high dose (liver, kidneys, and thyroid gland) and relevant gross lesions. Paraffin embedded tissues were sectioned approximately 6 μm thick, stained with hematoxylin and eosin and examined by a veterinary pathologist using a light microscope. The histopathological examination of the testes included a qualitative assessment of stages of spermatogenesis. A cross section through the approximate center of both testes of control and high-dose males were embedded in paraffin, sectioned at 5 μm and stained with modified periodic acid-Schiffs-hematoxylin. The presence and integrity of the stages of spermatogenesis were qualitatively evaluated following the criteria and guidance of Russell et al. (1990). Microscopic evaluation included qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross sections of the seminiferous tubules. The progression of these cellular associations defined the cycle of spermatogenesis. In addition, sections of both testes were examined for the presence of degenerative changes (e.g., vacuolation of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis).

Immunohistochemical staining of the kidneys for the detection of alpha 2μ globulin in all males given 0, 20, 100, or 500 mg/kg/day was performed by Charles River Laboratories – Pathology Associates (PAI), Frederick, Maryland. Methods for immunohistochemistry are documented in the report from Charles River Laboratories, and are included in the study file of the current report.

Selected histopathologic findings were graded to reflect the severity of specific lesions to evaluate: 1) the contribution of a specific lesion to the health status of an animal, 2) exacerbation of common naturally occurring lesions as a result of the test material, and 3) dose-response relationships for treatment-related effects. Very slight and slight grades were used for conditions that were altered from the normal textbook appearance of an organ/tissue, but were of minimal severity and usually with less than 25% involvement of the parenchyma. This type of change was neither expected to significantly affect the function of the specific organ/tissue nor have a significant effect on the overall health of the animal. A moderate grade would have been used for conditions that were of sufficient severity and/or extent (up to 50% of the parenchyma) that the function of the organ/tissue was adversely affected, but not to the point of organ failure. The health status of the animal may or may not be affected, depending on the organ/tissue involved, but generally lesions graded as moderate would not be life threatening. A severe grade was used for conditions that
were extensive enough to cause significant organ/tissue dysfunction or failure. This degree of change in a critical organ/tissue was life threatening.


Reference
Guengerich, F. P. (1982). Microsomal enzymes involved in toxicology - Analysis and separation. In, Principles and Methods of Toxicology (A. W. Hayes, Ed.), pp. 609-634, Raven Press, New York.

MacKenzie, P. I. and Hanninen, O. (1980). A sensitive kinetic assay for UDPGlucuronosyltransferase using 1-naphthol as substrate. Anal. Biochem. 109, 362-368.

Russell, L. D., Ettlin, R. A., SinhaHikim, A. P., and Clegg, E. D. (1990). Histological and Histopathological Evaluation of the Testis. Cache River Press, Clearwater, Florida.

Other examinations:

No additional information available.

Statistics:

Descriptive statistics only (means and standard deviations) were reported for RBC indices, and WBC differential counts. Parental body weights and parental body weight gains, litter mean body weights, feed consumption, urine volume, urine specific gravity, coagulation, clinical chemistry data, appropriate hematologic data, serum thyroid hormone concentrations, liver UGT, and organ weights (absolute and relative) were first evaluated by Bartlett's test (alpha = 0.01; Winer, 1971) for equality of variances. Based upon the outcome of Bartlett's test, either a parametric (Steel and Torrie, 1960) or nonparametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA) was performed. If the ANOVA was significant at alpha = 0.05, a Dunnett's test (alpha = 0.05; Winer, 1971) or the Wilcoxon Rank-Sum (alpha = 0.05; Hollander and Wolfe, 1973) test with Bonferroni's correction (Miller, 1966) was performed. Feed consumption values were excluded from analysis if the feed was spilled or scratched.

Gestation length, average time to mating, and litter size were analyzed using a nonparametric ANOVA. If the ANOVA was significant, the Wilcoxon Rank-Sum test with Bonferroni's correction was performed. Statistical outliers (alpha = 0.02) were identified by the sequential method of Grubbs (1969) and only excluded from analysis for documented, scientifically sound reasons. The mating, conception, fertility and gestation indices were analyzed by the Fisher exact probability test (alpha = 0.05; Siegel, 1956) with Bonferroni's correction. Evaluation of the neonatal sex ratio on PND 1 was performed by the binomial distribution test (alpha = 0.05; Steel and Torrie, 1960). Gender was determined for pups found dead on PND 0 and these data were included in sex ratio calculations.

Continued below.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation noted in some rats dosed with 100 mg/kg/day and higher

Mortality:

mortality observed, treatment-related

Description (incidence):

Salivation noted in some rats dosed with 100 mg/kg/day and higher

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Increased prothrobin time in males at 500 mg/kg/day

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Increased cholesterol in males and females at 500 mg/kg/day and decreased T3, T4 and TSH in males at 100 and 500 mg/kg/day and females at 500 mg/kg/day.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Decreased urinary pH in males at 100 and 500 mg/kg/day

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increased absolute and relative liver weights in males at 100 mg/kg/day. Increased absolute and relative liver, kidney and thyroid in males at 500 mg/kg/day & increased liver and thyroid in females at 500 mg/kg/day.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver of males at 100 mg/kg/day. Liver of males and females at 500 mg/kg/day. Kidney and thyroid of males at 500 mg/kg/day and

Histopathological findings: neoplastic:

no effects observed

Details on results:

Analytical Chemistry
Analyses of all dosing solutions from the initial mix revealed mean concentrations ranging from 98.4 to 107.7% of targeted concentrations. Analyses of aliquots for the low- and high-dose solutions indicated that the test material was homogeneously distributed. Trimethyl nonanol in corn oil was found to be stable for 107 days at concentrations ranging from 0.025 to 25%.

Mortality
All animals survived until scheduled termination.

Detailed Clinical Observations
Examinations performed on all animals pre-exposure revealed that all animals were in good health. Examinations performed on all rats revealed no treatment-related or statistically significant findings. A number of incidental observations bearing no relationship to treatment occurred during the study.

In-Life Observations
Oral administration of trimethyl nonanol resulted in increased salivation (clear perioral soiling) at doses ≥ 100 mg/kg/day in both sexes during all phases of the study (Text Tables 1 and 2). Increased salivation was transient as it was only observed immediately following dosing, suggesting a local response, perhaps to the taste of the test material. Salivation was observed intermittently in the high-dose group animals, with only a small number of isolated occurrences noted in the middle-dose animals. Salivation was not noted during the detailed clinical observations that were conducted prior to dosing. All other observations were incidental bearing no relationship to treatment. There were no notable observations made during the cage-side observations.

Functional Tests
Sensory Evaluation
Examinations performed on males and females at baseline and following treatment (Post-mating for males; LD 4 for females) revealed no treatment-related findings.
Rectal Temperature
Treatment did not affect rectal temperature in males (p = 0.7920) or females (p = 0.3458).
Grip Performance
There were no treatment-related effects on hindlimb grip performance either in males (p = 0.1994) or females (p = 0.2419). Similarly, there were no treatment-related effects on forelimb grip performance either in males (p = 0.8246) or females (p = 0.7808).
Motor Activity
Treatment did not affect motor activity total counts (treatment x time interaction) either in males (p = 0.9845) or in females (p = 0.6614). The distribution of the motor activity counts within session (treatment x time x epoch interaction) was not affected by treatment either in males (p = 0.7247) or in females (p = 0.7499).

Body Weights/Body Weight Gains
No significant differences in body weights were observed for males at any dose level. Similarly, there were no treatment-related differences in the body weights or body weight gains of females at any dose level tested during the pre-mating, gestation, or lactation periods.

Feed Consumption
There were no treatment-related differences in the amount of feed consumed by any of the dose groups when compared to their respective controls throughout the study. Pre-mating feed consumption of dams given 100 or 500 mg/kg/day was statistically identified as increased on TD 7-14 and gestation feed consumption of the high-dose females was statistically identified as increased on GD 0-7. These increases were considered to be spurious and unrelated to treatment.

Clinical Pathology
Hematology
There were no treatment-related alterations in hematologic parameters of males or females at any dose level.
Coagulation
The mean prothrombin time of males given 500 mg/kg/day was statistically identified as slightly higher than the control value (Text Table 3). This difference was interpreted to be treatment related, but was not associated with clinical evidence of hemorrhage and was not interpreted to be adverse.
Clinical Chemistry
Males and females given 500 mg/kg/day had higher mean cholesterol concentrations that were statistically identified in males (Text Table 4). These differences may be treatment related, but were interpreted not to be toxicologically significant due to the values being only slightly above (males) or within (females) historical control ranges.

Males given 100, or 500 mg/kg/day had treatment-related dose responsive lower mean concentrations of triiodothyronine (T3) and thyroxine (T4) that were statistically identified (Text Table 5). Males given 100 or 500 mg/kg/day also had treatment-related higher mean concentrations of thyroid stimulating hormone (TSH). Females given 500 mg/kg/day had treatment related lower mean concentrations of T3 and T4, and a higher mean concentration of TSH that was not statistically identified. The lower T3 and T4 concentrations in males given 20 mg/kg/day were not accompanied by higher concentrations of TSH or changes in thyroid weight and therefore the minor decrements in T3 and T4 concentrations were not considered treatment related. There were no treatment related alterations in thyroid hormone concentrations of females given 20 or 100 mg/kg/day. The potential association between alterations in T3, T4 and TSH and increased metabolism of T3 and T4 by the liver was explored by measuring liver glucuronosyltransferase (UGT) activity (results below), as an association between hepatic enzyme induction by a xenobiotic and thyroid hormone alterations has been recognized for a number of compounds (Capen et al., 2002).

Liver Glucuronosyltransferase (UGT)
There were no treatmentrelated effects on liver UGT at any dose level tested. This lack of change does not necessarily preclude a causal association between the thyroid hormone changes and hepatic enzyme induction because total UGT, rather than the specific isoform responsible for T4 glucuronidation (T4-UDP glucuronosyltransferase), was measured. In addition, the magnitude of thyroid hormone changes was slight. Therefore, the
change in T4-UDP glucuronosyltransferase may not have been sufficient to cause a detectable change in total UGT.

Urinalysis
The only treatment-related effect on urinalysis parameters was a dose-responsive decrease in urine pH, noted in males given 100 or 500 mg/kg/day(Text Table 6). This was most likely due to the excretion of acidic metabolites of TMN alcohol rather than a toxic effect per se.

Anatomic Pathology
Organ Weights
Males given 100 or 500 mg/kg/day and females given 500 mg/kg/day had treatment-related statistically identified higher mean absolute and/or relative liver weights (Text Table 7). The higher liver weights corresponded with centrilobular/midzonal hepatocellular hypertrophy in males given 100 mg/kg/day and females given 500 mg/kg/day, and with panlobular hepatocellular hypertrophy in males given 500 mg/kg/day. Males given 500 mg/kg/day had a treatment-related statistically identified higher mean absolute and relative kidney weight. This alteration corresponded with hyaline droplet formation in the proximal renal tubules. Males and females given 500 mg/kg/day had higher mean absolute and relative thyroid gland weights (statistically identified for only the female absolute thyroid weight at 500 mg/kg/day). The higher thyroid weights were interpreted to be treatment-related, and corresponded to slight hypertrophy of follicular epithelial cells in males, and slight alterations in T3, T4, and TSH concentrations for both sexes.

Gross Pathology
There were no treatment related gross pathologic observations of males and females at any dose level.

Histopathology
Males given 500 mg/kg/day had a treatment related increase in the incidence of very slight or slight multifocal degeneration of renal tubules (Text Table 8). The degeneration primarily involved the proximal tubules and was characterized by an increase in cytoplasmic basophilia of tubular epithelial cells, decreased size of epithelial cells, thickening of the tubular basement membrane, and the occasional presence of interstitial mononuclear inflammatory cells. Male rats given 100 or 500 mg/kg/day had treatment-related hyaline droplet formation in the renal proximal tubules. Although the hyaline droplets were most likely composed of proteinaceous material, the type of protein was not determined. Immunohistochemical staining of the kidneys by Charles River Laboratories showed that the hyaline droplets were negative for the presence of alpha 2μ globulin. Results from the Charles River immunohistochemical analyses are included in the study file of this report.

Males given 100 or 500 mg/kg/day and females given 500 mg/kg/day had treatment related hypertrophy of hepatocytes (Text Table 9). Hepatocellular hypertrophy was characterized by an increase in the size of hepatocytes in a panlobular (males at 500 mg/kg/day) or centrilobular/midzonal (males at 100 and females at 500 mg/kg/day) distribution and an increase in the eosinophilic staining of the hepatocytes. Hepatocellular hypertrophy was believed to be due to the induction of hepatic enzymes likely necessary to metabolize TMN alcohol.

Males given 500 mg/kg/day had a treatment-related increase in the incidence of hypertrophy of follicular epithelial cells of the thyroid gland (Text Table 10). Follicular cell hypertrophy was characterized by an increase in the amount of apical cytoplasm, giving the affected cells a columnar appearance. Microscopic thyroid alterations were not observed in female rats. Follicular cell hypertrophy was potentially the result of disruption in the hypothalamic-pituitary-thyroid axis due to the increased metabolism of T3 and T4 by the liver. Hepatic microsomal enzymes induced by TMN alcohol might have secondarily affected the metabolism of T3 and T4 resulting in the stimulation of the thyroid. A similar association of hepatic enzyme induction by a xenobiotic and thyroid hypertrophy has been recognized for a number of compounds (Capen et al., 2002).

Effect levels

1

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Effects noted related to liver and kidney in males at 100 mg/kg/day. Effects noted in females only at 500 mg/kg/day.

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

3

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Effects only noted in female rats dosed at 500 mg/kg/day.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Text Table 1. Salient Male Clinical Observations

Male Clinical Observations	Number of Animals Affected
mg/kg/day:	0	20	100	500
Perioral Soiling - Clear	0	0	2	12

Bold indicates effects considered to be treatment related.

Text Table 2. Salient Female Clinical Observations (all phases)

Female Clinical Observations	Number of Animals Affected
mg/kg/day	0	20	100	500
Perioral Soiling - Clear	0	0	2	12

Bold indicates effects considered to be treatment related.

Text Table 3. Prothrombin Time Effects

	Dose (mg/kg/day)
	0	Historical^1	20	100	500
Parameter	Males
Prothrombin Time (sec)	12.3	12.1 - 22.8	12.8	12.7	14.0^$
	Females
Prothrombin Time (sec)	13.0	11.7 - 14.7	13.1	12.7	13.0

^$ Statistically Different from Control Mean by Wilcoxon’s Test, Alpha = 0.05.

¹Historical controls group mean range from OECD 422 done since 2000.

Bold type indicates the effects judged to be treatment related.

Text Table 4. Serum Cholesterol Concentration Effects

	Dose (mg/kg/day)
	0	Historical^1	20	100	500
Parameter	Males
Cholesterol (mg/dl)	56	38 - 58	59	59	69*
	Females
Cholesterol (mg/dl)	59	42 - 73	53	63	70

*Statistically Different from Control Mean by Dunnett’s Test, Alpha = 0.05.

¹Historical controls group mean range from OECD 422 studies done since 2000.

Bold type indicates the effects judged to be treatment related.

Text Table 5. Serum Thyroid Hormone Effects

	Dose (mg/kg/day)
	0	20	100	500
Parameter	Males
T3 (ng/dl)	108.93	95.77	94.15*	91.71*
T4 (ug/dl)	6.65	5.71*	5.28*	4.70*
TSH (ng/dl)	3.89	3.03	5.05	5.86
	Females
T3 (ng/dl)	97.49	93.30	92.44	84.75
T4 (ug/dl)	3.70	3.93	3.76	3.42
TSH (ng/ml)	3.61	2.55	3.40	5.87

*Statistically Different from Control Mean by Dunnett’s Test, Alpha = 0.05.

Bold type indicates the effects judged to be treatment related.

Text Table 6. Urinary pH Effects

	Dose (mg/kg/day)
	0	20	100	500
Parameter	Males
Urine pH	6.5 (3)	6.0 (1)	6.5 (5)	5.5 (1)
	7.0 (4)	7.0 (7)	7.0 (7)	6.0 (2)
	7.5 (4)	7.5 (4)		6.5 (4)
	8.0 (1)			7.0 (5)

Urine pH data tabulated as the number of animals (N) with the stated value.

Text Table 7. Organ Weight Effects

	Dose (mg/kg/day)
	0	Historical^1	20	100	500
Parameter	Males
Final Body Weight (g)	408.5	354.1 - 447.2	409.2	398.4	411.6
Absolute Kidneys (g)	2.931	2.573 - 3.179	3.050	3.092	3.374*
Relative Kidneys (g/100g bw)	0.720	0.713 - 0.801	0.747	0.778	0.820*
Absolute Liver (g)	12.436	10.108 - 13.420	12.444	13.149	16.355*
Relative Liver (g/100g bw)	3.041	2.719 - 3.298	3.036	3.296*	3.966*
Absolute Thyroid (g)	0.0170	0.0164 - 0.0207	0.0185	0.0173	0.0203
Relative Thyroid (g/100g bw)	0.0042	0.0038 - 0.0050	0.0045	0.0043	0.0050
	Females
Final Body Weight (g)	271.5	251.2 - 290.4	268.5	274.0	276.9
Absolute Liver (g)	10.310	8.185 - 10.433	10.230	10.438	13.234*
Relative Liver (g./100g bw)	3.782	3.155 - 3.748	3.815	3.793	4.773*
Absolute Thyroid (g)	0.0133	0.013 - 0.0157	0.0143	0.0138	0.0158*
Relative Thyroid (g/100g bw)	0.0050	0.005 - 0.006	0.0053	0.0050	0.0057

*Statistically Different from Control Mean by Dunnett’s Test, Alpha = 0.05.

¹Historical controls group mean range from OECD 422 studies done since 2000.

Bold type indicates the effects judged to be treatment related.

Text Table 8. Treatment-Related Kidney Effects

Sex	Males				Females
Dose (mg/kg/day	0	20	100	500	0	20	100	500
Kidneys (# examined)	12	12	12	12	12	12	12	12
Degeneration, tubule, multifocal, very slight	1	1	2	9	1	1	0	0
Degeneration, tubule, multifocal, slight	0	0	0	1	0	0	0	0
Hyaline droplet formation, tubule, very slight	0	0	6	5	0	0	0	0
Hyaline droplet formation, tubule, slight	0	0	0	5	0	0	0	0

Bold type indicates the effects judged to be treatment related.

Text Table 9. Treatment-Related Liver Effects

Sex	Male				Female
Dose (mg/kg/day	0	20	100	500	0	20	100	500
Liver (# examined)	12	12	12	12	12	12	12	12
Hypertrophy, hepatocyte,centrilobular/midzonal, with alteredtinctorial properties, very slight	0	1	5	1	3	2	2	9
Hypertrophy, hepatocyte,centrilobular/midzonal, with alteredtinctorial properties, slight	0	0	0	0	0	0	0	2
Hypertrophy, hepatocyte, panlobular, withaltered tinctorial properties, very slight	0	0	0	2	0	0	0	0
Hypertrophy, hepatocyte, panlobular, withaltered tinctorial properties, slight	0	0	0	9	0	0	0	0

Bold type indicates the effects judged to be treatment related.

Text Table 10. Treatment-Related Thyroid Effects

Sex	Male				Female
Dose (mg/kg/day)	0	20	100	500	0	20	100	500
Thyroide gland (# examined)	12	12	12	12	12	12	12	12
Hypertrophy, follicular cell, slight	2	1	3	7	0	0	0	0

Bold type indicates the effect judged to be treatment related.

Applicant's summary and conclusion

Conclusions:

Based on the results of the conducted study, the no-observed effect level (NOEL) for general toxicity was 20mg/kg/day.

Executive summary:

Groups of 12 male and 12 female Crl:CD(SD) rats were administered trimethyl nonanol daily, by oral gavage at dose levels of 0 (control), 20, 100, or 500 mg/kg/day. Females were dosed once daily for two weeks prior to breeding, during breeding (two weeks),

gestation (three weeks), and lactation through postpartum day 4. Females were necropsied on post-partum day 5. Males were dosed two weeks prior to breeding and continuing through breeding (two weeks) until necropsy (test day 34). Effects on reproductive and neurological function as well as general toxicity were evaluated. In addition, post mortem examinations included a gross necropsy of the adults with collection of organ weights and histopathologic examination of tissues. Litter size, pup survival, sex, body weight, and the presence of gross external abnormalities were also assessed.

Administration of 100 or 500 mg/kg/day trimethyl nonanol resulted in parental systemic toxicity. The primary effect appeared to be an increase in absolute and relative liver weights and corresponding hepatocellular hypertrophy in males given 100 or 500 mg/kg/day and in females given 500 mg/kg/day. Treatment-related decreases in T3, T4 and increases in TSH concentrations were noted in the same groups, and were accompanied by increased thyroid weights in high-dose males and females and slight hypertrophy of follicular epithelial cells in males given 500 mg/kg/day. The thyroid changes may have been a consequence of hepatic enzyme induction leading to enhanced thyroid hormone metabolism, as this phenomenon has been demonstrated for a wide range of hepatic enzyme inducers. Males given 500 mg/kg/day also had increased kidney weights that corresponded with very slight or slight multifocal degeneration of renal tubules. Treatment-related hyaline droplet formation was noted in male rats given 100 or 500 mg/kg/day. Minor, non-adverse findings included transient post-dosing salivation (clear perioral soiling) noted intermittently in males and females at the 100 and 500 mg/kg/day dose levels, decreased urinary pH in males given 100 or 500 mg/kg/day, a slight increase in prothrombin time in males given 500 mg/kg/day, and increased serum cholesterol in males and females given 500 mg/k/day.

There were no adverse effects of trimethyl nonanol on neurological function.

The no-observed-effect level (NOEL) for general toxicity was 20 mg/kg/day. The NOEL for neurological effects was 500 mg/kg/day, the highest dose level tested.