Registration Dossier

Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Standard Guideline study conducted according to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test Material Name: 3-Amino-4-octanol
Chemical Name: 3-Amino-4-octanol
Synonyms: XU-12314.00
Supplier, City, State (Lot, Reference Number) : ANGUS Chemical Company, a wholly owned subsidiary of The Dow Chemical Company, Buffalo Grove, Illinois (lot # CEC-200700226-56)
Purity/Characterization (Method of Analysis and Reference): The purity of the test material was determined to be 97.9% on an anhydrous basis by gas
chromatography flame ionization detection with 0.279% water by Karl Fischer coulometric titration. Identification was determined by gas chromatography mass spectrometry and proton and carbon-13 nuclear magnetic resonance (Binkley, et. al., 2007).

Appearance (physical state, color): Semi-solid, opaque white
Molecular Formula: C8H19NO
Molecular Weight: 145.2

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
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 twothree 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:
After assignment to study, animals were housed singly in stainless steel cages, except during breeding (one male and one female) and during the littering phase. During littering, dams (and their litters) were housed in plastic cages provided with ground corncob nesting material from approximately GD 19 until completion of lactation. Cages had wire mesh floors and were suspended above catch pans. Cages contained a feed crock and a pressure activated lixit valve-type watering system.

Temperature: 22 +/-1 deg C
Humidity: 40-70%
Air changes: 12-15 times per hour
Photoperiod: 12 hour light/dark (on at 6.00 am, and off at 6.00 pm)

Randomization and Identification:
Before randomization, animals were stratified by body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform group mean weights and standard deviations at the start of the study. Animals placed on study were uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems, Seaford, Delaware) that were correlated to unique alphanumeric identification numbers

Feed and Water:
Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form. Feed and municipal water were 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 of the feed and water analyses indicated no contaminants that would interfere with the conduct of the study or interpretation of the results. Copies of these analyses were maintained at Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan.

Animal Welfare
In accordance with the U.S. Department of Agriculture animal welfare regulations, 9 CFR, Subchapter A, Parts 1-4, the animal care and use activities required for conduct of this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). The IACUC has determined that the proposed Activities were in full accordance with these Final Rules. The IACUC-approved Animal Care and Use Activities to be used for this study were DART 01, Subchronic Tox and Animal ID 01.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
Groups of 12 male and 12 female Crl:CD(SD) rats were administered 3-Amino-4-octanol in propylene glycol daily, by gavage, at dose levels of 0 (control), 15, 60, or 150 mg/kg/day. Female rats were dosed once daily for approximately two weeks prior to breeding, through breeding (two weeks), gestation (three weeks), and lactation up to postpartum day 4. Females were necropsied on postpartum day 5. Males were dosed for two weeks prior to breeding and continuing through breeding (two weeks) until necropsy (test day 33). Effects on general toxicity, 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. Litter size, pup survival, sex, body weight, and the presence of gross external abnormalities were also assessed. Pups were euthanized on PND 4.

Dose Preparation
The test material was administered in a propylene glycol vehicle, such that a dose volume of 6 ml/kg body weight yielded the targeted dose. Dose volumes were adjusted using the most current body weight. Dose volumes were prepared periodically throughout the study period based upon stability.

Details on mating procedure:
Breeding of the adults commenced after approximately two weeks of treatment. Each female was placed with a single male from the same dose level (1:1 mating) until pregnancy occurred or two weeks had elapsed. During the breeding period, daily vaginal lavage samples were evaluated for the presence of sperm as an indication of mating. The day on which sperm was detected or a vaginal copulatory plug was observed in situ was considered GD 0. The sperm- or plug-positive (presumed pregnant) females were then separated from the males and returned to their home cages. If a breeding male died, a substitute partner (from the same dose group) that had already completed mating was provided. If mating had not occurred after two weeks, the animals were separated without further opportunity for mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability
Stability of the test material in the vehicle was determined at concentrations ranging from 0.025 to 25% prior to the start of the main study.

Homogeneity and Concentration Verification
Analyses of all dosing solutions from the first mix of the main study were initiated prior to the start of dosing. The homogeneity of the low- and the high-dose levels was determined concurrent with dose confirmation. The analyses were conducted using LC/MS with internal standards.

Retainer Samples
A sample of the bulk test material was retained, but no samples of the dose formulations were retained.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
20 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
60 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
150 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Positive control:
None

Examinations

Parental animals: Observations and examinations:
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 could have been 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 also conducted on dams and their litters, at least twice daily. These examinations were conducted as described 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 exposure. 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.

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 premating 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, 20, and LD 1 and 4. 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 consumed 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) / (number of days in measurement cycle)

Litter observations:
Females were observed for signs of parturition beginning on or about GD 20. In so far as possible, parturition was observed for signs of difficulty or unusual duration. The day of parturition was recorded as the first day the presence of the litter was noted and was designated as LD 0. All litters were examined as soon as possible after delivery. The following information was recorded on each litter: date of parturition, litter size on the day of parturition (LD 0), the number of live and dead pups on days 0, 1, and 4, and the sex and the weight of each pup on LD 1 and 4. Any visible physical abnormalities or demeanor changes in the neonates were recorded as they were observed during the lactation period (see Daily In-Life Observations). In addition, pup clinical observations were recorded on each litter on PND 0 through 4. Any pups found dead were sexed and examined grossly, if possible, for external and visual defects and then discarded.
Postmortem examinations (parental animals):
Adult males (fasted) were submitted for necropsy after at least four weeks of exposure. Adult females (fasted) were terminated on LD 5-7, or at least 24 days after the end of the mating period for females not producing a litter. On the morning of the scheduled necropsy the animals were weighed. The animals were anesthetized by the inhalation of O2/CO2, their tracheas were exposed and clamped, and the animals were euthanized by decapitation.
A complete necropsy was conducted on all animals by a veterinary pathologist. 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 was 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, phosphatebuffered 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 were examined for the presence and number of implantation sites. After evaluation, uteri was gently rinsed with saline and preserved in neutral phosphate-buffered 10% formalin. Weights of the epididymides, kidneys, liver, testes and known target organs (spleen) were recorded, and organ:body weight ratios calculated.

Representative samples of tissues listed in below were collected and preserved in neutral, phosphate-buffered 10% formalin, with the exception of the testes and epididymides which were fixed in Bouin’s or another appropriate fixative.

Transponders were removed and placed in jars with the tissues. During routine working hours, any animals found dead or euthanized prior to the
scheduled necropsy was necropsied on that day. However, animals euthanized or found dead outside working hours were refrigerated until the next scheduled workday, at which time they were necropsied. Similar necropsy procedures were followed for these animals except that terminal body and organ weights were not recorded and the testes and epididymides were preserved in neutral, phosphatebuffered 10% formalin.

Tissues collected and preserved at necropsy:
ADRENALS (2) KIDNEYS (2)* PROSTATE (1)* AORTA (1) LACRIMAL/HARDERIAN GLANDS (2) RECTUM (1) AUDITORY SEBACEOUS GLANDS (0) LARYNX (1) SALIVARY GLANDS (1) BONE (1) (INCLUDING JOINT)(1) LIVER (3)* SEMINAL VESICLES (2)* BONE MARROW (1) LUNGS (5) SKELETAL MUSCLE (1) BRAIN (CEREBRUM, BRAINSTEM, CEREBELLUM) (3) MAMMARY GLAND - FEMALES ONLY(1)* SKIN AND SUBCUTIS (1) CECUM (1) MEDIASTINAL LYMPH NODE (1) SPINAL CORD (CERVICAL, THORACIC, LUMBAR) (3) CERVIX (1)* MEDIASTINAL TISSUES (1) SPLEEN (1)* COAGULATING GLANDS (2)* MESENTERIC LYMPH NODE (1) STOMACH (1) COLON (1) MESENTERIC TISSUES (1) TESTES (1)* CRANIAL NERVE – OPTIC (2) NASAL TISSUES/PHARYNX (2) THYMUS (1)
DUODENUM (1) ORAL TISSUES (2) THYROID GLAND (1) EPIDIDYMIDES (1)* OVARIES (2)* TONGUE (0) ESOPHAGUS (1) OVIDUCTS (2)* TRACHEA (1) EYES (2) PANCREAS (1) URINARY BLADDER (1) GROSS LESIONS (1)* PARATHYROID GLANDS (1) UTERUS (4)* HEART (1) PERIPHERAL NERVE -TIBIAL(1) VAGINA (1)* ILEUM (1) PITUITARY (1)* JEJUNUM (1)
* Tissues preserved for all surviving animals; examined histologically in high-dose and control animals (if treatment-related effects were seen; lower doses were examined).

Histopathology
Histologic examination of the tissues indicated above was conducted on all control and high-dose adult rats. Examination of tissues from the remaining groups was limited to those tissues that demonstrate treatment-related histologic effects at the high dose (testes) 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 both testes of all dose males was 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 a 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. 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). Testes from the low- and intermediate-dose groups were also prepared and histopathologically evaluated due to the presence of lesions in the high-dose males. 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 was 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 was not 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.

A complete set of tissues (listed above) was examined from rats found dead or moribund. Histological examination was conducted in a similar manner as described above, except that the testes were stained with hematoxylin and eosin.
Postmortem examinations (offspring):
All pups surviving to lactation day 4 were euthanized by oral administration of sodium pentobarbital solution (Veterinary Laboratories, Inc., Lenexa, Kansas), 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.
Reproductive indices:
Female mating index = (No. females with evidence of mating/No. paired) x 100
Male mating index = (No. males with evidence of mating/No. paired) x 100
Female conception index = (No. females with evidence of pregnancy/No. mated) x 100
Male conception index = (No. males siring a litter/No. mated) x 100
Female fertility index = (No. females with evidence of pregnancy/No. paired) x 100
Male fertility index = (No. males siring a litter/No. paired) x 100
Offspring viability indices:
Gestation index = (No. females delivering a viable litter/No. females delivering a litter) x 100
Gestation survival index = percentage of delivered pups alive at birth
Post-implantation loss = (No. implants – No. viable offspring)/(No. implants) x 100
Day 1 or 4 pup survival index = (No. viable pups on day 1 or 4/No. born live) x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

Examinations performed prior to study start revealed that all animals were in good health for study purposes. Three rats either died or were euthanized prior to the scheduled necropsy. Male rat (08A3885), in the 150 mg/kg/day group, had an apparent mechanical injury to the oral cavity associated with maloccluded incisors on TD 29 and was subsequently euthanized for animal welfare reasons due to the presence of a fracture of bone between the upper incisors (see Gross Pathology section). A second male rat (08A3860) in the 15 mg/kg/day dose group and one female rat (08A3931) in the 150 mg/kg/day dose group were found dead on TD 11 or 40, respectively. Gross and histopathologic examination of these animals revealed gavage error as the probable cause of death (see Gross Pathology section). All remaining animals survived until scheduled termination. No treatment-related effects on behavior or demeanor were observed in any phase of the study at any dose level. Although there was an increased incidence of noisy respiration in the high-dose females during the lactation phase of the study, these findings did not have a gross pathologic correlate and were likely associated with inadvertent aspiration of the dosing suspension during, or following, the oral gavage procedure.

Body Weights/Body Weight Gains
No significant differences in body weights or body weight gains were observed for males or females at any dose level or time point.

Feed Consumption
There were no treatment-related differences in the feed consumption of males or females at any dose level during the premating, gestation, or lactation phases of the study. Although there was a significant decrease in feed consumption during the TD 1-7 interval in females of the high-dose group, this decrease was not considered treatment related as there were no correlating effects on body weight or body weight gains of females at any
time point.

Reproductive Indices, Pup Survival, and Sex Ratio
There were no treatment-related effects at any exposure level on reproductive indices, time to mating, gestation length, post-implantation loss, pup survival or pup sex ratio. Although the PND 1 survival index was lower in the 60 mg/kg/day group and the PND 4 survival index was statistically identified as lower in the 15 and 60 mg/kg/day groups these differences were considered unrelated to treatment due to lack of a decrease in the high-dose group.

Organ Weights
There were no treatment-related effects on final body weights of males or females at any dose level.

Gross Pathology
There were no treatment-related gross pathologic observations of males and females at any dose level. Three rats either died or were euthanized prior to scheduled necropsy (see Daily In-Life and Clinical Observations section). Gross and histopathologic examination of male rat (08A3885) in the 150 mg/kg/day dose group indicated a recent fracture of the hard palate. A male rat (08A3860) in the 15 mg/kg/day dose group died spontaneously on TD 11. There were no clinical findings in this animal, but gross pathological observations indicated froth in the trachea, bloody facial soiling and mottled lungs consistent with probable gavage error.
Female rat (08A3931) in the 150 mg/kg/day dose group was found dead on TD 40 (GD 22) with no clinical observations. Gross pathological examination revealed mottled lungs and a pregnant uterus containing five normal appearing fetuses. Subsequent histopathologic examination of tissues indicated severe fibrinopurulent inflammation of the larynx and trachea consistent with probable gavage error as the cause of death.

Histopathology

There was a very slight degeneration of the testis germinal epithelium in 5 of 12 animals (3 unilateral; 2 bilateral) in the 150 mg/kg/day dose group, compared to an incidence of 2, 3, and 1 of 12 animals (all unilateral) in the 0, 15, and 60 mg/kg/day dose groups. This degeneration was characterized by loss and disruption of the germinal epithelial cells, and vacuolation of germinal and Sertoli cells. Although the incidence of this observation in the 150 mg/kg/day dose group was above concurrent and historical controls, this marginal (very slight) observation was deemed unrelated to treatment due to the fact that in a previous 28-day study in a different strain of rat (Otterdijk, 2007), a slightly higher dose did not produce similar effects. In addition, a follow-up 90-day study that administered 150 mg/kg/day 3-Amino-4-octanol to both previously used strains of rats (the current study and the previous 28-day study) did not reproduce the histopathologic findings despite the extended duration of the study, which allowed for completion of a full spermatogenic cycle (Rasoulpour et al., report in progress). Based on these data, this and all other histopathologic observations were interpreted to be spontaneous or iatrogenic alterations, unassociated with exposure to 3-Amino-4- octanol.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
>= 150 mg/kg bw/day (actual dose received)
Sex:
male/female

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Details on results (F1)

Litter Observations
Observations recorded in the offspring occurred at low frequency and bore no
relationship to treatment.

Litter Size and Pup Body Weights
There were no treatment-related effects on litter or pup body weights at any dose level tested.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 150 mg/kg bw/day (actual dose received)
Sex:
male/female

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Gavage administration of 3-Amino-4-octanol at dose levels up to, and including, 150 mg/kg/day produced no indication of reproductive toxicity at any dose level. There were no effects on prenatal/early neonatal growth and survival of the offspring. Based on these results, the no-observed-effect level (NOEL) for systemic and reproductive toxicity was 150 mg/kg/day, the highest dose level tested.