Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
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
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
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.
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.
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
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
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: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
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.

Dose descriptor:
NOAEL
Effect level:
>= 150 mg/kg bw/day (actual dose received)
Sex:
male/female
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
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.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 150 mg/kg bw/day (actual dose received)
Sex:
male/female
Reproductive effects observed:
not specified
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.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Good
Additional information

The Key study for this endpoint is the OECD 421 study, however additional information is also available from the 28 -day study and also a 90 -day study. In the OECD 421 study there were no effects observed in any of the reproductive parameters. However histopathology of the male rats in the high dose group indicated a very slight degeneration of the testis germinal epithelium in 5 of 12 animals. This was potentially considered as a treatment related effect, however neither a 28 -day study (higher dose in wistar rats) nor a 90- day study (using both SD and Wistar rats, same high dose) were able to replicate the findings of the OECD 421 study. 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.

In calculating a DNEL for reproductive toxicity, the starting point is 150 mg/kg bw/day. Whilst this NOEL is derived from a screening study, the lack of reproductive effects observed in other repeated dose studies support this NOEL Therefore in calculating a DNEL from this NOEL it is considered unnecessary to include an additional factor to take into account the lower sensitivity of this study design.


Short description of key information:
OECD 421 reproductive/developmental screening study in rats (oral Gavage)
28-day sub acute repeat dose toxicity study in rats (oral Gavage)
90-day sub chronic repeat dose toxicity study in rats (oral gavage)

Justification for selection of Effect on fertility via oral route:
OECD guideline, Reliability 1 study

Effects on developmental toxicity

Description of key information
OECD 421 reproductive/developmental screening study in rats (oral Gavage)
28-day sub acute repeat dose toxicity study in rats (oral Gavage)
90-day sub chronic repeat dose toxicity study in rats (oral gavage)
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted as per OECD TG 414, US EPA OPPTS 870.3700, EU Method B.31, JMAFF, Guideline 2-1-18 and in accordance with the Principles of Good Laboratory Practice (GLP).
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: JMAFF, Guideline 2-1-18, Teratogenicity Study
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (Portage, Michigan)
- Age at study initiation: sexually mature adults
- Weight at study initiation: approximately 200-250 grams
- Fasting period before study: not applicable
- Housing: individually housed in stainless steel, solid bottom cages
- Diet (e.g. ad libitum): ad libitum, animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form
- Water (e.g. ad libitum): ad libitum, drinking water obtained from the municipal water source through a pressure activated lixit valve-type watering system
- Acclimation period: for at least 4 days prior to dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C with a tolerance of ± 1°C (and a maximum permissible excursion of ± 3°C)
- Humidity (%): 40-70%
- Air changes (per hr): 10-15 times/hour (average)
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: All dosing solutions were prepared by mixing the test material in propylene glycol at concentrations of 0, 2.5, 10, or 25 mg/ml and administered at a dose volume of 6 ml/kg body weight to achieve the targeted dose levels. Dose solutions were not corrected for purity. Dose volumes were adjusted every day based on individual body weights. The control rats were dosed with propylene glycol at 6 ml/kg body weight.

VEHICLE
- Justification for use and choice of vehicle (if other than water): recommended by various regulatory agencies
- Concentration in vehicle: All dosing solutions were prepared by mixing the test material in propylene glycol at concentrations of 0, 2.5, 10, or 25 mg/ml and administered at a dose volume of 6 ml/kg body weight to achieve the targeted dose levels
- Amount of vehicle (if gavage): All dosing solutions were prepared by mixing the test material in propylene glycol at concentrations of 0, 2.5, 10, or 25 mg/ml and administered at a dose volume of 6 ml/kg body weight to achieve the targeted dose levels
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of all dosing solutions from the first mix revealed mean concentrations of 3-amino-4-octanol ranging from 97.2 to 99.7% of targeted concentrations. Analysis of aliquots for the low- and high-dose solutions indicated that the test material was homogeneously distributed based on standard deviations of 1.5 and 4.1%, respectively.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
days 6-20 of gestation
Frequency of treatment:
daily during days 6-20 of gestation
Duration of test:
5-6 months
Remarks:
Doses / Concentrations:
15 mg/kg/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
60 mg/kg/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
150 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
24 rats/dose level
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: on the basis of an acute oral study (estimated LD50 - 550 mg/kg) and a range-finding study, where five Crl:CD(SD) rats/sex were administered 3-amino-4–octanol in propylene glycol at dose levels of 0, 50, 100, 250, 350, 450 mg/kg/day by daily gavage, for up to 14 days at a dose volume of 6 ml/kg body weight. Dose levels of 250 mg/kg/day and above exceeded the maximum tolerated dose, as determined by clinical observations of noisy, labored respiration with or without mouth breathing in several animals in these three dose groups. Necropsy of the moribund animals revealed decreased amount of fat, darkened lungs, decreased ingesta and gas within the gastrointestinal tract. These findings were attributed to the physical and chemical properties of the test material causing irritation of the lungs likely from aspiration of the test material. Due to the prevalence and incidence, these were not considered gavage errors, but instead dose limiting characteristics of the 3-amino-4-octanol.
Based on these results of the range-finding study, in the OECD 421 study, 12 Crl:CD(SD) rats/sex/group were administered 0, 15, 60, or 150 mg/kg/day 3-amino-4-octanol, in propylene glycol by gavage, at a dose volume of 6 ml/kg body weight for two weeks pre-breeding, through gestation, and up to lactation day 4 (females) or 32 test days (males). 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.
In a 90-day oral gavage toxicity study, 3-amino-4-octanol was administered by gavage, at 6 ml/kg body weight dose volume in propylene glycol vehicle, to groups of ten male and ten female Crl:CD(SD) rats at dose levels of 0, 15, 60, or 150 mg/kg body weight/day and to groups of ten male Crl:WI(Han) rats at dose levels of 0 or 150 mg/kg body weight/day for at least 90 days to evaluate the potential for systemic toxicity. There were no treatment-related effects in clinical signs, functional tests, body weights, feed consumption, ophthalmic, hematology, prothrombin time, or clinical chemistry parameters in Crl:CD(SD) rats of either sex. Male Crl:WI(Han) rats had treatment-related decreases in body weight, body weight gain, and feed consumption with treatment-related clinical observations including reflux of test material, noisy respiration, slow respiration, labored respiration (without mouth breathing), and/or blood coming
from the nasal cavity in the 150 mg/kg/day, which were associated with irritancy of the test material. There were no treatment-related epididymal sperm parameters (motility, counts, or morphology), gross or histopathologic observations and no toxicologically significant effects in urinalysis parameters or organ weight effects in the Crl:CD(SD) or Crl:WI(Han) rats.
The no-observed-effect level (NOEL) for male reproductive effects in either strain was 150 mg/kg/day, the highest dose level tested. The no-observed-adverse-effect level (NOAEL) for Crl:CD(SD) rats of either sex was 150 mg/kg/day 3-amino-4-octanol
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily approximately at the same time each day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations were conducted on all animals at least once daily and 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 WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on GD 0 (by the supplier) and daily during GD 6 through 21.

FOOD CONSUMPTION: Yes
- Feed consumption was recorded and statistically analyzed for all animals every three days from GD 3-21 by weighing feed containers at the start and end of a measurement cycle (3-6, 6-9, 9-12, 12-15, 15-18, 18-21)

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: On day GD 21, all surviving females (not fasted) were euthanized by carbon dioxide inhalation and a limited gross pathologic examination (necropsy) was performed. The sequence of the maternal necropsies was counterbalanced across groups (e.g., control, high, middle, low) to control for potential confounding influences of timing on fetal growth and skeletal ossification.
The maternal necropsy included an examination of the external tissues and all orifices. The skin was reflected from the carcass, the thoracic and abdominal
cavities were opened and the viscera were examined. The stomach, liver, and kidneys were dissected from the carcass and were incised. Any obvious gross
pathologic alterations were recorded, and the weight of the liver, kidneys, and gravid uterus were recorded. The ratios of liver and kidney weights to terminal
body weight were calculated. Representative sections of liver, kidneys, and gross lesions were preserved in neutral, phosphate-buffered 10% formalin. Microscopic examination of tissues was not conducted. Transponders were removed and placed in jars with the tissues.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- A detailed examination of the reproductive tract was performed and the number and position of implantations, viable fetuses, dead fetuses, and resorptions were recorded. Resorptions were classified as either “early” or “late” based on the presence (late resorption) or absence (early resorption) of grossly recognizable embryonic/fetal form, while a “dead fetus” indicated a very recent death as evidenced by a lack of external degenerative changes. For females with one or
more viable fetuses, the number of ovarian corpora lutea was counted. The uteri of females lacking visible implantations was stained with a 10% aqueous solution of ammonium sulfide and examined for evidence of early resorptions in order to verify pregnancy status.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
- The sex of all fetuses was recorded and the body weight of all viable fetuses was recorded. All fetuses were given an external examination that included
observations on body proportions, the head and face (including closure of the palate), abdomen, spine, extremities, genitalia, rectum and tail. All viable fetuses were euthanized by sublingual oral administration of sodium pentobarbital solution. Approximately one half of all the fetuses in each litter were chosen randomly via computer for visceral examination conducted by dissection under a low power stereomicroscope for evidence of visceral alterations. The visceral examination included observation of the thymus, trachea, esophagus, lungs, great vessels, heart (external and internal), liver, gastrointestinal tract, pancreas, spleen, kidney (sectioned), adrenal glands, ureters, bladder and reproductive organs. The heads of these fetuses were removed, placed in Bouin’s fixative and serially sectioned to allow for inspection of the eyes, brain, nasal passages and tongue. The remaining fetuses not selected for visceral examination were skinned, eviscerated, preserved in alcohol and double stained with Alcian Blue and Alizarin Red S for cartilage and bone. A thorough evaluation of the fetal skeleton was conducted on the remaining fetuses not selected for visceral examination.
Statistics:
Standard statistical methods were employed
Indices:
Pre- and post-implantation loss
Historical control data:
refer to attachment
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
- In-life Observations - Examinations performed on all animals revealed no treatment-related findings.
- Body weights/body weight gains - Although body weights in the treated groups were similar to controls, body weight gain in animals given 60 and 150 mg/kg/day from GD 6-9 was decreased in by 33.6 and 50.4%, respectively. There were no treatment-related effects on body weight gain in the 15 mg/kg/day dose level.
- Food consumption - Animals given 60 and 150 mg/kg/day had treatment-related decreases in feed consumption from GD 9-12 and 6-12, respectively, (ranging from 8.6 to 19.1%) relative to controls. There were no treatment-related effects on feed consumption in the 15 mg/kg/day dose level.
- Organ weights - There were no treatment-related differences in any of the measured parameters for any treated groups when compared to controls.
- Gross pathology - There were no treatment-related gross pathologic observations. The small number of gross pathologic observations either occurred at low frequencies and/or lacked a dose-response relationship.
Dose descriptor:
NOEL
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- Reproductive parameters - There were no treatment-related effects on pregnancy rates, resorption rates, litter size, numbers of corpora lutea or implantations, percent pre-implantation loss, percent postimplantation loss, fetal sex ratios, fetal body weight or gravid uterine weights in any treatment group.
- Fetal examinations - There were no treatment-related differences in the incidence of any fetal alteration in any treatment group compared to controls. Malformations or variations observed in fetuses from dams administered 3-amino-4-octanol either occurred at low frequencies and/or lacked a dose-response relationship.
- External examination - There were no treatment-related external malformations or variations in any treatment group. Incidental findings bearing no relationship to treatment included subdermal hematoma and slight forelimb flexure (variations), and forelimb flexure (malformation). These observations occurred at low frequencies and lacked a dose response relationship.
- Craniofacial examination - There were no treatment-related external malformations or variations in any treatment group. Incidental findings bearing no relationship to treatment included dilated perinasal area (variation) and hydrocephaly (malformation).
- Visceral examination - There were no treatment-related visceral malformations or variations in any treatment group. Incidental findings bearing no relationship to treatment included enlarged atrium, double carotid artery, missing caudal lung lobe, discolored liver, torsion strangulation of the liver, bifurcated renal vein, and convoluted ureter in individual fetuses. These observations occurred at low frequencies and/or lacked a dose-response relationship. In addition, there was a single fetus in the high-dose group with ventricular septal defect, ventricular double outlet, and situs inversus. Although this fetus was in the high-dose group, the findings were deemed spurious and unrelated to treatment as they were isolated to a single fetus from a single dam.
- Skeletal examination - There were no treatment-related external malformations or variations in any treatment group. Incidental findings bearing no relationship to treatment included delayed ossification (DO) frontal, DO interparietal, DO parietal, DO occipital, DO sternebrae, extra site of ossification sternebrae, DO thoracic centra, class I wavy ribs, class II wavy ribs, calloused ribs, and extra first lumbar rib. These observations occurred at low frequencies, lacked a dose-response relationship, and/or were within the range of recent historical control values.
Abnormalities:
not specified
Developmental effects observed:
not specified

None

Conclusions:
Under the conditions of this study, the no-observed-effect level (NOEL) for maternal toxicity was 15 mg/kg/day, and the developmental toxicity NOEL was 150 mg/kg/day, the highest dose tested.
Executive summary:

The purpose of this study was to evaluate the maternal and developmental toxicity of 3 -amino-4-octanol in Crl:CD(SD) rats following repeated gavage administration. Groups of 24 time-mated female rats were administered 3-amino-4-octanol in propylene glycol

by gavage at dose levels of 0, 15, 60, or 150 mg/kg/day on gestation day (GD) 6 through 20. In-life maternal study parameters included clinical observations, body weight, body weight gain, and feed consumption. On GD 21, all rats were euthanized and examined

for gross pathologic alterations. Liver, kidneys, and gravid uterine weights were recorded, along with the number of corpora lutea, uterine implantations, resorptions and live/dead fetuses. All fetuses were weighed, sexed and examined for external alterations. Approximately one half of the fetuses were examined for visceral and craniofacial alterations, while skeletal examinations were conducted on the remaining fetuses.

Treatment-related effects were limited to decreases in body weight gain and feed consumption in dams given 60 and 150 mg/kg/day. Maternal body weight gain in these groups was decreased by 33.6 and 50.4%, respectively, from GD 6-9. Feed consumption was decreased in dams given 60 and 150 mg/kg/day from GD 9-12 and 6-12, respectively. Body weight gain and feed consumption were similar to controls for the remainder of the study.

Gavage administration of 3-amino-4-octanol at dose levels up to, and including, 150 mg/kg/day produced no treatment-related developmental toxicity.

Therefore, under the conditions of this study, the no-observed-effect level (NOEL) for maternal toxicity was 15 mg/kg/day, and the developmental toxicity NOEL was 150 mg/kg/day, the highest dose tested.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
good
Additional information

Two studies are available for this substance. A standard OECD Guideline 414 Developmental toxicity study and an OECD 421 reproductive/developmental screening study.

The OECD 421 study did not find any gross abnormalities in pups at any dose level (highest dose was 150 mg/kg bw), nor was there an increase in post implantation loss compared to control in any dose group. Therefore this substance does not appear to be associated with developmental toxicity under the conditions of this test system.

In the OECD 414 treatment related effects were limited to decreases in bodyweight gain (GD 6 -9) in dams dosed with the mid and high doses, (60 and 150 mg/kg bw) and decreases in food consumption between GD 9 -12 and 6 -12 in dams dosed with the mid and high doses respectively. These parameters weere not affected in the control and low dose group (15 mg/lg bw/day) or at other periods in during the study. Based on these findings the Maternal NOEL is 15 mg/kg bw/day. There were no developmental toxicity findings at any of the dose levels in this study. Therefore the Developmental toxicity NOEL is the highest dose, 150 mg/kg bw/day.

It was not possible to test at doses higher than 150 mg/kg bw/day due to the corrosivity of the test material.

Based on the available data there is no evidence that this substance is a developmental toxicant.

Regarding a second species developmental toxicity study:

According to column 2 of Annex IX of the REACH legal text, a decision whether to perform a second developmental toxicity study using a second species at this tonnage band or the next must be made based on the outcome of the first study and all other available information. Unfortunately, the guidance on how to make this decision is unclear and there is little clarity within the legal text.

Comparing the available data with the CLP criteria for developmental toxicity leads to the conclusion that this substance does not require classification for developmental toxicity. Within the CLP text there is no requirement that developmental toxicity data be generated in 2 species in order to assess developmental toxicity therefore it is concluded that there are adequate information available to determine the classification for this endpoint.

In addition, this substance is corrosive, readily biodegradable and used in applications where oral, dermal or inhalation exposure are low. Taking into consideration the risk management measures implemented to minimise potential for exposure and the supported uses it is considered unlikely that a pregnant women would be exposed either directly or indirectly to this substance at a toxicologically relevant level.

As such it is proposed to delay the conduct of a developmental toxicity study in a second species until the next tonnage band is reached.


Justification for selection of Effect on developmental toxicity: via oral route:
OECD guideline, Reliability 1 study

Justification for classification or non-classification

In the OECD 421, 414, 28 day and 90 -day study, there were no indications of an effect on developmental or reproductive toxicity at the highest dose tested (150 mg/kg bw). Therefore the no effect level for developmental and reproductive toxicity is considered to be 150 mg/kg bw/day. No classification for developmental or reproductive toxicity is required.