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Neurotoxicity

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Description of key information

The subchronic NOAEL for systemic toxicity, including neurotoxicity, is considered to be 1000 mg/kg bw/day.

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records
Reference
Endpoint:
neurotoxicity: sub-chronic oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline subchronic toxicity study; GLP compliant; no deficiencies
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: approximately 37 days old at receipt
- Housing: animals were housed 2 to 3 per cage by sex in clean, solid bottom cages containing ground corncob bedding material (Bed O’Cobs®)
- Diet (e.g. ad libitum): PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 (meal) ad libitum
- Water (e.g. ad libitum): Reverse osmosis treated (on site) drinking water ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 69.1°F to 72.0°F (20.6°C to 22.2°C)
- Relative Humidity (%): 35.2% to 64.7%
- Air changes (per hr): a minimum of 10 fresh air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour light (0600 hours to 1800 hours)/12 hour dark photoperiod
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored refrigerated (2°C to 8°C), protected from light, under nitrogen. The test item formulations were stirred continuously throughout the preparation, sampling, and for at least 30 minutes prior to the start of the dose administration procedures.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses to determine stability and homogeneity of dosing formulations were conducted prior to initiation of the 90-day treatment period. Samples for concentration analysis were collected during study weeks 0, 3, 7, and 12 from the middle stratum of each dosing formulation (including the control group). All analyses were conducted by the WIL Research Analytical Chemistry Department using a validated gas chromatography with flame ionization detection method.
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 males and 10 females per group
Recovery animals: 5 males and 5 females in the control and high-dose groups
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The high dosage level was the limit dose based on the absence of findings reported by the Sponsor in earlier studies of the test item (14- and 28-day gavage) at 1000 mg/kg/day). The low dosage level was included to help assure a no observed adverse effect level.
Observations and clinical examinations performed and frequency:
SURVIVAL
All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity.

CLINICAL OBSERVATIONS
Clinical examinations were performed at the time of dose administration and 1-2 hours following dose administration. During the recovery period, the animals were observed once daily. Detailed physical examinations were conducted on all animals 1 week (± 2 days) prior to randomization, on the day of randomization, weekly (± 2 days) during the study period, and on the days of the scheduled necropsies.

BODY WEIGHTS
Individual body weights were recorded 1 week (± 2 days) prior to randomization, on the day of randomization, study day 0 (prior to dosing), weekly (± 2 days) during the study period, and on the day prior to the first day of the scheduled necropsies (nonfasted).

FOOD CONSUMPTION
Cage food weights were recorded weekly (± 2 days) beginning following randomization and throughout the study period. Food consumption was normalized to the number of animals/cage and calculated as g/animal/day. The last food weights were collected on the day prior to the final day of study week 12 (primary necropsy) or 16 (recovery necropsy).

CLINICAL PATHOLOGY
Blood and urine samples for clinical pathology evaluations (hematology, coagulation, serum chemistry, and urinalysis) were collected from all animals assigned to the scheduled necropsies (study weeks 12/13 and 16/17; designated as study weeks 12 and 16, respectively, for report presentation purposes). The animals were fasted overnight prior to blood collection while in metabolism cages for urine collection. Blood was collected for hematology and serum chemistry evaluation via the retro-orbital sinus of animals anesthetized by inhalation of isoflurane. Blood was collected for coagulation parameters at the time of euthanasia via the vena cava of animals euthanized by inhalation of carbon dioxide. The following parameters were evaluated:

HEMATOLOGY AND COAGULATION - Total leukocyte count, Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Platelet count, Prothrombin time, Activated partial thromboplastin time, Reticulocyte count (Percent, Absolute), Mean platelet volume, Differential leukocyte count, Red cell distribution width, Hemoglobin distribution width, Platelet estimate, Red cell morphology.

SERUM CHEMISTRY – Albumin, Total protein, Globulin [by calculation], Albumin/globulin ratio (A/G Ratio) [by calculation], Total bilirubin, Urea nitrogen, Creatinine, Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma glutamyltransferase, Glucose, Total cholesterol, Calcium, Chloride, Phosphorus, Potassium, Sodium, Sorbitol dehydrogenase, Triglycerides, Appearance.

URINALYSIS - Specific gravity, pH, Urobilinogen, Total volume, Color, Clarity, Protein, Glucose, Ketones, Bilirubin, Occult blood, Leukocytes, Nitrites, Microscopy of sediment.

OPHTHALMIC EXAMINATIONS
Ocular examinations were conducted on all animals during acclimation (29-Jan-2015; study week -1) and near the end of the dosing period (30-Apr-2015; study week 12). All ocular examinations were conducted using an indirect ophthalmoscope and slit lamp biomicroscope preceded by pupillary dilation with an appropriate mydriatic agent.
Neurobehavioural examinations performed and frequency:
FUNCTIONAL OBSERVATIONAL BATTERY ASSESSMENTS
Functional observational battery (FOB) assessments were recorded for all animals during study week 11/12 (designated as study week 12 for report presentation purposes). Assessments were conducted prior to dose administration. The FOB utilized at WIL Research is based on previously developed protocols (Gad, 1982; Haggerty, 1989; Irwin, 1968; Moser et al., 1988; Moser et al., 1991; and O’Donoghue, 1989). Testing was performed by the same biologists, whenever possible, without knowledge of the animal’s group assignment. The FOB was performed in a sound attenuated room equipped with a white noise generator set to operate at 70 ± 10 dB. All animals were observed for the following parameters: HOME CAGE OBSERVATIONS – Posture, Convulsions/tremors, Feces consistency, Biting, Palpebral (eyelid) fissure; HANDLING OBSERVATIONS - Ease of removal from cage, Lacrimation/chromodacryorrhea, Piloerection, Palpebral closure, Eye prominence, Red/crusty deposits, Ease of handling animal in hand, Salivation, Fur appearance, Respiratory rate/character, Mucous membranes/eye/skin color. Muscle tone; OPEN FIELD OBSERVATIONS – Mobility, Rearing, Convulsions/tremors, Grooming, Bizarre/stereotypic behavior, Time to first step (seconds), Gait, Arousal, Urination/defecation, Gait score, Backing (Note: Open field observations were evaluated over a 2-minute observation period); SENSORY OBSERVATIONS - Approach response, Startle response, Pupil response, Forelimb extension, Air righting reflex, Touch response, Tail pinch response, Eyeblink response, Hindlimb extension, Olfactory orientation; NEUROMUSCULAR OBSERVATIONS - Hindlimb extensor strength, Hindlimb foot splay, Grip strength hind and forelimb, Rotarod performance; PHYSIOLOGICAL OBSERVATIONS – Catalepsy, Body temperature, Body weight.

MOTOR ACTIVITY
Motor activity was assessed for all animals during study week 11/12. The motor activity assessment was conducted prior to dose administration. Motor activity, recorded after the completion of the FOB, assessment was conducted using a personal computer controlled system that utilizes a series of infrared photobeams surrounding an amber, plastic rectangular cage to quantify each animal’s motor activity. Four-sided black plastic enclosures were used to surround the transparent plastic boxes and decrease the potential for distraction from extraneous environmental stimuli or activity by biologists or adjacent animals. The motor activity assessment was performed in a sound attenuated room equipped with a white noise generator set to operate at 70 ± 10 dB. Each animal was tested separately. Data were collected in 5 minute epochs (print intervals) and the test session duration was 60 minutes. These data were compiled as six, 10-minute subintervals for tabulation. Data for ambulatory and total motor activity were tabulated. Total motor activity was defined as a combination of fine motor skills (i.e., grooming, interruption of 1 photobeam) and ambulatory motor activity (interruption of 2 or more consecutive photobeams).
Sacrifice and (histo)pathology:
A complete necropsy was conducted on all animals.

ORGAN WEIGHTS
The following organs were weighed from all animals at the scheduled necropsies: Adrenals. Brain, Epididymides, Heart, Kidneys, Liver, Ovaries with oviducts, Pituitary, Prostate with seminal vesicles, Spleen, Testes, Thymus, Thyroid with parathyroids, Uterus.

MICROSCOPIC EXAMINATION
Microscopic examination was performed on tissues from all animals in the control and 1000 mg/kg/day groups at the primary necropsy. In addition, gross lesions were prepared from all animals in the 100 and 300 mg/kg/day groups at the primary necropsy and from all animals at the recovery necropsy.
Positive control:
no
Statistics:
Body weight, body weight change, food consumption, continuous FOB, clinical pathology, and organ weight data were subjected to a parametric one way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test item treated groups to the control group. Functional observational battery parameters that yielded scalar or descriptive data were analyzed using Fisher’s Exact Test (Steel and Torrie, 1980).

All repeated measures analysis of variance (RANOVA) statistical analyses for total and ambulatory motor activity counts recorded during study week 11/12 were conducted by BioSTAT Consultants, Inc., Portage, MI, using SAS® version 9.2 software (SAS Institute, Inc., 2002-2008). Each analysis endpoint was analyzed, by sex and session, with a RANOVA.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Ophthalmological findings:
no effects observed
Clinical biochemistry findings:
not examined
Behaviour (functional findings):
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Details on results:
All animals survived to the scheduled necropsies. There were no test item-related effects on body weights, food consumption, functional observational battery parameters, motor activity, or organ weights. Hematology, coagulation, serum chemistry, and urinalysis parameters were unaffected by test item administration. There were no test item-related ophthalmic, macroscopic, or microscopic findings.
Test item-related, nonadverse findings were noted at 1-2 hours post-dosing during the 90 day treatment period and included clear material around the mouth and/or ventral neck in the 300 and 1000 mg/kg/day group males and clear material around the mouth in the 1000 mg/kg/day group females. These findings were not noted during the recovery period.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: oral administration of test substance for a minimum of 90 days was well tolerated at dosage levels of 100, 300, and 1000 mg/kg/day.
Critical effects observed:
no
Conclusions:
Based on the results of this study, oral administration of 9-Decenoic Acid, Methyl Ester (9DAME) to Sprague Dawley rats for a minimum of 90 days was well tolerated at dosage levels of 100, 300, and 1000 mg/kg/day. Treatment-related findings were limited to nonadverse clinical observations in the 300 and 1000 mg/kg/day group males and 1000 mg/kg/day group females. Therefore, the no-observed-adverse-effect level (NOAEL) was considered to be 1000 mg/kg/day.
Executive summary:

9-Decenoic Acid, Methyl Ester (9DAME), in the vehicle (corn oil) was administered orally by gavage once daily for a minimum of 90 consecutive days to Crl:CD(SD) rats. Dosage levels were 100, 300, and 1000 mg/kg/day. A concurrent control received the vehicle (corn oil). The dose volume was 4 mL/kg for all groups. Following a minimum of 90 days of dose administration, 10 animals/sex/group were euthanized; the remaining 5 animals/sex in the control and high-dose groups were euthanized following a minimum 28 day nondosing (recovery) period.

 

All animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily, and detailed physical examinations were performed weekly (± 2 days). Individual body weights were recorded weekly (± 2 days). Cage food weights were recorded weekly (± 2 days) beginning following randomization. Functional observational battery (FOB) and motor activity data were recorded for all animals during study week 11/12. Ophthalmic examinations were performed during study weeks -1 and 12. Clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis) were analyzed for all animals assigned to the primary (study week 12/13) and recovery (study week 16/17) necropsies. Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsies. Selected tissues were examined microscopically from all animals in the control and high-dose groups at the primary necropsy. Gross lesions were examined microscopically from all animals.

 

All animals survived to the scheduled necropsies. There were no test item-related effects on body weights, food consumption, functional observational battery parameters, motor activity, or organ weights. Hematology, coagulation, serum chemistry, and urinalysis parameters were unaffected by test item administration. There were no test item-related ophthalmic, macroscopic, or microscopic findings.

 

Test item-related, nonadverse findings were noted at 1-2 hours post-dosing during the 90 day treatment period and included clear material around the mouth and/or ventral neck in the 300 and 1000 mg/kg/day group males and clear material around the mouth in the 1000 mg/kg/day group females. These findings were not noted during the recovery period.

 

Based on the results of this study, oral administration of 9-Decenoic Acid, Methyl Ester (9DAME) to Sprague Dawley rats for a minimum of 90 days was well tolerated at dosage levels of 100, 300, and 1000 mg/kg/day. Treatment-related findings were limited to nonadverse clinical observations in the 300 and 1000 mg/kg/day group males and 1000 mg/kg/day group females. Therefore, the no-observed-adverse-effect level (NOAEL) was considered to be 1000 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Additional information

A 28 day repeat dose oral toxicity study combined with a reproduction/ developmental toxicity screening test was performed in the rat in accordance with GLP and OECD Guideline 422 (Harlan Laboratories Ltd, 2012). The test item, 9-decenoic acid, methyl ester was administered by gavage to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 30, 300 and 1000 mg/kg bw/day. There were no treatment related effects observed on behavioural assessment, functional performance tests or sensory reactivity assessments. Since no treatment-related effects were observed, a NOAEL for systemic toxicity was considered to be 1000 mg/kg bw/day.

A 90 day repeat dose oral toxicity study was performed in the rat in accordance with GLP and OECD Guideline 408 (WIL Research, 2015). 9-Decenoic Acid, Methyl Ester (9DAME), was administered orally by gavage once daily for a minimum of 90 consecutive days to Crl:CD(SD) rats. Dosage levels were 100, 300, and 1000 mg/kg/day. Mortality, clinical signs, body weight, food consumption, neurobehavioral parameters, ophthalmology, and clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis) were monitored during the study. Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsies. Selected tissues were examined microscopically. The subchronic oral administration of 9-decenoic acid, methyl ester (9DAME) to rats by gavage did not result in any toxicologically significant effects. There were no treatment related effects observed on functional observational battery parameters or on motor activity. ‘The subchronic NOAEL for systemic toxicity, including neurotoxicity, is therefore considered to be 1000 mg/kg bw/day.

Justification for classification or non-classification

No systemic toxicological findings, including no evidence of neurotoxicity, were detected in rats after repeated administration of 9-decenoic acid, methyl ester (9DAME) by the oral route at dose levels up to 1000 mg/kg bw/day for periods of 28 or 90 days. Based on these results, 9-decenoic acid, methyl ester (9-DAME) does not warrant classification for neurotoxicity according to the criteria described in Regulation (EC) No 1272/2008.