Registration Dossier

Administrative data

Description of key information

• Repeat dose oral (OECD 422 and OECD 408): No toxicologically significant effects were observed during the course of these studies at doses up to 1000 mg/kg/day. The NOAEL for subchronic systemic toxicity is considered to be 1000 mg/kg bw/day.

• Repeat dose dermal: No studies are available

• Repeat dose inhalation: No studies are available

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: 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 study design; GLP compliant; no deficiencies
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
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 oral 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)
Remarks:
control
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.
Positive control:
no
Observations and 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).

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).

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.
Sacrifice and 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.
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
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
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
Histopathological findings: neoplastic:
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 (nominal)
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:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 23 November 2011 and 15 May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to GLP and guideline
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Oxon, UK
- Age at study initiation: approximately 12 weeks
- Weight at study initiation: 308-335 g (male); 193-219 g (female)
- Fasting period before study: no
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to
their original cages. Mated females were housed individually during gestation and lactation, in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet: free access to a pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories UK Ltd, Oxon, UK)
- Water: free access to mains drinking water supplied from polycarbonate bottles attached to the cage.
- Acclimation period: 7 days during which health status of each animal was assessed

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 2 ºC
- Humidity: 55 ± 15 %
- Air changes: at least 15 per hour
- Photoperiod: 12 hours continuous light and 12 hours dark via low intensity fluorescent lighting


IN-LIFE DATES: From: 29 December 2011 (first day of treatment) To: 11 February 2012 (final necropsy)
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF TEST ITEM
- Test material was prepared at the appropriate concentrations as a solution in arachis oil BP.
- The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least twenty-one days. Formulations were therefore prepared twice monthly and stored at +4ºC in the dark.


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of 9-decenoic acid, methyl ester (9DAME) in the test item formulations was determined by gas chromatography (GC) using an external standard technique.

The standard and sample solutions were analysed by GC using the following conditions:
GC system : Agilent Technologies 5890, incorporating autosampler and workstation
Column : ZB-5 (30 m x 0.53 mm id x 5 μm film)
Oven temperature program : initial 150 ºC for 1 mins
rate 10 ºC/min
final 340 ºC for 6 mins
Injection temperature : 300 ºC
Flame ionisation detector temperature: 300 ºC
Injection volume : 1 μl
Retention time : ~ 5.4 mins
Duration of treatment / exposure:
Up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females).
Frequency of treatment:
Test item was administered daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle control

Dose / conc.:
30 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
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen based on the results of previous work (Harlan Laboratories Ltd., Project Number 41103600).
Positive control:
No.
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS:
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends and public holidays (except for females during parturition where applicable). All observations were recorded.


FUNCTIONAL OBSERVATIONS:
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

BEHAVIOURAL ASSESSMENTS:
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed: Gait, Tremors, Twitches, Convulsions, Bizarre/Abnormal/Stereotypic behaviour, Salivation, Pilo-erection, Exophthalmia, Lachrymation, Hyper/Hypothermia, Skin colour, Respiration, Palpebral closure, Urination, Defecation, Transfer arousal, Tail elevation.

MOTOR ACTIVITY:
Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The Tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail, 1979).

FORELIMB/HINDLIMB GRIP STRENGTH
An automated meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).


SENSORY REACTIVITY:
Each tested animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988). The following parameters were observed: Grasp response, Vocalisation, Toe pinch, Tail pinch, Finger approach, Touch escape, Pupil reflex, Blink reflex, Startle reflex.

BODY WEIGHT:
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and

FOOD CONSUMPTION
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum. Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

FOOD EFFICIENCY (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated for females during gestation and lactation.

WATER CONSUMPTION
Water intake was measured daily throughout the pre-pairing phase of the study

LABORATORY INVESTIGATIONS
Haematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 post partum for females). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.

HAEMATOLOGY
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant: Haemoglobin (Hb), Erythrocyte count (RBC), Haematocrit (Hct), Erythrocyte indices, Total leucocyte count (WBC), Differential leucocyte count, Platelet count (PLT), Reticulocyte count (Retic), Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/l).

BLOOD CHEMISTRY
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant: Urea, Glucose, Total protein (Tot.Prot.), Albumin, Albumin/Globulin (A/G) ratio (by calculation), Sodium (Na+), Potassium (K+), Calcium (Ca++), Inorganic phosphorus (P), Aspartate aminotransferase (ASAT), Alanine aminotransferase (ALAT), Alkaline phosphatase (AP), Creatinine (Creat), Total cholesterol (Chol), Chloride (Cl-), Triglycerides, Total bilirubin (Bili), Bile acids.

PATHOLOGY
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

ORGAN WEIGHTS
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Prostate, Seminal vesicles, Spleen, Testes, Thymus, Thyroid (weighed post-fixation with Parathyroid), Uterus (weighed with Cervix).





















Sacrifice and pathology:
Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum.

HISTOPATHOLOGY:
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated:
Adrenals, Ovaries, Aorta, (thoracic) Pancreas, Bone & bone marrow (femur including stifle joint), Pituitary Bone & bone marrow (sternum), Prostate, Brain, (including cerebrum, cerebellum and pons), Oesophagus, Caecum, Rectum, Coagulating gland, Salivary glands (submaxillary), Colon, Sciatic nerve, Duodenum, Seminal vesicles, Epididymides♦, Skin (hind limb), Eyes* Spinal cord (cervical, mid-thoracic and lumbar), Gross lesions, Heart, Spleen,
Ileum (including peyer’s patches), Stomach, Jejunum, Thyroid/parathyroid, Kidneys, Trachea, Liver, Testes♦, Lungs (with bronchi)#, Thymus, Lymph nodes (cervical and mesenteric), Urinary bladder, Mammary gland, Uterus/Cervix, Muscle (skeletal) and Vagina

* =E yes fixed in Davidsons fluid
♦ = preserved in Bouin’s fluid then transferred to 70% Industrial Methylated Spirits (IMS) approximately forty-eight hours later
# = = lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative

All tissues were despatched to the Test Site (Propath UK Ltd., Willow Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU) for processing (Principal Investigator: N Fower). The tissues from five selected control and 1000 mg/kg bw/day dose group animals, any animals dying during the study, and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination.




Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters: Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Haematology, Blood Chemistry, Absolute Organ Weights, Body Weight- Relative Organ Weights

Data were analysed using the decision tree from the ProvantisTM Tables and Statistics Module

Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analysed using Bartlett’s test. Intergroup variance were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data shows non-homogeneity of means, the data were analysed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was
performed using the Student t-test (parametric) or the Mann-Whitney U test (nonparametric).

Data not analysed by the Provantis data capture system were assessed separately using the SPSS statistical package.
Probability values (p) were calculated as follows:
p<0.001 ***
p<0.01 **
p<0.05 *
p0.05 (not significant)




















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
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
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
Histopathological findings: neoplastic:
no effects observed
Details on results:
MORTALITY:
There were no treatment related deaths.
One female treated with 300 mg/kg bw/day was killed in extremis on Day 41 due to difficulties encountered during parturition. Macroscopic examination revealed a total of 13 dead foetuses in the uterus and histopathological evaluations revealed incidental lesions which were not also present in females treated with 1000 mg/kg bw/day. Therefore in isolation this interim death was considered not to be related to test item toxicity.

CLINICAL OBSERVATIONS:
There were no toxicologically significant clinical signs detected in treated animals.
Clinical signs were confined to transient instances of increased salivation detected in animals of either sex treated with 1000 mg/kg bw/day.

A swollen ano-genital region, pilo-erection and pallor of the extremities was recorded for the female that was killed in extremis on Day 41 following the start of parturition.

BEHAVIOURAL ASSESSMENTS:
Weekly open field arena observations did not reveal any treatment-related effects for treated animals when compared to controls All inter and intra group differences in behavioural scores were considered to be a result of normal variation for rats of the strain and age used, and the differences were of no toxicological importance.

FUNCTIONAL PERFORMANCE TESTS:
There were no treatment related changes in functional performance. Statistical analysis of the data did not reveal any significant intergroup differences.

SENSORY REACTIVITY ASSESSMENTS:
There were no treatment-related changes in sensory reactivity.

BODY WEIGHT:
There were no toxicologically significant effects detected in body weight development. Males treated with 1000 and 300 mg/kg bw/day showed a statistically significant reduction in body weight gain during the third week of treatment whilst females from these treatment groups showed a statistically significant reduction in cumulative body weight gain between Days 1 and 14 of gestation. Recovery was evident thereafter (identified as a statistically significant increase in males at 1000 mg/kg bw/day during Week 4) and in the absence of any significant effect on overall body weight development, the intergroup differences were considered not to be of toxicological significance.

FOOD CONSUMPTION:
There were no treatment related effects detected on mean food consumption or mean food efficiency during this study, when compared to the control. Females treated with 300 mg/kg bw/day showed a statistically significant reduction in food consumption during Days 0 - 7 of gestation. In the absence of a similar effect in 1000 mg/kg bw/day females the intergroup difference was considered not to be of toxicological importance.

WATER CONSUMPTION:
There were no adverse effects detected in water consumption for treated animals, when compared to control animals.

HAEMATOLOGY
There were no toxicologically significant effects detected in the haematological parameters examined. Males treated with 1000 and 300 mg/kg bw/day showed a statistically significant increase in erythrocyte count. In the absence of a true dose related response the intergroup difference was considered not to be of toxicological importance.

BLOOD CHEMISTRY:
There were no toxicologically significant effects detected in the blood chemical parameters examined. Females treated with 1000 mg/kg bw/day showed a statistically significant reduction in cholesterol. The majority of individual values were within the normal range for rats of the strain and age used, and in the absence of any histology correlates the intergroup difference was considered not to be of toxicological importance.

PATHOLOGY:
The female that was killed in extremis due to parturition difficulties had nine dead foetuses in the left horn and four dead foetuses in the right horn
No toxicologically significant macroscopic abnormalities were detected in terminal kill animals.
One male treated with 30 mg/kg bw/day had increased pelvic space in the right kidney. A further male from this treatment group also showed increased pelvic space in both kidneys and the right kidney was fluid filled. One female treated with 30 mg/kg bw/day had reddened lungs at necropsy. In the absence of similar effects seen at 1000 mg/kg bw/day animals the intergroup differences were considered not to be of toxicological significance. One control male had enlarged kidneys, increased renal pelvic space in both kidneys and fluid present in the left kidney. In the absence of treatment this was considered to be incidental.

ORGAN WEIGHTS:
There were no toxicologically significant effects detected in the organ weights measured.
Males from all treatment groups showed a non-dose related statistical significant reduction in thyroid weight both absolute and relative to terminal body weight. In the absence of a true dose related response or any histology correlates the intergroup difference was considered not to be of toxicological importance.

HISTOPATHOLOGY
No treatment related microscopic findings were detected.


























Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The oral administration of the test substance at dose levels of 30, 300 and 1000 mg/kg mg/day did not result in any toxicologically significant effects.
Critical effects observed:
no
Conclusions:
The oral administration of 9-decenoic acid, methyl ester (9DAME) to rats by gavage, at dose levels of 30, 300 and 1000 mg/kg bw/day did not result in any toxicological significant effects. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 1000 mg/kg bw/day.




Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP compliant studies have been conducted according to OECD Guideline 422 and OECD Guideline 408 and are adequately reported. As such the studies are assigned reliability 1.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeat dose oral toxicity:

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. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, behavioural assessments, body weight change, food and water consumption were monitored during the study. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

The oral administration of 9-decenoic acid, methyl ester (9DAME) to rats by gavage did not result in any toxicologically significant effects. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore 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. The subchronic ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity was therefore considered to be 1000 mg/kg bw/day.

 

No studies are currently available that have evaluated the long-term repeated dose oral toxicity of 9-decenoic acid, methyl ester (9DAME). However, based on the lack of systemic toxicity observed in all of the toxicity studies that have been performed, further oral studies are considered scientifically unjustified.

 

Justification for classification or non-classification

No systemic toxicological findings 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 a period of 28 days or 90 days. Therefore, classification of 9-decenoic acid, methyl ester as STOT RE is not justified according to the criteria described in Regulation (EC) No 1272/2008.