Registration Dossier

Administrative data

Description of key information

In conclusion, there was no evidence of toxicity after oral gavage administration of 1000 mg/kg/day of Coconut Oil, Reaction Products with Polyethylene Glycol and Trimethylolpropane for at least 90 consecutive days to Han Wistar rats. Based on these results, the no-observed-effect level (NOEL) was considered to be at least 1000 mg/kg/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 2013 to January 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
Histology and histopathology were conducted in accordance with the United States Code Code of Federal Regulation, 21, Part 58: Good Laboratory Practice Standards for Non-Clinical Laboratory Studies
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Han-Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Ltd., Margate, Kent
- Age at study initiation: approx. 7-8 weeks
- Weight at study initiation: 228 - 233 g (males); 157 - 163 g (females)
- Fasting period before study: no
- Housing: in groups of up to 3 per cage in appropriately sized suspended polypropylene cages with stainless steel grid tops and solid bottom, sterilized white wood shaving were used as bedding
- Diet: Rat and Mouse (modified) No. 1 Expanded SQC diet (supplied by Special Diets services limited, Essex, UK) ad libitum
- Water: water from the public supply from water bottles ad libitum
- Acclimation period: approx. 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 30-69 %
- Air changes (per hr): minimum of 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2013-04-03 To: 2013-07-03
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item dosing formulations were prepared weekly based on a method established at the Test Facility (Analytical Procedure No. 2971; Formulation process document 998849-13-014).
The appropriate amount of test item was weighed and added to the appropriate amount of control item, corn oil, to produce the required concentration. The formulation was stirred magnetically until visibly homogeneous, dispensed into daily aliquots which were stored in a refrigerator set to maintain 4°C until use.
control item: dose level: 0 mg/kg/day, dose volume: 2.5 mL/kg vehicle
dose level: 100 mg/kg/d; dose concentration: 40 mg/mL; dose volume: 2.5 mL/kg
dose level. 500 mg/kg/d; dose concentration: 200 mg/mL; dose volume: 2.5 mL/kg
dose level: 1000 mg/kg/d; dose concentration: 400 mg/mL; dose volume: 2.5 mL/kg

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed by gas chromatography using an analytical procedure validated at the Test Facility (Study No. 429713, Report No. 33880, Analytical Procedure 2971).


Stability analysis was not performed during this study. The dosing formulations prepared within the Charles River Dispensary at ca 0.500 mg/mL and ca 400 mg/mL Coconut Oil, Reaction Products with Polyethylene Glycol and Trimethylolpropane were found to be stable for at least 8 days when stored at 2-8°C in the dark.

Dose formulation samples for analysis of concentration and homogeneity were collected in weeks 1, 6, 8 and 12. Duplicate sets of top, middle and bottom samples (1 mL) for each sampling point were provided to the analytical laboratory at the Test Facility. Triplicate top, middle and bottom samples (1 mL) were retained at the Test Facility as backup samples in a refrigerator set to maintain 4°C. Concentration results were considered acceptable if sample concentration results were within or equal to 10% of theoretical concentration. Homogeneity results were considered acceptable if the relative standard deviation (RSD) of the mean value at each sampling location was ≤ 5%. During Week 12 the homogeneity of the formulations prepared at 200 mg/mL (Group 3) had a coefficient of variation of 6.8%. The formulations were stirred during dosing and as the mean found concentration (201 mg/ml) was within the acceptance criteria it was considered that the animals received their appropriate dosage.
Duration of treatment / exposure:
90 days
Frequency of treatment:
once daily
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10

Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels were selected on the basis of the findings of a 14 day oral (gavage) toxicity study in rats (Charles River No. 523891).
Results of the 14 day oral (gavage) toxicity study in rats:
The objective of this study was to determine the potential toxicity of Coconut Oil, Reaction Products with Polyethylene Glycol and Trimethylolpropane when given by oral gavage for 14 days to Han Wistar rats. These data provided information to allow the selection of suitable dosages for further repeat dose studies.
The study design was as follows: 5 animals per Sex and group, dose levels: 0, 100, 500 and 1000 mg/kg bw/day. Coconut Oil, Reaction Products with Polyethylene Glycol and Trimethylolpropane was administered using corn oil as the vehicle and a constant dose volume of 2.5 mL/kg. The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight changes, food consumption, and on Day 15, gross necropsy findings and organ weights. There were no unscheduled deaths or adverse clinical signs that were considered to be related to treatment. Body weights, food consumption and organ weights were considered to be unaffected by treatment. In conclusion, there was no evidence of toxicity after 14 days of oral gavage administration of 1000 mg/kg/day of Coconut Oil, Reaction Products with Polyethylene Glycol and Trimethylolpropane to Han Wistar rats. Based on these results, 1000 mg/kg/day was considered to be a suitable high dose level for further repeat dose studies in this species using this test item.
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Mortality / Moribundity check: twice daily (in the morning and as late as practical each day)
Additional clinical observations: examination for reaction to treatment particular during and for the first hour after dosing.
On first approach to the cage, the technician checked the posture/condition of the animal for signs of prostration, lethargy, writhing, circling, breathing abnormalities, gait abnormalities, tremors, fasciculation, convulsions, biting (of cage components or self-mutilation), vocalisations and piloerection.
Body temperature was recorded from the electronically implanted microchip. Where the data failed to collect on the data capture system, a rectal temperature was recorded.
The technician also checked the ease at which the animal was removed from the cage, the condition of its eyes (for pupillary function, miosis, mydriasis, exophthalmos, encrustation and lacrimation), the condition of the coat, the presence of salivation and the overall ease of handling.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once each week from week-1

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded twice during pretrial and daily during dosing.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: once during pretrial and once weekly during dosing. With the exception of cage 2 holding Group 1 males control and cage 10 holding Group 3 males receiving 500 mg/kg/day, food consumed during pretrial was measured from Day -7 to Day 0. For the 2 cages detailed food consumed was measured over a 4-day period.

WATER CONSUMPTION: Water consumption was monitored regularly throughout the study by visual inspection of the water bottles. There were no inter-group differences recorded.

OPHTHALMOSCOPIC EXAMINATION: Yes
Ophthalmic examinations were conducted on all animals once during pretrial. After this examination, one female (Animal 359F) was replaced with Animal 503F due to findings noted. The controls (Group 1) and animals in Group 4 were examined once during Week 13. During this latter examination a haemorrhage was noted in the retina of one female in Group 4 (Animal 454F), consequently, the animals allocated to Groups 2 and 3 were examined. The anterior, lenticular and fundic areas of the eyes were examined using an indirect ophthalmoscope after the application of a mydriatic agent (1% Tropicamide, Mydriacyl®).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Samples were collected during Week 13, blood was collected from the orbital sinus under isoflurane anaesthesia.
- Animals fasted: No
- How many animals: all of control and high dose group
- Parameters examined: haemoglobin, red blood cell count, haematocrit, mean cell haemoglobin, mean cell volume, mean cell haemoglobin concentration, red cell distribution width, platelets, reticulocytes (%), Reticulocyte count (absolute), white blood cell count, neutrophils, lymphocytes, monocytes, eosinophils, basophils, large unstained cells, Fibrinogen, activated partial thromboplastin time, prothrombin time.
Other: A blood smear was prepared from each haematology specimen. The smears were labelled, stained, stored and archived.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Samples were collected during Week 13
- Animals fasted: No
- How many animals: all of control and high dose group
- Parameters examined: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, creatine phosphokinase, urea, glucose, total bilirubin, cholesterol, total protein, albumin, globulin, albumin globulin ratio, sodium, potassium, chloride, inorganic phosphate, calcium, creatinine.

URINALYSIS: Yes
- Time schedule for collection of urine: Samples were collected during Week 13
- Metabolism cages used for collection of urine: no data
- Animals fasted: no
- Parameters examined: colour, specific gravity, volume, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood pigments, leukocytes, microscopic evaluation of spun deposit, turbidity

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during pretrial and once during Week 12 at an approximately standardised time of day during pretrial and once during Week 12 at an approximately standardised time of day
- Dose groups that were examined: all
- Battery of functions tested: cage side observations (signs of prostration, lethargy, writhing, circling, breathing abnormalities, gait abnormalities, tremors, fasciculation, convulsions, biting (of cage components or self-mutilation), vocalisations and piloerection), Body temperature was recorded from the electronically implanted microchip, Where the data failed to collect on the data capture system, a rectal temperature was recorded,condition of its eyes (for pupillary function, miosis, mydriasis, exophthalmos, encrustation and lacrimation), the condition of the coat, the presence of salivation and the overall ease of handling,latency (time to first locomotory movement), level of mobility, rearing, grooming, urination/defecation, arousal (level of alertness), posture, tremor/convulsions, vocalisation, piloerection, palpebral closure, gait abnormalities and stereotypy and/or unusual behaviours,reactions to a sudden sound (click above the head) and the reaction to touch on the rump with a blunt probe. Tests were performed for grip strength, pain perception, landing foot splay and motor activity. Any other physical/functional abnormalities were recorded.

OTHER: none
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. All animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. A veterinary pathologist was available for consultation.
The organs (see table 1) were weighed at necropsy for all animals. Paired organs were weighed separately and are reported together. With the exception of the thyroid glands, all organs were weighed before fixation. Representative samples of the tissues identified were collected from all animals and preserved in 10% neutral buffered formalin, unless otherwise indicated.

HISTOPATHOLOGY: Yes. Tissues identified in Table 2, with the exception of testes, were processed at the Test Site. Testes samples were trimmed and processed to wax impregnation at the Test Facility. Further processing of the testes was carried out at the Test Site (Charles River Pathology Associates). All other tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with haematoxylin and eosin.
Histopathological evaluation was performed by a veterinary pathologist at the Test Site (Charles River Pathology Associates) with training and experience in laboratory animal pathology.
Other examinations:
The bone marrow smears collected from the femur were stained with May-Grunwald-Giemsa stain at the Test Facility.
Statistics:
Unless otherwise stated, all statistical tests were two-sided and performed at the 5% significance level using in-house software. Males and females have been analysed separately.
The following pairwise comparisons were performed against the control group (Group 1):
Control Group v Group 2
Control Group v Group 3
Control Group v Group 4
Body weight, food consumption, selected functional observational battery and motor activity data, haematology, coagulation, clinical chemistry and selected urinalysis data were analysed for homogeneity of variance using the ‘F-Max’ text. Where the group variances appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F protected LSD method via Student’s t test i.e. pairwise comparisons were only made if the overall F-test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilise the variances. If the variances remained heterogeneous, a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (via z tests, the non-parametric equivalent of Student’s t test).
In circumstances where it was not possible for perform the ‘F-Max’ test due to zero standard deviation in at least one group, the non-parametric ANOVA results have been reported.
Organ weights were analysed using ANOVA as above and by analysis of covariance (ANCOVA) using terminal kill body weight as covariate. In addition, organ weights as a percentage of terminal body weight were analysed using ANOVA.

In circumstances where the variances in the ANCOVA remained heterogeneous following log or square root transformations, the data were subjected to a rank transformation prior to analysis.
In the ANOVA and ANCOVA summary tables, the results of the analysis were reported indicating the level of statistical significance (p<0.05, p<0.01 and p<0.001) of each pairwise comparison.

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:
not specified
Water consumption and compound intake (if drinking water study):
not examined
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
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths during the observation period.

There were no adverse signs noted during the observation period that were considered to be related to treatment with Coconut Oil, Reaction Products with Polyethylene Glycol and Trimethylolpropane.

Signs noted such as fur staining or scabbing on the neck area were considered to represent those commonly seen in this species at these laboratories. They were also noted in the controls. Therefore, they were considered to be unrelated to treatment.


BODY WEIGHT AND WEIGHT GAIN
No test item related changes in body weight occurred during the study.

FOOD CONSUMPTION
Food consumption was unaffected by treatment with Coconut Oil, Reaction Products with Polyethylene Glycol and Trimethylolpropane.

OPHTHALMOSCOPIC EXAMINATION
There were no changes in the eye that were considered to be related to treatment with Coconut Oil, Reaction Products with Polyethylene Glycol and Trimethylolpropane.

HAEMATOLOGY
Haematology and coagulation was unaffected by treatment with Coconut Oil, Reaction Products with Polyethylene Glycol and Trimethylolpropane.

CLINICAL CHEMISTRY
Plasma chemistry was unaffected by treatment with Coconut Oil, Reaction Products with Polyethylene Glycol and Trimethylolpropane.
There were statistically significant differences but none of these were considered to be related to treatment. Chloride was higher in males receiving 100 or 500 mg/kg/day of Coconut Oil, Reaction Products with Polyethylene Glycol and Trimethylolpropane, when compared with controls (p<0.05). There was higher globulin and a lower albumin:globulin ratio in females receiving 100 mg/kg/day of Coconut Oil, Reaction Products with Polyethylene Glycol and Trimethylolpropane, when compared with controls (p<0.05). These differences were considered minor, there was no evidence of a relationship with dosage and there were no differences in associated parameters. Therefore, these differences were considered unrelated to treatment.

Urine volume and composition were unaffected by treatment with Coconut Oil, Reaction Products with Polyethylene Glycol and Trimethylolpropane.
There was a higher volume noted in females that received Coconut Oil, Reaction Products with Polyethylene Glycol and Trimethylolpropane, when compared with controls (p<0.05). Inspection of the individual data indicated that there was wide variation within the data with the volume for the controls ranging between 2.0-6.7 mL and for the females receiving 1000 mg/kg/day the volume range being 3.4-9.2 mL with 7/10 values being within the control range. There was no relationship with dosage and the group mean value for females receiving 100 mg/kg/day was higher than that of the females receiving 1000 mg/kg/day, but did not achieve statistical significance due to variation within the data. This difference in urine volume was considered unrelated to treatment.


NEUROBEHAVIOUR
There were no changes in behaviour, movement or physical function in any animal that was considered to be related to treatment with Coconut Oil, Reaction Products with Polyethylene Glycol and Trimethylolpropane.
There was an apparent increase in fore and hind grip during Week 12, when values recorded were compared with those pretrial. For example, fore grip group mean values for males ranged between 253-274 g during pretrial and 332-432 g during Week 12, and for females the values were 246-277 g during pretrial and 305-398 g during Week 12. This effect was noted throughout the groups, including controls, and was considered to be due to the natural muscular development of the animals. The group mean values for fore grip in females receiving 100 or 1000 mg/kg/day was lower during Week 12, when compared with controls. Inspection of the data indicated a wide variation within the data, but broadly values within the groups are similar. There was no evidence of a relationship with dosage. This statistically significant difference was considered unrelated to treatment with Coconut Oil, Reaction Products with Polyethylene Glycol and Trimethylolpropane.

ORGAN WEIGHTS
Organ weights were unaffected by treatment with Coconut Oil, Reaction Products with Polyethylene Glycol and Trimethylolpropane.
There were 2 isolated organ weight values that were statistically different from the controls. These were a higher brain weight (covariate analysis) and a higher thymus weight (relative and covariate analysis) in females that received 100 mg/kg/day. There was no evidence of any relationship with dosage and inspection of the individual data indicated that broadly the values were similar to controls. Therefore, the organ weight differences observed were considered incidental and unrelated to treatment.

GROSS PATHOLOGY
There were no gross findings that were considered related to treatment with Coconut Oil, Reaction Products with Polyethylene Glycol and Trimethylolpropane. Those that were observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence in control and treated animals.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no histopathological changes that were considered to be related to treatment with Coconut Oil, Reaction Products with Polyethylene Glycol and Trimethylolpropane. The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to treatment.

OTHER FINDINGS
none
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Conclusions:
In conclusion, there was no evidence of toxicity after oral gavage administration of 1000 mg/kg/day of Coconut Oil, Reaction Products with Polyethylene Glycol and Trimethylolpropane for at least 90 consecutive days to Han Wistar rats. Based on these results, the no-observed-effect level (NOEL) was considered to be at least 1000 mg/kg/day.
Executive summary:

The objective of this study was to determine the potential toxicity of Coconut Oil, Reaction Products with Polyethylene Glycol and Trimethylolpropane when given by oral gavage for at least 90 days to rats, to provide data to allow hazard classification to be assigned. The study was designed to be compliant with OECD Guideline No. 408 and in accordance with the OECD Principles of Good Laboratory Practice.

Three goups of 10 male and 10 female Han Wistar rats were administered Coconut Oil, Reaction Products with Polyethylene Glycol and Trimethylolpropane orally at dose levels of 100, 500 or 1000 mg/kg/day for a period of 90 days. A similarly constituted control group received the vehicle corn oil.

The following parameters and end points were evaluated in this study: clinical signs, body weights, food consumption, selected functional observations and motor activity data, ophthalmology, clinical pathology parameters (haematology, coagulation, clinical chemistry, and urinalysis), organ weights, and histopathologic examinations. There were no unscheduled deaths during the observation period. There were no in-life findings or organ weight differences that were considered to be related to treatment. There were no microscopic changes that were related to treatment. In conclusion, there was no evidence of toxicity after oral gavage administration of 1000 mg/kg/day of Coconut Oil, Reaction Products with Polyethylene Glycol and Trimethylolpropane for at least 90 consecutive days to Han Wistar rats. Based on these results, the no-observed-effect level (NOEL) was considered to be at least 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
Quality of whole database:
well conducted OECD 408-study according to GLP

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

In conclusion, there was no evidence of toxicity after oral gavage administration of 1000 mg/kg/day of Coconut Oil, Reaction Products with Polyethylene Glycol and Trimethylolpropane for at least 90 consecutive days to Han Wistar rats. Based on these results, the no-observed-effect level (NOEL) was considered to be at least 1000 mg/kg/day.

Justification for classification or non-classification

The available data on repeated dose toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008.