Registration Dossier

Administrative data

Description of key information

Based on the changes noted in liver of males and females and the changes in kidney of males at 300 mg/kg b.w./day, a NOAEL of 40 mg/kg b.w./daywas established for CAE (Heat-treated distillate of avocado oil).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: oral, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD No. 407
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
The study integrity was not adversely affected by the deviations
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
yes
Remarks:
The study integrity was not adversely affected by the deviations
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
Identification: CAE (Heat-treated distillate of avocado oil)
Appearance: Green-brown viscous liquid
Batch: 1797
Purity/Composition: See Certificate of Analysis
Test item storage: At room temperature protected from light, container flushed with nitrogen
Stable under storage
conditions until: 31 October 2016
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Sprague Dawley Crl:CD (SD) (outbred, SPF-Quality) from Charles River Deutschland, Sulzfeld, Germany.
Recognized by international guidelines as the recommended test system (e.g. EPA, FDA, OECD and EC).
Sex:
male/female
Details on test animals and environmental conditions:
30 males, 30 females (females were nulliparous and non-pregnant) with an age at start of treatment of approximately 6 weeks.
Group housing of 5 animals per sex in Macrolon cages with sterilized sawdust as bedding material and paper as cage-enrichment. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage-enrichment, bedding material, food and water.

Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals did not have access to food for a maximum of 2 hours.
Water: Free access to tap-water.
Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

Conditions: Environmental controls for the animal room are set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle. The light/dark cycle was interrupted for study related activities. Any variations to these conditions were evaluated and maintained in the raw data.
Route of administration:
oral: gavage
Details on route of administration:
Oral gavage, using a plastic feeding tube.
Formulations were placed on a magnetic stirrer during dosing.
Vehicle:
corn oil
Remarks:
specific gravity 0.92
Details on oral exposure:
Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated weekly according to the latest body weight.
Analytical verification of doses or concentrations:
no
Remarks:
see below
Details on analytical verification of doses or concentrations:
Analysis of test item in vehicle for concentration, stability, homogeneity was not performed during this study at request of the Sponsor. The test item CAE (Heat-treated distillate of avocado oil) is a Substance of Unknown or Variable composition (UVCB). This substance is known to be stable and homogeneous when storage conditions are properly respected. Moreover the batch to be tested was synthetized especially for this repeated dose 28-day oral toxicity study (01/10/2015). Therefore no analysis was performed by Charles River Den Bosch since the Sponsor had provided a certificate of analysis which confirmed the nature of the test item.
To limit the impact, the test item preparation was performed with approved procedures and documented in detail. Preparations were visually inspected for homogeneity prior to use and all preparations were used within 4 hours after adding vehicle to the test item.
Duration of treatment / exposure:
At least 28 days. Main animals were dosed up to the day prior to necropsy, and Recovery animals were dosed up to the day prior to start of the recovery period.
Frequency of treatment:
Once daily, 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (main test)
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (recovery)
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
Group 2 (main test)
Dose / conc.:
40 mg/kg bw/day (nominal)
Remarks:
Group 3 (main test)
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Group 4 (main test)
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Group 4 (recovery)
No. of animals per sex per dose:
5 rats/sex/dose
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
Mortality / Viability: At least twice daily.

Clinical signs: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals and were recorded. Once prior to start of treatment and at weekly intervals during the treatment phase, this was also performed outside the home cage in a standard arena. The clinical observations were conducted immediately (0-30 min) after dosing and 3 hours (± 30 min) after dosing (based on results of the dose-range-finding study).
The time of onset, grade and duration of any observed signs was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.

Functional Observations: During Week 4 of treatment, the following tests were performed in all Recovery Group 1 and 4 animals and all Main Group 2 and 3 animals:
- hearing ability, pupillary reflex and static righting reflex (score 0 = normal/present, score 1 = abnormal/absent),
- fore- and hind-limb grip strength, recorded as the mean of three measurements per animal.
- locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system). Total movements and ambulations are reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.
Functional observation tests were conducted after observation for clinical signs (incl. arena observation, if applicable) 3 hours after dosing.
Since the above mentioned measurements did not reveal treatment-related effects, the functional observation tests were not performed at the end of the recovery phase.

Body weights: Weekly.

Food consumption: Weekly.

Water consumption: Subjective appraisal was maintained daily during the study, but no quantitative investigation introduced as no effect was suspected.

Clinical laboratory investigations
There were conducted at the end of treatment, Day 29 (Main and Recovery animals) and at the end of recovery, Day 43 (Recovery animals). Blood samples were collected (under anaesthesia using isoflurane) for the usual haematological parameters (0.5 mL), for the usual clinical biochemistry parameters (0.5 mL) and for determination of bile acids.
Sacrifice and pathology:
Animals surviving to the scheduled day of necropsy were deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in 10% buffered formalin:
IIdentification marks: not processed
Adrenal glands, (Aorta), Brain-cerebellum, midbrain, cortex (7 levels), Caecum, Cervix, (Clitoral gland), Colon, Duodenum, Epididymides, Eyes (including optic nerve and [if detectable] harderian gland), (Female mammary gland area), Femur including joint, Heart, Ileum, Jejunum, Kidneys, (Larynx), (Lacrimal gland, exorbital), Liver, Lung, infused with formalin, Lymph nodes - mandibular, mesenteric, (Nasopharynx), (Oesophagus), Ovaries, (Pancreas),
Peyer's patches [jejunum, ileum] if detectable, (Pituitary gland), (Preputial gland), Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, Seminal vesicles including coagulating gland, Skeletal muscle, (Skin), Spinal cord -cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid including parathyroid [if detectable], (Tongue), Trachea, Urinary bladder, Uterus, Vagina, All gross lesions.
Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

Other examinations:
The following organ weights and terminal body weight were recorded from the animals on the scheduled day of necropsy:
Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus, Uterus (including cervix) , Prostate , Seminal vesicles including coagulating glands , Thyroid including parathyroid.

Histopathology:
- all tissues collected at the scheduled sacrifice from all Main Group 1 and 4 animals,
- brain (level 6 and 7), liver, mesenteric lymph nodes, prostate, seminal vesicles and coagulation gland and kidneys of all male animals of Groups 2 and 3 and Recovery group animals and liver and adrenal glands of all female animals of Groups 2 and 3 and Recovery group animals, based on (possible) treatment-related changes in these organs in Group 4,
- kidneys stained with PAS staining of all male animals (main and recovery) based on (possible) treatment-related changes,
- all gross lesions.
All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
Histopathology was subjected to a peer review.
Statistics:
All statistical tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicological relevant clinical signs were noted during the observation period. And no abnormalities were noted during the weekly arena observations in this study.
Salivation seen after dosing among the animals treated at 300 mg/kg b.w./day and one animal at 40 mg/kg b.w./day was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.
Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of treated animals and body weight gain of treated males remained in the same range as controls over the study period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after correction for body weight remained similar to the control level over the study period.

Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males and females at 300 mg/kg b.w./day showed some changes at the end of treatment and recovery period, which could be related to high turnover of red blood cells (lower red blood cell count, higher reticulocyte count, higher red blood cell distribution width, lower haemoglobin, lower haemacocrit levels, lower mean corpuscular volume and/or lower mean corpuscular haemoglobin level).
Any other statistically significant changes in haematology parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend and/or remained within the range considered normal for rats of this age and strain.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes were noted in clinical biochemistry parameters of treated rats.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength and motor activity was similar between control and high dose animals.
Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
The lower grip strength of the fore limbs in animals at 40 mg/kg b.w./day (achieving statistical significance) was considered not toxicologically relevant in absence of a dose response relation.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Test item-related higher liver weights compared to control animals (absolute and relative to body weight) were noted in 300 mg/kg b.w./day treated males (51 and 53% resp.) and females (48 and 44% resp.) at the end of treatment and in females only (21 and 10% respectively) at the end of recovery.
Test item-related higher seminal vesicles weights (absolute and relative to body weight) were noted in males at the end of treatment at 300 mg/kg b.w./day (33 and 35% resp). Based on the absence of correlating test-item related histopathologic findings in any male reproductive organs, it was considered not to be toxicological relevant.
All other organ weight differences observed in both Main and Recovery groups, including those that reached statistical significance, were considered incidental and unrelated to the test item.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related macroscopic findings were noted in the liver of males at the end of treatment, which consisted of:
- Enlarged liver in 3/5 males at 300 mg/kg b.w./day. The microscopic correlate was hepatocellular hypertrophy.
- Pale discoloration in 1/5 males at 10 mg/kg b.w./day, 1/5 males at 40 mg/kg b.w./day and in 1/5 males at 300 mg/kg b.w./day. The microscopic correlate was periportal hepatocellular vacuolation.
- Accentuated lobular pattern in 1/5 males at 10 mg/kg b.w./day, in 1/5 males at 40 mg/kg b.w./day and in 1/5 males at 300 mg/kg b.w./day. The microscopic correlate was periportal hepatocellular vacuolation.
This finding was also noted in 1/5 Main control females and 1/5 Main females at 300 mg/kg b.w./day. Since incidences did not distinguish treated females from controls, this finding was considered not to be treatment-related in females.
There were no test item-related macroscopic findings in rats of the Recovery groups.
All of the remaining recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were noted in the kidneys and mesenteric lymph node of males, adrenal glands of females and liver of males and females at the end of treatment and females at the end of recovery.
In the kidney, glomerulopathy was observed at slight degree in 5/5 Main males at 300 mg/kg b.w./day. This finding was absent after a 14-day treatment-free period.
In the mesenteric lymph node, sinus histiocytosis was observed at increased incidence (4/5) and/or severity (up to slight) in Main males at 300 mg/kg b.w./day, compared to minimal degrees in a few males of the other groups. This finding was present at background incidence/severity after a 14-day treatment-free period.
In adrenal glands, vacuolation of the zona glomerulosa was observed at increased incidence (4/5, minimal degree) in Main females at 300 mg/kg b.w./day. This finding was present at background incidence/severity after a 14-day treatment-free period.
In the liver, a dose-related increase in incidence and/or severity (up to slight) of hepatocellular hypertrophy was observed in Main males from 10 mg/kg b.w./day onward and Main females from 40 mg/kg b.w./day onward.
After a 14-day treatment-free period, hepatocellular hypertrophy was observed at minimal degree in 1/5 Recovery males and 3/5 Recovery females at 300 mg/kg b.w./day.
In addition, a dose-related increase in incidence and/or severity (up to moderate) of periportal hepatocellular vacuolation was observed in the liver of Main males from 10 mg/kg b.w./day onward and Main females from 40 mg/kg b.w./day onward.
After a 14-day treatment-free period, periportal hepatocellular vacuolation was observed in 3/5 Recovery males (minimal) and 4/5 Recovery females (up to slight) at 300 mg/kg b.w./day.
All of the remaining recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
Sprague Dawley rats were treated with CAE (Heat-treated distillate of avocado oil) for 28 consecutive days by daily oral gavage at dose levels of 10, 40 and 300 mg/kg b.w./day, followed by a 14-day treatment-free recovery period.
No toxicologically significant changes were noted in clinical appearance, functional observations, body weight, food consumption and in clinical biochemistry parameters.
At macroscopic examination, test item-related findings were present in the liver of Main group males and consisted of low incidences of pale discoloration and/or accentuated lobular pattern starting at 10 mg/kg b.w./day (both with the microscopic correlate periportal hepatocellular vacuolation) and enlarged livers in the majority of males at 300 mg/kg b.w./day (with microscopic correlate hepatocellular hypertrophy). The enlarged liver was also confirmed by the increased liver weights observed at 300 mg/kg b.w./day. In addition, the increased liver weight was also observed in Main Group females at 300 mg/kg b.w./day and Recovery group females (the microscopic correlate was hepatocellular hypertrophy).
At microscopic examination, a dose-related increase in incidence and/or severity was seen in hepatocellular hypertrophy (up to slight) and/or periportal hepatocellular vacuolation (up to moderate) which was observed in males from 10 mg/kg b.w./day onward and females from 40 mg/kg b.w./day onwards. Based on statistical higher liver weights at 300 mg/kg b.w./day correlating with hepatocellular hypertrophy and the presence of periportal hepatocellular vacuolation at high incidence and mean severity at 300 mg/kg b.w./day, the liver findings were considered to be adverse at 300 mg/kg b.w./day in both sexes.
After the 14-day treatment-free period, test item-related microscopic findings were present in the liver. Hepatocellular hypertrophy (in females related with increased liver weight) and/or periportal hepatocellular vacuolation was noted in Recovery group males and females at 300 mg/kg b.w./day. Incidences and mean severities of these findings were lower compared to Main group rats treated at 300 mg/kg b.w./day, suggesting partial recovery.
Furthermore, histopathology revealed glomerulopathy in the kidneys in all males at 300 mg/kg b.w./day (slight degree) which was considered to be adverse.
Other test item related microscopic findings in mesenteric lymph nodes and adrenal glands were considered not adverse. This included an increased incidence and/or severity of sinus histiocytosis in the mesenteric lymph nodes, as observed in males at 300 mg/kg b.w./day up to slight degree. This was regarded to be an adaptive response to the oral gavage of test substance. And although an increased incidence of vacuolation of the zona glomerulosa in the adrenal glands was observed in females at 300 mg/kg b.w./day, at this low severity (only minimal degree) it was considered as non-adverse.
Haematological investigation revealed some changes in males and females at 300 mg/kg b.w./day, which could be related to high turnover of red blood cells (lower red blood cell count, higher reticulocyte count, higher red blood cell distribution width, lower haemoglobin, lower haematocrit levels, lower mean corpuscular volume and/or lower mean corpuscular haemoglobin level). Since these changes were very slight in nature and occurred in absence of morphological changes, these changes were considered not toxicologically relevant.
Key result
Dose descriptor:
NOAEL
Remarks:
of 40 mg/kg b.w./day
Effect level:
> 40 mg/kg bw/day (nominal)
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
System:
other: liver and kidney
Organ:
liver
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
Based on the changes noted in liver of males and females and the changes in kidney of males at 300 mg/kg b.w./day, a NOAEL of 40 mg/kg b.w./daywas established for CAE (Heat-treated distillate of avocado oil).
Executive summary:

Rationale for dose levels

Based on the results of a 7-day range finding study , the dose levels for this 28-day oral gavage study were selected to be 0, 10, 40 and 300 mg/kg b.w./day.

 

Study outline

The test item, formulated in corn oil, was administered daily for 28 days by oral gavage to SPF-bred Sprague Dawley rats. One control group and three treated groups were tested, each consisting of 5 males and 5 females. An extra 5 animals per sex, respectively in the control and high dose groups, were allowed 14 days of recovery.

 

Evaluated parameters

The following parameters were evaluated: clinical signs daily; functional observation tests in Week 4; body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

 

Results

No toxicologically significant changes were noted in clinical appearance, functional observations, body weight, food consumption and clinical laboratory investigations.

 

At macroscopic examination, test item-related findings were present in the liver of Main group males and consisted of low incidences of pale discoloration and/or accentuated lobular pattern starting at 10 mg/kg b.w./day (both with the microscopic correlate periportal hepatocellular vacuolation) and enlarged livers in the majority of males at 300 mg/kg b.w./day (with microscopic correlate hepatocellular hypertrophy). The enlarged liver was also confirmed by the increased liver weights observed at 300 mg/kg b.w./day. In addition the increased liver weight was observed in Main Group females at 300 mg/kg b.w./day and Recovery group females (the microscopic correlate was hepatocellular hypertrophy).

At microscopic examination, a dose-related increase in incidence and/or severity in hepatocellular hypertrophy(up to slight)and/orperiportal hepatocellular vacuolation(up to moderate)were observed in males from 10 mg/kg b.w. /day onward and females from 40 mg/kg b.w. /day onwards. Based on statistical higher liver weights at 300 mg/kg b.w./day correlating with hepatocellular hypertrophy and the presence of periportal hepatocellular vacuolation at high incidence and mean severity at 300 mg/kg b.w./day, the liver findings were considered to be adverse at 300 mg/kg b.w./day in both sexes.

After the 14-day treatment-free period, test item-related microscopic findings remained present in the liver. Hepatocellular hypertrophy (in females related with increased liver weight) and/orperiportal hepatocellular vacuolationwas noted in Recovery group males and females at 300 mg/kg b.w./day. Incidences and mean severities of these findings were lower compared to Main group rats treated at 300 mg/kg b.w./day,suggesting partial recovery.

Furthermore, histopathology revealed glomerulopathy in the kidneys in all males at 300 mg/kg b.w./day (slight degree) which was considered to be adverse.


Other test item related microscopic findings observed in mesenteric lymph nodes and adrenal glands were considered not adverse. This included an increased incidence and/or severity of sinus histiocytosis in the mesenteric lymph nodes, as observed in males at 300 mg/kg b.w./day up to slight degree. This was regarded to be an adaptive response to the oral gavage of test substance. And although an increased incidence of vacuolation of the zona glomerulosa in the adrenal glands was observed in females at 300 mg/kg b.w./day, at this low severity (only minimal degree) it was considered as non-adverse.

 

Conclusion

Based on the changes noted in liver of males and females and the changes in kidney of males at 300 mg/kg b.w./day, a NOAEL of 40 mg/kg b.w./day was established for the test item CAE (Heat-treated distillate of avocado oil).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
40 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Organ:
liver
kidney

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

Justification for classification or non-classification