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Description of key information

The repeated dose toxicity potential of the test substance ‘squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation’ can be deduced based on information available for its individual constituents. Studies conducted with the major constituents, glycerides, fatty acids and unsaponifiable matters (including tocopherols, sterols, squalene and hydrocarbons) indicate overall low toxicity. The sterols, sterol esters and squalene are found to be poorly absorbed both orally and dermally, therefore unlikely to contribute significantly to toxicity. Chronic and sub-chronic repeated dose studies with sterols and squalene identified NOAELs at 2500 and 3300 mg/kg bw/day respectively. Tocopherols, on the other hand, were well absorbed and although beneficial at nutritionally relevant doses, may antagonise the function of other fat-soluble vitamins at higher doses. Further, several safe limits (in the form of ULs or ADIs) have been derived for tocopherols by the different regulatory authorities (SCF/EFSA, IOM, EVM). Considering the overall low inherent toxicity potential of the constituents, the latest EFSA re-evaluated upper limit based on a NOAEL of 540 mg/day or 7.7 mg/kg bw/day for a 70 Kg adult, has been considered further hazard and risk assessment.

Considering the intermediate use and physico-chemical properties of the test substance and/or its constituents, the absorption potential via the dermal route is not expected to be higher than oral route. Exposure via the inhalation route is not expected considering the intermediate use and low vapour pressure of the substance. Therefore, testing via these routes is unlikely to result in any additional hazard identification and hence further testing involving vertebrate animals may be omitted, in accordance with Annex XI (1.2) of the REACH regulation. The risk assessment for the likelihood of any potential dermal or inhalation exposure in workers has been conducted based on the conservative NOAEL identified from the studies with the constituents and by using appropriate route-to-route extrapolation assessment factors as per the ECHA Guidance R.8.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

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:
weight of evidence
Study period:
1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Groups of 25 weanling Sprague-Dawley rats of each sex were fed diets containing caprenin at dietary concentrations of 0, 5.23, 10.23 or 15.00% (w/w) for 91 d. Survival, clinical signs, body weight, feed consumption, feed efficiency, organ weights, organ-to-bodyweight ratios, organ-to-brain-weight ratios, haematological values and clinical chemistry parameters were evaluated in all groups. Histopathology of a full complement of tissues was evaluated in the corn oil and MCT oil control groups as well as the high-dose caprenin group.


GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC, USA
- Age at study initiation: 4 wk
- Weight at study initiation: 71 to 98 g (male) and from 68 to 88 g (female)
- Housing: Housed individually in suspended wire-bottomed cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23.8°C
- Humidity (%): 50 + 20%
- Photoperiod: 12 h light/12 h dark

Route of administration:
oral: feed
Details on route of administration:
0, 0, 5.23, 10.23 and 15.00 % (w/w of the diet) caprenin
Vehicle:
corn oil
Details on oral exposure:
DIET PREPARATION
- Mixing appropriate amounts with (Type of food): All diets were prepared to provide about 4000 kcal/kg, based on the assumption that the
caloric densities (or physiological fuel values) of corn oil, MCT oil and caprenin were 9, 7 and 5 kcal/g, respectively


Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Not applicable
Duration of treatment / exposure:
91 d
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0, 0, 5.23, 10.23 and 15.00 % (w/w of the diet) caprenin
Basis:
nominal in diet
No. of animals per sex per dose:
25 rats/dose
Control animals:
yes, concurrent vehicle
Details on study design:
No data
Positive control:
No data
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before initiation of the study and at week 13

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the study (Day 91)
- Anaesthetic used for blood collection: Yes (CO2/02 mix)
- Animals fasted: Yes
- How many animals: 20

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the study (Day 91)
- Animals fasted: Yes
- How many animals: 20


Sacrifice and pathology:
At terminal necropsy, organ weights were determined for brain, spleen, liver, kidneys, heart, gonads, adrenals, caecum (after rinsing with saline) and colon (after rinsing with saline) were determined and expressed as absolute and relative (organ-to-body weight and organ-to-brain weight)
values, and a histopathological examination was performed.
Other examinations:
Additional sections from the liver and heart were stained with Oil Red O and graded under microscopic evaluation for fat content.
Extra sections of kidney were stained with alizarin red and graded for incidence and severity of nephrocalcinosis.
Statistics:
- ANOVA
- Bartlett's test of homogeneity
- t-test
- Wilcoxon's rank sum test
- Fisher-Irwin exact test
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
Description (incidence and severity):
Lower feed efficiency in the high-dose group females in comparison with rats fed the MCT oil diet
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
No significant treatment related effects were observed
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No significant treatment related effects were observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No significant treatment related effects were observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
No significant treatment related effects were observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No significant treatment related effects were observed
Histopathological findings: neoplastic:
not specified
Details on results:
No treatment-related effects on growth, mortality, haematology and serum chemistry values, or in anatomical or microscopical pathology

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 15 other: % in diet (i.e., ca. 13200 mg/kg bw/day)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Absence of treatment-related effects in growth, mortality, haematology and serum chemistry values, or in anatomical or microscopical pathology
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 15 other: % in diet (i.e., ca. 14600 mg/kg bw/day)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Absence of treatment-related effects in growth, mortality, haematology and serum chemistry values, or in anatomical or microscopical pathology
Key result
Critical effects observed:
no

None

Conclusions:
Under the test conditions, the NOAEL of the substance was determined to be 13,200 and 14,600 mg/kg bw/day (the highest tested dose) for males and females, respectively.
Executive summary:

A study was conducted to evaluate the sub-chronic repeated dose toxicity of the constituent caprenin (a randomized triglyceride primarily comprising caprylic (C8:0), capric (C 10:0), and behenic (C22:0) acids) in rats. Groups of 25 rats of each sex were fed diets containing 5.23, 10.23 or 15.00% (w/w) of the substance for 91 d. Corn oil was added at 8.96, 5.91 and 3.00%, respectively, to provide essential fatty acids and digestible fat calories. Corn oil alone (12.14%) and a blend of medium-chain triglyceride (MCT) oil plus corn oil (11.21 and 3.13%, respectively) served as controls. All diets were formulated to provide about 4000 kcal/kg of diet and 26.8% of digestible calories from fat by assuming that corn oil, MCT oil, and caprenin provided 9, 7 and 5 kcal/g respectively. Survival, clinical signs, body weight, feed consumption, feed efficiency, organ weights, organ-to-bodyweight ratios, organ-to-brain-weight ratios, haematological values and clinical chemistry parameters were evaluated in all groups. Histopathology of a full complement of tissues was evaluated in the corn oil and MCT oil control groups as well as the high-dose caprenin group. Additional rats (5/sex/group) were included in the study to determine the levels of C22:0 in heart, liver or perirenal fat at the end of the 91 d feeding period. No significant differences in body weight gain were measured with the balanced caloric diets, although feed conversion efficiency was reduced in the high-dose group. No adverse effects from the ingestion of the substance were detected, nor were significant amounts of C22: 0 present in the fat extracted from the selected fat depot sites. Under the test conditions, the NOAEL of the substance was determined to be >15% (w/w) in the diet (or more than 83% of total dietary fat), which is equal to a mean exposure level of > 13,200 and 14,600 mg/kg/day for male female rats, respectively (Webb, 1993).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1960
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Qualifier:
no guideline followed
Principles of method if other than guideline:
Groups of 5 Osborne-Mendel male rats were fed diets containing lauric acid at dietary concentration of 10% (w/w) for 18 wk. Survival, clinical signs, body weight, organ weight, haematological values and clinical chemistry parameters and histopathological evaluation was performed in experimental animals.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Osborne-Mendel
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 40 to 50 g
- Housing: Housed individually in cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum


Route of administration:
oral: feed
Details on route of administration:
10 % in diet

Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Diets were prepared in quantities sufficient for 2 wk by blending the basal diet of a ground commercial rat biscuit with the substance so that the desired percentage by weight of the total diet was achieved.

Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Not applicable
Duration of treatment / exposure:
18 wk
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
10 % in diet
Basis:
nominal in diet
No. of animals per sex per dose:
5 rats/dose
Control animals:
yes, concurrent no treatment
Details on study design:
No data
Positive control:
No data
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes


BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the study

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the study



Sacrifice and pathology:
At terminal necropsy, organ weights were determined and a histopathological examination was performed.
Other examinations:
None
Statistics:
No data
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):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No significant treatment related effects were observed
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No significant treatment related effects were observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No significant treatment related effects were observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
No significant treatment related effects were observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No significant treatment related effects were observed
Histopathological findings: neoplastic:
not specified
Details on results:
No treatment-related effects on clinical signs, weight gain, mortality, gross pathology and individual organ weights
Key result
Dose descriptor:
NOAEL
Effect level:
10 other: % of diet (i.e., equivalent to 5142 mg/kg bw/day)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no treatment-related effects observed
Key result
Critical effects observed:
no

None

Conclusions:
Under the test conditions, the NOAEL of the substance in rats was determined to be 10% (equivalent to 5142 mg/kg bw/day).
Executive summary:

A study was conducted to evaluate the sub-chronic repeated dose toxicity of the constituent lauric acid in rats. Five male rats were fed the substance at the level of 10% of their diet for 18 weeks. A control group of 5 males was fed concurrently. Survival, clinical signs, body weight, haematological values and clinical chemistry parameters were evaluated in all experimental animals. Organ weights and gross pathology were recorded for the sacrificed animals at the end of the experiment. No significant treatment-related effects in any of the experimental animals. Under the test conditions, the NOAEL of the substance was determined to be 10% (equivalent to 5,142 mg/kg bw/day) in the diet (Fitzhugh, 1960).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes
Remarks:
FDA Good Laboratory Practices Regulations (21 CFR 58)
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Simonsen Laboratories, Gilroy, CA
- Age at study initiation: 6 wk
- Fasting period before study: No
- Housing: 5 per cage in polycarbonate cages lined with heat-treated hardwood chips and covered with polyester filter sheets. The cages were stored on stainless steel racks equipped with an automatic watering system
- Diet: NIH 07; available ad libitum
- Water: Ad libitum
- Acclimation period: 14 d
- Other: Feed hoppers in the animal cages were changed twice weekly


ENVIRONMENTAL CONDITIONS
- Temperature: 68-76°F
- Humidity: 42-72%
- Air changes: 10/h
- Photoperiod: 12 h dark/12 h light

Route of administration:
oral: feed
Details on route of administration:
0, 0.62, 1.25, 2.5, 5.0 or 10% in diet
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Method of mixing: Formulated diets were prepared by blending the appropriate amount of castor oil with a small quantity of feed to prepare a premix. The premix then was layered between the required amounts of feed in a twin-shell blender and blended for 15 min to achieve a uniform mix.
- Mixing appropriate amounts with (Type of food): 10% (100 mg/g) determined by gravimetric analysis, and blends at 0.5% (5 mg/g) determined by HPLC analysis.
- Storage temperature of food: Stored for no longer than 3 weeks at 5°C
- Stability under test conditions: 0.5% dose level is stable for at least 21 d when stored in the dark at 5°C and for 3 d when stored open to air and light in a rodent cage.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Determined by HPLC at the study and analytical chemistry laboratories in duplicates. The results of the analyses for all dose mixtures given to the animals ranged from 97% to 106% of the target concentrations.
Duration of treatment / exposure:
13 wk or 90 d
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0, 0.62, 1.25, 2.5, 5.0 or 10% in diet
Basis:
actual ingested
No. of animals per sex per dose:
10 rats per sex per dose
Control animals:
yes, plain diet
Observations and examinations performed and frequency:
BODY WEIGHT: Yes
- Time schedule for examinations: Weighed initially and thereafter once weekly


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 5, 21 and 90
- Anaesthetic used for blood collection: Yes (CO2 anesthesia)
- Animals fasted: No
- How many animals: 10 per sex at each dose level
- Parameters examined: Red blood cell (RBC) count, red blood cell morphologic assessment, hematocrit (HCT), hemoglobin concentration
(HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), white blood cell count (WBC), white blood cell differential count, reticulocyte count (absolute), and platelet counts (absolute).


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 5, 21 and 90
- Animals fasted: No
- How many animals: 10 per sex at each dose level
- Parameters examined: Alkaline phosphatase activity (ALP), albumin (ALB), urea nitrogen (UN), creatinine (CREA), alanine aminotransferase activity
(ALT), total bile acids (TBA), sorbitol dehydrogenase activity (SDH), total protein (TP), and creatinine kinase (CK).


OTHER: ORGAN WEIGHTS:
- Organ weights were determined to the nearest milligram for the liver, right kidney, right testicle, heart, thymus, and lungs. All tissues were preserved in 10% neutral buffered formalin.



Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
- Necropsy and Histologic Examinations: Complete histopathology examinations were conducted on all rats from the control and 10% dose groups. The following tissues were routinely processed for preparation of histologic sections and microscopic examination:
adrenal glands, brain, cecum, colon, duodenum, epididymis/seminal vesicles/prostate/testes or ovaries/uterus, esophagus, eyes (if grossly abnormal), femur (including marrow), heart, ileum, jejunum, kidneys, liver, lungs and main stem bronchi, mammary gland, mandibular and mesenteric lymph nodes, nasal cavity and turbinates, pancreas, parathyroid glands, pituitary gland, preputial or clitoral glands, rectum, salivary glands, skin, spinal cord and sciatic nerve (if neurologic signs present), spleen, forestomach and glandular stomach, thymus, thyroid gland, trachea, urinary bladder, zymbal glands, and all gross lesions and tissue masses including regional lymph nodes.
Liver was examined from male rats in all other dose groups, and histologic sections of gross lesions were examined from all rats.
Other examinations:
REPRODUCTIVE TOXICITY SCREEN:
- Parameters examined: Sperm motility and morphology were evaluated at necropsy and vaginal cytology
- Time schedule for examinations: Sperm motility and morphology: At necropsy; Vaginal cytology: At 12 wk


Statistics:
Statistically analyzed within each sex by one-way Analysis of Variance tests, followed by Dunnett's t-test for pair-wise comparisons (p < 0.05)

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
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Details on results:
MORTALITY: No effect on survival up to 10%


BODY WEIGHT AND WEIGHT GAIN: No significant diference in group mean body weights of rats were observed. However, mean body weights of exposed female rats were slightly lower than the mean body weights of controls but the differences were not dose-related.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No significant differences in average food consumption among each sex were observed, although food consumption of male and female rats receiving diets containing 10% castor oil was slightly lower than that of controls.


HAEMATOLOGY: None of the observed changes was considered biologically significant.
However, the observed effects were: slight decrease in MCHC in male rats at Day 21, receiving 10% diet; a statistically significant decrease in MCV among the 10% group; a decrease in MCH among the 5% and 10% groups; and an increase in platelets among the 1.25, 5, and 10% groups. The only change observed among female rats was a statistically significant decrease in reticulocyte counts at Day 5 in groups receiving the 0.62% or 10% diets.


CLINICAL CHEMISTRY:
- A treatment- and dose-related increase in the activity of serum alkaline phosphatase was observed in male and female rats at Days 5 and 21 and at study termination.
- Total bile acids were increased among males receiving the higher dietary levels at Days 5 and 21 but were not increased at study termination.
- Other minor changes included increases in albumin observed at study termination in males receiving 5% diets and at Day 5 in females receiving 10% diets, and an increase in urea nitrogen at study termination in males that received 0.62% diets and a decrease at Day 5 in females that received castor oil at 10% in the diet.


ORGAN WEIGHTS:
- Absolute liver weights and the liver-to-body-weight ratio was increased in male rats, that received 10% castor oil.
- Heart-to-body-weight ratios were increased in groups of male rats receiving 0.62%, 2.5%, and 10% diets; however, absolute heart weights were not increased, and the differences in body weight ratios were small and not considered treatment related.


GROSS PATHOLOGY: No morphologic changes were observed.


HISTOPATHOLOGY: NON-NEOPLASTIC: Histopathologic examination revealed an absence of compound-related lesions in any organ or tissue of rats exposed to castor oil in the diet.


OTHER FINDINGS: REPRODUCTIVE TOXICITY SCREEN: No evidence of any reproductive toxicity.
However, the observed effects were: In male rats, there was a slight decrease in epididymal weight (6-7%) which occurred in the middle- and high-dose groups, but this was not dose-related. There were no effects on any other male rat reproductive endpoint, or on any female rat reproductive endpoint.
Dose descriptor:
NOAEL
Effect level:
10 other: % in diet (i.e. ca. 5,700 - 6,500 mg/kg bw/d)
Sex:
male/female
Basis for effect level:
other: overall effects
Critical effects observed:
not specified

None

Conclusions:
Under the conditions of this study, the NOAEL of the substance in rats was determined to be 10% in diet (i.e. 5,800 mg/kg bw/day based on actual feed consumption and body weight data).
Executive summary:

A 90 d oral repeated dose study was conducted in F344/N rat to evaluate the sub-chronic and reproductive toxicity of the constituent ‘glycerides, C16 and C18-unsatd. and C18-unsatd. hydroxy’ (as castor oil). Rats (10 /sex / group) were exposed for 13 weeks to 0, 0.62, 1.25, 2.5, 5.0 or 10% of the substance mixed in diet. Mortality, bodyweight, food consumption, hematology and clinical chemistry parameters were recorded throughout the study. Organ weights were determined and gross pathology and histopathology conducted at termination. Additionally, sperm motility and morphology were evaluated at necropsy, and vaginal cytology during the week preceding necropsy. Exposure to the substance at dietary concentrations as high as 10% did not affect survival or bodyweight gain. There were no biologically significant effects noted in hematologic analyses. Mild increases in total bile acids and serum alkaline phosphatase were recorded at various times in the high dose groups. Liver weights were increased in male rats at this dose and in females as of 5% in diet. However, there were no histopathologic lesions associated with these liver changes, nor were there any compound-related morphologic changes in any organ. No significant changes were noted in a screening for male reproductive endpoints, including sperm count and motility, and no changes were observed in the length of female estrous cycles. Thus, no significant adverse effects of the substance were noted. Under the conditions of this study, the NOAEL of the substance in rats was determined to be 10% in diet (i.e. ca. 5,700 - 6,500 mg/kg bw/day based on actual feed consumption and body weight data) (Irwin, 1992).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes
Remarks:
FDA Good Laboratory Practices Regulations (21 CFR 58)
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Simonsen Laboratories, Gilroy, CA
- Age at study initiation: 6 wk
- Fasting period before study: No
- Housing: Individually caged in polycarbonate cages lined with heat-treated hardwood chips and covered with polyester filter sheets. The cages were stored on stainless steel racks equipped with an automatic watering system
- Diet: NIH 07; available ad libitum
- Water: Ad libitum
- Acclimation period: 15 d
- Other: Feed hoppers in the animal cages were changed twice weekly


ENVIRONMENTAL CONDITIONS
- Temperature: 68-76°F
- Humidity: 42-72%
- Air changes: 10/h
- Photoperiod: 12 h dark/12 h light
Route of administration:
oral: feed
Details on route of administration:
0, 0.62, 1.25, 2.5, 5.0 or 10% in diet

Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Method of mixing: Formulated diets were prepared by blending the appropriate amount of castor oil with a small quantity of feed to prepare a premix. The premix then was layered between the required amounts of feed in a twin-shell blender and blended for 15 min to achieve a uniform mix.
- Mixing appropriate amounts with (Type of food): 10% (100 mg/g) determined by gravimetric analysis, and blends at 0.5% (5 mg/g) determined by HPLC analysis.
- Storage temperature of food: Stored for no longer than 3 weeks at 5°C
- Stability under test conditions: 0.5% dose level is stable for at least 21 d when stored in the dark at 5°C and for 3 d when stored open to air and light in a rodent cage.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Determined by HPLC at the study and analytical chemistry laboratories in duplicates. The results of the analyses for all dose mixtures given to the animals ranged from 97% to 106% of the target concentrations.
Duration of treatment / exposure:
13 wk or 90 d
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0, 0.62, 1.25, 2.5, 5.0 or 10% in diet
Basis:
actual ingested
No. of animals per sex per dose:
10 mice per sex per dose
Control animals:
yes, plain diet
Observations and examinations performed and frequency:
BODY WEIGHT: Yes
- Time schedule for examinations: Weighed initially and 1 x wk thereafter


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes


OTHER: ORGAN WEIGHTS:
- Organ weights were determined to the nearest milligram for the liver, right kidney, right testicle, heart, thymus, and lungs. All tissues were preserved in 10% neutral buffered formalin.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
- Necropsy and Histological Examinations: Complete histopathology examinations were conducted on all mice from the control and 10% dose groups. The following tissues were routinely processed for preparation of histological sections and microscopic examination:
adrenal glands, brain, cecum, colon, duodenum, epididymis/seminal vesicles/prostate/testes or ovaries/uterus, esophagus, eyes (if grossly abnormal), femur (including marrow), heart, ileum, jejunum, kidneys, liver, lungs and main stem bronchi, mammary gland, mandibular and mesenteric lymph nodes, nasal cavity and turbinates, pancreas, parathyroid glands, pituitary gland, preputial or clitoral glands, rectum, salivary glands, skin, spinal cord and sciatic nerve (if neurologic signs present), spleen, forestomach and glandular stomach, thymus, thyroid gland, trachea, urinary bladder, zymbal glands, and all gross lesions and tissue masses including regional lymph nodes.
Other examinations:
REPRODUCTIVE TOXICITY SCREEN:
- Parameters examined: Sperm motility and morphology were evaluated at necropsy and vaginal cytology
- Time schedule for examinations: Sperm motility and morphology: At necropsy; Vaginal cytology: At 12 wk
Statistics:
Statistically analyzed within each sex by one-way Analysis of Variance tests, followed by Dunnett's t-test for pair-wise comparisons (p < 0.05)
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
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Details on results:
MORTALITY: No effect on survival up to 10% concentration of castor oil in diet.


BODY WEIGHT AND WEIGHT GAIN: No significant effects were observed.
- However the results observed were: Mean body weights of exposed male mice generally were lower than controls, while mean body weights of exposed females generally were higher. These differences were not related to dietary concentrations of castor oil, except that mean body weights of male mice receiving the 10% castor oil diet were consistently lower than those of control mice from week 3 through the end of the study.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No statistically significant differences in average food consumption were observed, although food consumption by female mice receiving diets containing 10% castor oil was slightly lower than controls.


ORGAN WEIGHTS:
- Liver weights were increased in male and female mice at both 5% or 10% of castor oil.
- Kidney weights were increased in female mice at both 5 % and 10 % of castor oil.


GROSS PATHOLOGY: No morphologic changes were observed


HISTOPATHOLOGY: NON-NEOPLASTIC: No compound-related lesions were observed in any organ or tissue of mice exposed to castor oil in the diet.


OTHER FINDINGS: REPRODUCTIVE TOXICITY SCREEN: No adverse effects were observed in any male (testes weight, epididymal sperm motility, density, or testicular spermatid head count) or female mice (estrual cycle length, or time spent in each phase of the cycle) reproductive parameters.
Dose descriptor:
NOAEL
Effect level:
10 other: % in diet (i.e. ca. 14,600 - 20,000 mg/kg bw)
Sex:
male/female
Basis for effect level:
other: no treatment-related effects observed
Key result
Critical effects observed:
no
Conclusions:
Under the conditions of this study, the NOAEL of the substance in mice was determined to be 10% in diet (i.e. ca. 14,600 - 20,000 mg/kg bw/day based on actual feed consumption and body weight data).
Executive summary:

A 90 d oral repeated dose study was conducted in male and female B6C3F1 mice in order to evaluate the sub-chronic and reproductive toxicity of the constituent castor oil. Mice were exposed for 13 weeks to castor oil at 0, 0.62, 1.25, 2.5, 5.0 or 10% in the diet. Mortality, bodyweight and food consumption were recorded throughout the study. Organ weights were determined, and gross pathology as well as histopathology conducted at termination. Sperm motility, morphology and vaginal cytology were evaluated at various time intervals. Exposure to castor oil at dietary concentrations as high as 10% did not affect survival or body weight gains. Liver weights were increased in male and female mice as of 5% castor oil in the diet. However, there were no histopathological lesions associated with these liver changes, nor were there any compound-related morphologic changes in any organ. No significant changes were noted in a screening for male reproductive endpoints, including sperm count and motility, and no changes were observed in the length of the female estrous cycles. Thus, no significant adverse effects of castor oil were noted. Under the conditions of this study, the NOAEL of the substance in mice was determied to be 10% in diet (i.e. ca. 16000 mg/kg bw/day based on actual feed consumption and body weight data) (Irwin, 1992).

Endpoint:
sub-chronic toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1977
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Groups of 10 male and 30 female rats were fed for 13 weeks with diets containing 15% crude palm oil. Other groups received diets with heated palm oil, crude/heated soy oil, crude/heated peanut oil, or crude/heated sunflower oil at the same concentration. Clinical signs and bodyweight were recorded throughout the study. After 13 weeks, hematology, clinical chemistry and urinalysis parameters were assessed.Ten males and ten females were sacrificed for gross and microscopic pathology. After 10 weeks of treatment, 10 males and 20 females (15 - 16 weeks of age) were mated for 18 d. The females were then allowed to deliver the offsprings. Maternal bodyweight and reproductive parameters (e.g. % implantation, % surviving young, % embryo loss etc.) were recorded. At 5 weeks of age, the young were sacrificed. One male and 2 females of each litter was autopsied. Liver and kidneys were weighed. For 5 males and 5 females selected randomly, these organs were examined microscopically.
GLP compliance:
not specified
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Own breeding
- Age at study initiation: ca. 6 weeks
- Weight at study initiation: 150 g
- Housing: 5 per sex per cage
- Diet (e.g. ad libitum): 40% wheat, 20% maize, 12% fish meal, 7% blood powder, 15% oil and 6% vitamin/minerals complement
- Water (e.g. ad libitum): yes
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): every 4 - 5 d
- Storage temperature of food: 4°C
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily
Dose / conc.:
15 other: %
Remarks:
Basis:
nominal in diet
No. of animals per sex per dose:
10 males, 30 females for the 13 week feeding study
10 males, 20 females for the reproduction screening
Observations and examinations performed and frequency:
13 WEEK FEEDING STUDY

Clinical signs and bodyweight were recorded throughout the study. After 13 weeks, hematology, clinical chemistry and urinalysis parameters were assessed.Ten males and ten females were sacrificed for gross and microscopic pathology.

REPRODUCTION SCREENING

Maternal bodyweight and reproductive parameters (e.g. % implantation, % surviving young, % embryo loss etc.) were recorded. At 5 weeks of age, the young were sacrificed. One male and 2 females of each litter was autopsied. Liver and kidneys were weighed. For 5 males and 5 females selected randomly, these organs were examined microscopically.
Sacrifice and pathology:
Gross and microscopic pathology at the end of the 13 week screening.
Microscopic pathology of liver and kidney of young rats from the reproduction screening at 35 d of age.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Details on results:
OTHER FINDINGS
- No digestive intolerance noted during the 13 week feeding study
- During the reproductive screening study, no effects on maternal bodyweight evolution, reproductive parameters, pup liver and kidney weights, and pup liver and kidney histopathology
Key result
Dose descriptor:
NOAEL
Effect level:
15 other: % in diet (i.e. ca. 7,500 mg/kg bw/day)
Sex:
male/female
Basis for effect level:
other: treatment related effects observed
Key result
Critical effects observed:
no
Conclusions:
Under the conditions of this study, the substance did not show any adverse effects on male and female rats compared to other crude or heated vegetable oils when administered for 13 weeks at 15% in diet. Furthermore, no signs of toxicity were observed on maternal rats or pups in a follow-up reproductive screening trial.
Executive summary:

A study was conducted to determine the effect of ‘glycerides, C16-18 and C18-unsatd.’ (as crude palm oil) on rats when administered for 13 weeks at 15% in diet. Results were compared to those obtained with heated palm oil, crude/heated soy oil, crude/heated peanut oil, or crude/heated sunflower oil at the same concentration. Clinical signs and bodyweight were recorded throughout the study. After 13 weeks, hematology, clinical chemistry and urinalysis parameters were assessed, as well as gross and microscopic pathology. After 10 weeks of treatment, 10 males and 20 females were mated for 18 days. Maternal bodyweight and reproductive parameters were recorded. At 5 weeks of age, the young were sacrificed. Liver and kidneys weights were recorded and these organs were examined microscopically. The test substance did not show any adverse effects on male and female rats compared to other crude or heated vegetable oils when administered for 13 weeks at 15% in diet. Furthermore, no signs of toxicity were observed on maternal rats or pups in the follow-up reproductive screening trial. Under the conditions of this study, the NOAEL of the substance can be considered to be 15% in diet (Coquet, 1977).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Groups of 30 (15 male and 15 female) weanling rats were fed diets containing 10% of either the constituent crude palm oil, groundnut oil or refined palm olein oil and adequate amounts of all other nutrients for 90 d. Food intake and bodyweight were monitored weekly. At the end of the experiment, cholesterol and triglycerides of serumm, liver and heart of all animals were analysed and compared statistically.
GLP compliance:
not specified
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
The National Research Council's guide for the care and use of laboratory animals was followed.
Diet and water were given ad libitum.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
90 d exposure and the rats were divided as followed:
Group 1: Protein free group
Group 2: 20% casein protein with 10% groundnut oil
Group 3: 20% casein protein with 10% crude palm oil
Group 4: 20% casein protein with 10% refined palm olein oil
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
No
Duration of treatment / exposure:
90 d
Frequency of treatment:
daily
Dose / conc.:
10 other: %
Remarks:
Basis:
nominal in diet
No. of animals per sex per dose:
15
Control animals:
yes
Observations and examinations performed and frequency:
Body weights and food intake were recorded weekly
Sacrifice and pathology:
Cholesterol and triglycerides of serum, liver and heart of the animals were analyzed and statistically compared amongst groups.
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
Haematological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
not specified
Details on results:
The crude palm oil and refined palm oil-fed animals showed higher concentrations of cholesterol than the groundnut oil-fed animals, though these difference were not found to be statistically significant. The triglyceride concentrations of the crude palm oil and refined groups remained higher, whereas all other parameters had comparable values among different groups.
Key result
Dose descriptor:
NOAEL
Effect level:
10 other: % in diet (i.e. ca. 5,000 mg/kg bw/day)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related effects
Key result
Critical effects observed:
no

Groundnut oil, gain in body weight in 28 d (n=10): 215.6 ± 14.8

Crude palm oil, gain in body weight in 28 d (n=10): 196.5 ± 14.8

Palm-olein oil, gain in body weight in 28 d (n=10): 196.2 ± 13.8

Groundnut oil, feed efficiency ratio: 19.8

Crude palm oil, feed efficiency ratio: 18.5

Refined palm-olein oil, feed efficiency ratio: 18.9

Serum, Cholesterol (mmol/L)

Groundnut oil: 2.39 ± 0.18

Crude palm oil: 2.38 ± 0.46

Refined palm oil: 2.15 ± 0.53

Serum, Triglycerides (mmol/L)

Groundnut oil: 1.08 ± 0.18

Crude palm oil: 1.43 ± 0.23

Refined palm oil: 1.39 ± 0.24

Liver, Cholesterol (mmol/L of total lipid)

Groundnut oil: 114.8 ± 2.84*

Crude palm oil: 132.4 ± 5.17*

Refined palm oil: 173.8 ± 2.84

Liver, Triglycerides (mmol/L of total lipid)

Groundnut oil: 19.8 ± 2.03

Crude palm oil: 20.8 ± 2.60

Refined palm oil: 20.8 ± 2.82

Heart, Cholesterol (mmol/L of total lipid)

Groundnut oil: 60.3 ± 0.78*

Crude palm oil: 68.0 ± 0.52*

Refined palm oil: 44.9 ± 0.26

Heart, Triglycerides (mmol/L of total lipid)

Groundnut oil: 25.4 ± 1.02

Crude palm oil: 29.0 ± 0.90

Refined palm oil: 25.5 ± 0.79

* Significantly different from refined palm oil value, P < 0.05.

Conclusions:
Under the study conditions, the test substance showed adequate nutritional value compared to groundnut and palm olein oil. A NOAEL of 10% for the palm oil in diet was established.
Executive summary:

A study was conducted to evaluate the nutritional effects and repeated dose toxicity effects of the constituent ‘glycerides, C16 -18 and C18 -unsatd.’ (as palm oil) in rat. The substance was administered through the diet to groups of fifteen male and female rats for up to 90 d at dose levels of 10%. No adverse effects compared to controls were observed as judged by growth rate, feed efficiency ratio, protein efficiency ratio, net protein utilization, digestibility, fat absorption, nitrogen balance, phosphorous and calciul retention, serum enzymes and hematology. There was also no difference in lipid concentrations compared to controls. Under the study conditions, the substance showed adequate nutritional value compared to groundnut and palm olein oil. A NOAEL of 10% for the plam oil in diet was established (Manorama and Rukmini, 1991).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
209
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:
no
GLP compliance:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Breeding centre Charles River Deutchland, Sulzfeld, Germany)
- Age at study initiation: 6 weeks
- Housing: in group of five in Macrolon cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 d


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.9-22.0 deg. C
- Humidity (%): 31-84%
- Air changes (per hr): 15/h
- Photoperiod (hrs dark / hrs light): 12 h/12 h
Route of administration:
oral: feed
Details on route of administration:
0, 1, 5 and 15% in diet
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Accuracy of the diet preparations ranged from 87% to 95% of nominal value, which was considered as an acceptable level of accuracy
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical analyses revealed that experimental diets were homogenously prepared; accuracy of the diet ranged from 87%-95% of nominal value
Duration of treatment / exposure:
98 and 100 d for female and male rats, respectively
Frequency of treatment:
Daily
Dose / conc.:
1 other: %
Remarks:
Basis: nominal in diet
Dose / conc.:
5 other: %
Remarks:
Basis: nominal in diet
Dose / conc.:
15 other: %
Remarks:
Basis: nominal in diet
Remarks:
Doses / Concentrations:
15%
Basis:
nominal in diet
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: No higher dose level than 15% was selected as non-specific effects of high fat intake in rats can be expected to appear at dose levels above 15%
Positive control:
None
Observations and examinations performed and frequency:
All animals were observed twice daily for mortality/viability and at least once daily for clinical observations and signs for toxicity, feed intake and body weight were evaluated once a week
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
Where the variables were assessed to follow a normal distribution, a Dunnett-test based on a pooled variance estimate, was applied to compare the treated groups and the control group of each gender. The Steel-test was applied where the data could not be assumed to follow a normal distribution. The exact Fisher test was applied to frequency data. All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data.
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
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
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:
HAEMATOLOGY: Statistically significant changes in haematology parameters were considered to be of no toxicological relevance as they occurred in the absence of a dose related response and/or were of a very slight size. These changes included lower white blood cell counts (WBC) and prothrombin times in males fed 15% of teh substance, higher relative neutrophil and lower relative lymphocyte count in females fed 1% and 5% substance but not in females of the 15% dose group, and lower haemoglobin and haematocrit levels in females at 1% but not in females at 5% or 10%.

CLINICAL CHEMISTRY: Higher alkaline phosphatase activity levels (ALP) in females at 15%, lower total bilirubin levels in males at 5% and 15%, lower total protein levels in females at 1%, 5% and 15%, lower cholesterol levels in males at 15%, and in females at 1%, and 15%, lower calcium levels in males and females fed 1%, 5% and 15%, lower inorganic phosphate levels in males at 15% and increased creatinine and sodium levels in the females of 5% group. Although some statistical significant changes in biochemical parameters were observed it is important to note that compared to rats on a normal fat diet, most changes fall within the historical control values.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 15 other: % in diet (i.e., ca. 8866 mg/kg bw/day)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no toxicologically significant effects
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 15 other: % in diet (i.e., ca. 10242 mg/kg bw/day)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no toxicologically significant effects
Key result
Critical effects observed:
no
Conclusions:
Under the study conditions, the substance is considered to have a NOAEL of 15% in diet, i.e. 8,866 and 10,242 mg/kg bw/d for male and female rats, respectively.
Executive summary:

A study was conducted to determine the subchronic oral toxicity of the constituent ‘glycerides, C16 -18 and C18 -unsatd.’ (as pine nut oil) in rats according to OECD Guideline 408 and GLP. The substance was administered through the diet to three groups, each of ten male and ten female Wistar Crl:Wi(Han) strain rats, for up to 98 and 100 d, at dose levels of 0, 1, 5 and 15%. In Week 12 a functional observation test was carried out. At Week 13 ophthalmoscopic examinations were carried out. Clinical signs, bodyweight development, food intake and mortality/viability were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the treatment period. All animals were subjected to gross necropsy and macroscopic examination and a complete histopathological examination was performed. No toxicologically significant changes were observed at any of the dietary levels. A NOAEL of 15% in diet was established, which corresponds to 8,866 and 10,242 mg/kg bw/d for male and female rats, respectively (Speijers, 2009).

Endpoint:
sub-chronic toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1977
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Groups of 10 male and 30 female rats were fed during 13 weeks with diets containing 15% crude soy oil. Other groups received diets with heated soy oil, crude/heated palm oil, crude/heated peanut oil, or crude/heated sunflower oil at the same concentration. Clinical signs and bodyweight were recorded throughout the study. After 13 weeks, hematology, clinical chemistry and urinalysis parameters were assessed.Ten males and ten females were sacrificed for gross and microscopic pathology. After 10 weeks of treatment, 10 males and 20 females (15 - 16 weeks of age) were mated for 18 d. The females were then allowed to produce young. Maternal bodyweight and reproductive parameters (e.g. % implantation, % surviving young, % embryo loss etc.) were recorded. At 5 weeks of age, the young were sacrificed. One male and 2 females of each litter was autopsied. Liver and kidneys were weighed. For 5 males and 5 females selected randomly, these organs were examined microscopically.
GLP compliance:
not specified
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Own breeding
- Age at study initiation: ca. 6 weeks
- Weight at study initiation: 150 g
- Housing: 5 per sex per cage
- Diet (e.g. ad libitum): 40% wheat, 20% maize, 12% fish meal, 7% blood powder, 15% oil and 6% vitamin/minerals complement
- Water (e.g. ad libitum): yes
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:


DIET PREPARATION
- Rate of preparation of diet (frequency): every 4 - 5 d
- Storage temperature of food: 4°C
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily
Dose / conc.:
15 other: %
Remarks:
Basis:
nominal in diet
No. of animals per sex per dose:
10 males, 30 females for the 13 week feeding study
10 males, 20 females for the reproductive toxicity screening
Observations and examinations performed and frequency:
13 WEEK FEEDING STUDY

Clinical signs and bodyweight were recorded throughout the study. After 13 weeks, hematology, clinical chemistry and urinalysis parameters were assessed.Ten males and ten females were sacrificed for gross and microscopic pathology.

REPRODUCTION SCREENING

Maternal bodyweight and reproductive parameters (e.g. % implantation, % surviving young, % embryo loss etc.) were recorded. At 5 weeks of age, the young were sacrificed. One male and 2 females of each litter was autopsied. Liver and kidneys were weighed. For 5 males and 5 females selected randomly, these organs were examined microscopically.
Sacrifice and pathology:
Gross and microscopic pathology at the end of the 13 week screening.
Microscopic pathology of liver and kidney of young rats from the reproduction screening at 35 d of age.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Details on results:
OTHER FINDINGS
- No digestive intolerance noted during the 13 week feeding study
- During the reproductive screening study, no effects on maternal bodyweight evolution, reproductive parameters, pup liver and kidney weights, and pup liver and kidney histopathology
Key result
Dose descriptor:
NOAEL
Effect level:
15 other: in diet (i.e. ca. 7,500 mg/kg bw/day)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment-related effects observed
Key result
Critical effects observed:
no
Conclusions:
Under the conditions of this study, the substance did not show any adverse effects on male and female rats compared to other crude or heated vegetable oils when administered for 13 weeks at 15% in diet.
Executive summary:

A study was conducted to determine the effect of the constituent ‘glycerides, C16 -18 and C18 -unsatd.’ (as crude soy oil) in rats when administered for 13 weeks at 15% in diet. Results were compared to those obtained with heated palm oil, crude/heated soy oil, crude/heated peanut oil, or crude/heated sunflower oil at the same concentration. Clinical signs and bodyweight were recorded throughout the study. After 13 weeks, hematology, clinical chemistry and urinalysis parameters were assessed, as well as gross and microscopic pathology. After 10 weeks of treatment, 10 males and 20 females were mated for 18 d. Maternal bodyweight and reproductive parameters were recorded. At 5 weeks of age, the offsprings were sacrificed. Liver and kidneys weights were recorded and these organs were examined microscopically. The substance did not show any adverse effects in male and female rats compared to other crude or heated vegetable oils when administered for 13 weeks at 15% in diet. Furthermore, no signs of toxicity were observed on maternal rats or pups in the follow-up reproductive screening trial. Under the study conditions, the NOAEL of the substance can be considered to be 15% in diet (Coquet, 1977).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
; study at single dose; not all parameters measured
Principles of method if other than guideline:
The effect of dietary soybean oil was investigated (as control) in a subchronic toxicity study.

GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, MA
- Housing: The animals were housed in individual stainless steel wire-mesh cages
- Diet: Ad libitum
- Water: Ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23±1
- Humidity (%): 50±5
- Photoperiod (h dark / h light): 12/12
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Mixing appropriate amounts with (Type of food): Normal diet without any other fat source
Duration of treatment / exposure:
91 d
Frequency of treatment:
Daily ad libitum in food
Dose / conc.:
19 other: %
Remarks:
Basis:
nominal in diet
No. of animals per sex per dose:
20 animals/sex/group


Control animals:
other: Soybean oil group was itself used as control to study effect of 7.5% fully hydrogenated soybean oil
Details on study design:
- Rationale for animal assignment: Random


Observations and examinations performed and frequency:
BODY WEIGHT: Yes
- Time schedule for examinations: Weekly


FOOD CONSUMPTION (if feeding study): Yes
- Time schedule for examinations: Weekly


HAEMATOLOGY: Yes
- Time schedule for collection of blood: At time of sacrifice (end of study)
- Anaesthetic used for blood collection: Yes
- Animals fasted: No data
- How many animals: 10 animals/sex/group
- Parameter examined: Standard hemograms done


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At time of sacrifice (end of study)
- Animals fasted: No data
- How many animals: 10 animals/sex/group
- Parameter examined: Serum cholesterol and phospholipids


URINALYSIS: Yes
- Time schedule for collection of urine: 24 h collection at 11th wk of study
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameter examined: Volume, nitrogen, ketones, glucose, bilirubin, albumin and pH

OTHER: During the 3rd and 11th wks, feces were collected from 10 animals/sex/group for analysis of unabsorbed fat.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
(The heart, liver, kidneys and gonads were removed and weighed. Sections of these organs and of lung, pancreas, stomach, jejunum, adrenals, spleen, mesenteric lymph nodes and gastrocnemius muscle were examined)
Statistics:
All of the data were analyzed by the Analysis of Variance and partitioned by the Tukey minimum significant difference method.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis 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:
No adverse effects were observed on any of the parameters in soybean oil group (control) as mentioned above.
Key result
Dose descriptor:
NOAEL
Effect level:
19 other: % in diet (i.e. ca. 9,500 mg/kg bw/day)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects on any of the parameters recorded.
Key result
Critical effects observed:
no

 Fat Absorbability: High absorption was observed for soybean oil (95±1% - males; 98±1% - females).

Conclusions:
Under the conditions of the study, the NOAEL of the substance in rats was found to be 19% in diet.

Executive summary:

A study was conducted to determine the repeated dose toxicity of the constituent ‘glycerides, C16-18 and C18-unsatd.’ (as soybean oil) as control in a subchronic toxicity study through diet. Diet containing 7.5% of the substance (plus 11.5% soybean oil as normal fat source) was fed to 20 Sprague-Dawley rats/sex for 91 d. The control group was fed with 19% soybean oil for same duration. There was no indication of any systemic toxicity (including body weight gain, food consumption, organ weights, urinalysis, clinical chemistry, haematology, gross and histopathology) in the treated group. Hence, under the conditions of this study, the NOAEL of the substance in rats was found to be 19% in diet (Nolen, 1981).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
; only single dose level used; not all parameters examined
Principles of method if other than guideline:
Not applicable
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, MA
- Housing: The animals were housed in individual stainless steel wire-mesh cages
- Diet: Ad libitum
- Water: Ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23±1
- Humidity (%): 50±5%
- Photoperiod (h dark / h light): 12/12

Route of administration:
oral: feed
Details on route of administration:
0 and 7.5%
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Mixing appropriate amounts with (Type of food): Normal diet containing 11.5% soybean oil as fat source


Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Not applicable
Duration of treatment / exposure:
91 d
Frequency of treatment:
Daily ad libitum in food
Remarks:
Doses / Concentrations:
7.5%
Basis:
nominal in diet
No. of animals per sex per dose:
20 animals/sex/group
Control animals:
other: yes, 19% soybean oil
Details on study design:
- Rationale for animal assignment: Animals were distributed into groups of 20 rats per sex so that the litter mates were distributed evenly among the groups and the mean body weights did not vary more than 0.5 g.
Positive control:
Not applicable
Observations and examinations performed and frequency:
BODY WEIGHT: Yes
- Time schedule for examinations: Weekly


FOOD CONSUMPTION (if feeding study): Yes
- Time schedule for examinations: Weekly


HAEMATOLOGY: Yes
- Time schedule for collection of blood: At time of sacrifice (end of study)
- Anaesthetic used for blood collection: Yes
- Animals fasted: No data
- How many animals: 10 animals/sex/group
- Parameter examined: Standard hemograms done


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At time of sacrifice (end of study)
- Animals fasted: No data
- How many animals: 10 animals/sex/group
- Parameter examined: Serum cholesterol and phospholipids


URINALYSIS: Yes
- Time schedule for collection of urine: 24 h collection in 11th wk of study
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameter examined: Volume, nitrogen, ketones, glucose, bilirubin, albumin and pH

OTHER: During the 3rd and 11th wks, feces were collected from 10 animals/sex/group for fat absorption analysis.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
(The heart, liver, kidneys and gonads were removed and weighed. Sections of these organs and of lung, pancreas, stomach, jejunum, adrenals, spleen, mesenteric lymph nodes and gastrocnemius muscle were examined)

Other examinations:
None
Statistics:
All of the data were analyzed by the Analysis of Variance and partitioned by the Tukey minimum significant difference method.
Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
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:
There were no significant differences in growth and weight gain; however, food intake and caloric efficiency (kcal/g gain) was slightly increased in treatment group after 4 wks. The mean caloric efficiency of treatment group (18.9 and 26.9 kcal/g gain in males and females respectively) was significantly higher than that of control group (16.8 and 23.6 kcal/g gain in males and females respectively). This was attributed to the lower absorbability of the fully hydrogenated soybean oil. No effects on any other parameter as mentioned above.


Key result
Dose descriptor:
NOAEL
Effect level:
7.5 other: % in diet (i.e. ca. 3,750 mg/kg bw/day)
Sex:
male/female
Basis for effect level:
other: no treatment-related effects observed
Key result
Critical effects observed:
no

Fat Absorbability: Low absorption was observed for experimental fat i.e. fully hydrogenated soybean oil (17±8% - males; 17±7% - females). The total fat absorption was significantly lower in treatment group (64±4% - males; 68±3% - females) with respect to control group (95±1% - males; 98±1% - females).

Conclusions:
Under the conditions of this study, the NOAEL of the substance in rats was determined to be 7.5% in diet.
Executive summary:

A subchronic toxicity study was conducted in rats to investigate the effect of the consituent 'glycerides, C16-18' (as fully hydrogenated soybean oil) in diet. Diets containing 7.5% of the substance (plus 11.5% soybean oil as normal fat source) were fed to 20 Sprague-Dawley rats/sex for 91 d. A control group was fed with 19% soybean oil for same duration. There was no indication of any systemic toxicity (including body weight gain, organ weights, urinalysis, clinical chemistry, haematology, gross and histopathology). Only observable effect was slightly increased feed consumption (and thus increased caloric efficiency) in treatment group after 4 wks which was attributed to the lower absorbability of the substance. Hence, under the conditions of this study, the NOAEL of the substance in rats was determined to be 7.5% in diet (Nolen, 1981).

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1968
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
A 47 week repeated dose study was conducted in rats to compare the effects of the constituent coconut oil with various constituents of dietary fat (oleo oil, butter fat, corn oil and safflower oil). Body weight gain and food intake were measured during the course of the study. Fecal samples were also collected daily for fat absorption analysis. At specified intervals, blood was obtained for cholesterol determination. At termination of study various organs were weighed, and liver and intestine were examined histologically. The total lipid, phospholipids and cholesterol levels were also determined in liver.

GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Housing: Individual screen-bottom cages
- Diet: Normal diet differing in source of dietary fat; ad libitum
- Water: Ad libitum


ENVIRONMENTAL CONDITIONS
Air-conditioned animal rooms

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Mixing appropriate amounts with (Type of food): 18.5% of the substance along with other constituents of dietary fat (oleo oil, butterfat, corn oil, and safflower oil) were mixed with diet containing additional 2.5% safflower oil to ensure adequacy of the essential fatty acids in all diets.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Not applicable
Duration of treatment / exposure:
47 wks
Frequency of treatment:
Daily ad libitum in diet
Dose / conc.:
18.5 other: %
Remarks:
Basis: nominal in diet
No. of animals per sex per dose:
15 animals/sex/group
Control animals:
yes
Observations and examinations performed and frequency:
BODY WEIGHT: Body weight was determined during the study and body weight gains were compared at 4, 8 and 47 wks.


FOOD CONSUMPTION: Records of food intake were kept for the calculation of caloric efficiency and net absorption of fat.


CLINICAL CHEMISTRY: Only plasma cholesterol level was determined (at 7, 14, 21, 35 and 47 wks)


FECAL ANALYSIS: At intervals during the study, feces were collected daily, pooled in weekly samples, and analyzed for fat absorption.
Sacrifice and pathology:
At the termination of the study the rats were killed, and the weights of the livers, kidneys, spleen, heart, adrenals, femurs, testes and epididymal fat pads were determined. Liver and intestine were examined histologically. The total lipid, phospholipids and cholesterol levels were also determined in liver.
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
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Body weight and food intake: No effects on body weight gain and calorific efficiency with respect to other groups. The weight gain in the coconut oil group was in the upper range of the recorded values.

Mortality: Mortality was not markedly different with respect to other groups.

Organ weights and histopathology: No effect on various organ weights and histopathology of liver and intestine as compared to other groups.

Fat absorption: The net fat absorption, calculated from dietary intakes and fecal excretion was 96%.

Lipid levels: The plasma cholesterol and liver lipid, phospholipids and cholesterol level were not markedly different from other groups.
Key result
Dose descriptor:
NOAEL
Effect level:
18.5 other: % in diet (i.e. ca. 9,250 mg/kg bw/day)
Based on:
test mat.
Basis for effect level:
other: No effects on body weight gain, calorific efficiency, mortality, organ weights and histopathology of liver and intestine. The plasma cholesterol and liver lipid, phospholipids and cholesterol level were also not markedly different from other groups.
Key result
Critical effects observed:
no
Conclusions:
Under the test conditions, supplementation of 18.5% of the substance in diet for 47 weeks did not produce any significant adverse effects in rats as compared to other normal dietary fat sources.
Executive summary:

A chronic study was conducted in rats to compare the repeated dose toxicity of the constituent ‘glycerides, C8 -18 and C18 -unsatd.’ (as coconut oil) in rats according to an unspecified method. Various constituents of dietary fat (oleo oil, butter fat, corn oil and safflower oil) were included at 18.5% level in diets of 15 wistar rats/sex/group for 47 weeks. Additional 2.5% safflower oil was included to insure adequacy of the essential fatty acids in all diets. Body weight gain and food intake were measured during the course of the study. Fecal samples were also collected daily for fat absorption analysis. At specified intervals, blood was obtained for cholesterol determination. At termination of study various organs were weighed, and liver and intestine were examined histologically. The total lipid, phospholipids and cholesterol levels were also determined in liver. No effects were observed on body weight gains and calorific efficiency in the substance group with respect to other groups. Mortality was not markedly different with respect to other groups. No effects were observed on various organ weights and histopathology of liver and intestine. The plasma cholesterol and liver lipid, phospholipids and cholesterol level were not markedly different from other groups. Under the test conditions, supplementation of 18.5% of the substance in diet for 47 weeks did not produced any significant adverse effects in rats as compared to other normal dietary fat sources (Harkins, 1968).

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1967
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
A chronic study was conducted in mice to investigate the effect of feeding hydrogenated coconut oil on longevity, hepatic lipid peroxidation and hepatic fatty acid composition.
GLP compliance:
not specified
Limit test:
no
Species:
mouse
Strain:
other: LAFJ and C3H1HeJ
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Jackson Laboratory, Bar Harbor, Maine
- Age at study initiation: 4 wks
- Diet (ad libitum): Purina Laboratory Chow


Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): The sucrose and hydrogenated coconut oil were blended with the powdered diet (Purina Chow) in an electric mixer.
- Storage temperature of food: Under refrigeration

Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
Entire lifespan
Frequency of treatment:
Daily ad libitum in diet
Dose / conc.:
15 other: %
Remarks:
Basis:
nominal in diet
No. of animals per sex per dose:
60
Control animals:
other: 15% sucrose in diet
Observations and examinations performed and frequency:
BODY WEIGHT: Yes
- Time schedule for examinations: At monthly intervals

LONGEVITY: Yes
Sacrifice and pathology:
When the animals were sacrified 12-24 h before anticipated death, a portion of liver was extracted to determine hepatic peroxide values and hepatic fatty acid compositions.
Statistics:
The probabilities that differences in the data were due to chance were calculated by the t test.
Body weight and weight changes:
no effects observed
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
No difference in longevity, body weights, hepatic peroxide values and hepatic fatty acid compositions between the groups in both the strains.
Key result
Dose descriptor:
NOAEL
Effect level:
15 other: % in diet (i.e. ca. 15,000 - 30,000 mg/kg bw/day).
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No effect on longevity, body weight, hepatic peroxide values and hepatic fatty acid compositions
Key result
Critical effects observed:
no
Conclusions:
Under the test conditions, supplementation of 15% of the substance in diet did not alter lifespan, body weight and hepatic peroxide/fatty acid values of mice. Under the test conditions, the NOEAL was determined to be equivalent to 15% of the substance in the diet.
Executive summary:

A chronic study was conducted in LAFJ and C3H1HeJ mice to determine the repeated dose toxicity potential of the constituent ‘glycerides, C8-18 and C18-unsatd.’ (as fully hydrogenated coconut oil) on longevity, hepatic lipid peroxidation and hepatic fatty acid composition. 15% of the substance or sucrose was fed to 60 animals/strain/group for lifetime. Body weights were recorded at monthly intervals. When the animals were sacrificed 12 -24 h before the anticipated death, a portion of the liver was extracted to determine hepatic peroxide values and hepatic fatty acid compositions. No differences were observed in longevity, body weights, hepatic peroxide values and hepatic fatty acid compositions between the groups in both the strains. No information was provided about the general toxicological effects. Under the test conditions, the NOEAL was determined to be equivalent to 15% of the substance in the diet (Morin, 1967).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
not specified
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
other: F344 and Sprague-Dawley
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Kingston, New York
- Age at study initiation: 6 weeks
- Weight at study initiation: F344:84 to 104 grams; Sprague-Dawley: 132 to 166 grams
- Housing: Individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24
- Humidity (%): 40% to 70%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light

IN-LIFE DATES: Not reported
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Not reported
- Mixing appropriate amounts with Purina certified rodent chow, Purina mills.
- Storage temperature of food: Not reported
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Diet concentration verification was conducted on weeks 1, 5, 9, and 13. All concentrations were stated to be between 85% and 110% of the nominal concentration. Recovery from the diet at the 0.2% and 2.0% dose levels was 99 ± 4.2% and 96.5 ± 1.5%, respectively. There were also periodic measurements for stability and homogeneity.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Continuous in feed
Remarks:
Doses: 0, 2000, 20,000 ppm (0, 0.2, 2% respectively)
Basis: nominal in diet
Remarks:
Equivalent doses: 0, 161, 1582 mg/kg bw/day for F344 rats and 0, 158, 1624 mg/kg bw/day for SD rats
Basis: nominal in diet
No. of animals per sex per dose:
Fifteen animals per treatment group, an additional 10 F344 rats in the control and high-dose group were sacrificed on day 30 or 61.
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Doses were selected based on previous publications.
- Rationale for animal assignment (if not random): Not reported
- Rationale for selecting satellite groups: Not reported
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observation details were not provided.

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at scheduled sacrifice
- Anaesthetic used for blood collection: Yes (methoxyflurane)
- Animals fasted: Yes
- How many animals: Ten per group
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at scheduled sacrifice
- Animals fasted: Yes
- How many animals:Ten per group
- Parameters checked in table 2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes, although it is stated that an extensive list of tissues were preserved, it states that only the liver, mesenteric lymph nodes, and gross lesions were processed for histology.
Other examinations:
The heart, liver, kidneys, ovaries, and spleen were weighed.
Statistics:
For parametric data, a one way analysis of variance with F distribution followed by a Dunnett's test were used. A standard linear regression analysis was also performed. For nonparametric data, a Kruskal-Wallis test followed by Dunn's summed rank test were used. A Jonckheere's test for monotonic trends was used to test for a dose response.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs in either rat strain at any dose level during the study.
Mortality:
no mortality observed
Description (incidence):
There were no effects on mortality.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Apart from a transient decrease in body weight for one day at one dose level in one strain of rat, there were no effects on growth rate.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no effects on food consumption. There were no data on the compound intake.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Description (incidence and severity):
There were no treatment-related effects on ophthalmology.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Hematological effects were only noted in F344 rats and included an increase in white cell count. At day 30 this was observed in the 2.0% group, but by 90 days was seen in both dose groups in a dose-related manner with a significant increase observed in the high-dose group (21.1%, 24.5%, and 36.1% in control, 0.2% and 2.0% groups).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
There were also marginal differences in some serum chemistry values of F 344 rats compared to controls, but these changes were not considered clinically significant.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute and relative liver weights of the F344 rats in the 2% group were increased by 1.2- and 1.3-fold respectively and in the 0.2% group both absolute and relative liver weights were increased 1.1-fold. The mesenteric lymph nodes (absolute and relative) were increased in the 2% group by 1.4 to 3.7 fold depending on the time of sacrifice. There were no changes in organ weight in the Sprague-Dawley rat.
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological changes in the livers of the treated F344 rats consisted of the occurrence of an increased incidence of microgranuloma. These were not present at day 30, but were present in more than 80% of the animals in the 2% dose group after 61 days. By 90 days microgranulomas were also observed in the 0.2% dose group (incidence not stated in the publication). Central necrosis and/or the presence of Langhan's type cells were seen in occasional microgranuloma. The incidence and severity were both dose and temporally-related as the appearance of granuloma occurred at 61 days in the high-dose group and was present in both dose groups at 92 days. Histiocytosis occurred in the mesenteric lymph nodes of the F 344 rats in the 2% dose group after 30 days. Microgranulomas were observed at 61 and 92 days in the 2% and also in the 0.2% groups at 92 days. The incidence and severity of the microgranulomas were increased in a dose related manner. Histopathological examination of the livers of the Sprague Dawley rats revealed that in contrast to the F 344 rats there were no microgranulomas in either dose group, but there was a slightly increased incidence of minimal multifocal chronic inflammation in the 2% dose group. No histological changes were observed in the mesenteric lymph node of the Sprague Dawley rats at either dose level.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
There was a dose and strain related increase in mineral hydrocarbon in the liver and mesenteric lymph nodes.
Key result
Dose descriptor:
NOAEL
Remarks:
Sprague-Dawley rats
Effect level:
ca. 2 other: % in diet (i.e., ca. 1600 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: overall effects
Key result
Dose descriptor:
LOAEL
Remarks:
F344 rats
Effect level:
ca. 0.2 other: % in diet (i.e., ca.160 mg/kg bw/day)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
not specified
Conclusions:
Under the study conditions, the NOAEL in SD female rats and LOAEL in F344 female rats were determined to be ≥1624 mg/kg bw/day and 161 mg/kg bw/day, respectively.

Executive summary:

A subchronic dietary toxicity study was conducted to determine the repeated dose toxicity of food-grade white oil in female Fischer-344 (F-344) and Sprague-Dawley-derived (CRL:CD) rats. In a study, groups of 15 female rats of two strains (F344 and Sprague-Dawley) were fed diets containing 0, 2000 or 20,000 (0, 0.2 or 2.0% respectively) P15(H) white oil. Ten animals were sacrificed after 90 days for toxicological evaluation and the remaining 5 rats were sacrificed after 91 days for a tissue analysis for mineral hydrocarbon content. In addition 20 extra female F-344 rats were fed diet containing 0 or 2% white oil. Ten of these animals were sacrificed after 30 days treatment and the remaining ten animals after 61 days treatment. These extra animals were included to follow the time course of the development of any lesions that developed. All animals were observed daily and body weights and food consumption were recorded weekly. At each scheduled sacrifice blood samples were taken for a wide range of haematological and serum chemistry determinations. There were no clinical signs in either rat strain at any dose level during the study. There were no effects on mortality. Apart from a transient decrease in body weight for one day at one dose level in one strain of rat, there were no effects on growth rate. There were no effects on food consumption. There were no data on the compound intake. Hematological effects were only noted in F344 rats and included an increase in white cell count. At day 30 this was observed in the 2.0% group, but by 90 days was seen in both dose groups in a dose-related manner with a significant increase observed in the high-dose group (21.1%, 24.5%, and 36.1% in control, 0.2% and 2.0% groups). There were also marginal differences in some serum chemistry values of F 344 rats compared to controls, but these changes were not considered clinically significant. The absolute and relative liver weights of the F344 rats in the 2% group were increased by 1.2- and 1.3-fold respectively and in the 0.2% group both absolute and relative liver weights were increased 1.1-fold. The mesenteric lymph nodes (absolute and relative) were increased in the 2% group by 1.4 to 3.7 fold depending on the time of sacrifice. There were no changes in organ weight in the Sprague-Dawley rat. Histopathological changes in the livers of the treated F344 rats consisted of the occurrence of an increased incidence of microgranuloma. These were not present at day 30, but were present in more than 80% of the animals in the 2% dose group after 61 days. By 90 days microgranulomas were also observed in the 0.2% dose group (incidence not stated in the publication). Central necrosis and/or the presence of Langhan's type cells were seen in occasional microgranuloma. The incidence and severity were both dose and temporally-related as the appearance of granuloma occurred at 61 days in the high-dose group and was present in both dose groups at 92 days. Histiocytosis occurred in the mesenteric lymph nodes of the F 344 rats in the 2% dose group after 30 days. Microgranulomas were observed at 61 and 92 days in the 2% and also in the 0.2% groups at 92 days. The incidence and severity of the microgranulomas were increased in a dose related manner. Histopathological examination of the livers of the Sprague Dawley rats revealed that in contrast to the F 344 rats there were no microgranulomas in either dose group, but there was a slightly increased incidence of minimal multifocal chronic inflammation in the 2% dose group. No histological changes were observed in the mesenteric lymph node of the Sprague Dawley rats at either dose level. There was a dose and strain related increase in mineral hydrocarbon in the liver and mesenteric lymph nodes. White mineral oil was more toxic to F344 rats than to Sprague-Dawley rats. There was no NOAEL in the F344 rats and the LOAEL was 2000 ppm (equivalent to 161 mg/kg bw/day), based on the histopathology in the liver and mesenteric lymph nodes accompanied by changes in the weight of those organs. The NOAEL in the Sprague-Dawley rat was greater than or equal to 20,000 ppm (equivalent to 1624 mg/kg bw/day), based on the lack of any toxic effects at the highest dose. Under the study conditions, the NOAEL in SD rats and LOAEL in F344 rats were determined to be 1624 mg/kg bw/day and 161 mg/kg bw/day, respectively (Firriolo, 1995).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc., Indianapolis, IN or Harlan Olac, Ltd, UK.
- Age at study initiation: approximately 4 weeks
- Weight at study initiation: Not reported
- Fasting period before study: Not reported
- Housing: 5 rats of the same sex per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 24 degrees Celsius
- Humidity (%): 45% to 70%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 hour dark/ 12 hours light.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Prepared by serial dilution from a 10% (100,000 ppm) premix

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Storage temperature of food: room temperature

VEHICLE
- No vehicle used
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical concentrations were determined as described by Walters et al., 1994. The detection limit by this method was 1 microgram/gram (0.0001% or 1 ppm) in the diet, and the control diet was found to contain around 0.003% (w/w, 30 ppm) background
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:0, 20, 200, 2000, 20,000 ppm
Basis: nominal in diet
Remarks:
Doses / Concentrations:0, 1.7, 17, 173, 1815 mg/kg bw/day (males) and 0, 2, 19, 190, 1951 mg/kg bw/day (females)
Basis: actual ingested
No. of animals per sex per dose:
Main group: 20 rats/sex/dose for treated group, 60 rats/sex/dose for untreated control group
Reversal group: 10 rats/sex for treated groups, 20 rats/sex for untreated control group at high dose only
Control animals:
yes, concurrent no treatment
Details on study design:
The reversal group was added at the high dose only (20,000) to assess whether or not effects observed at 90 days would reverse following 28 days without treatment. The tissue level and tissue level reversal group were satellite groups (5 rats/sex) added for untreated controls and the high dose only to evaluate whether or not nonpolar hydrocarbons were accumulated in selected tissues.
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: at pretest, twice daily during treatment and at necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during the week preceding treatment and the during the last week of treatment.
- Dose groups that were examined: high dose and untreated controls

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: all animals.
- Parameters checked: mean cell volume, total erythrocyte count, hematocrit, hemoglobin concentration, differential leukocyte count, total leukocyte count, platelet count, prothrobin time, and reticulocyte count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Not reported
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: albumin, albumin/globulin ratio, total bilirubin, creatinine, electrolytes (calcium, chloride, phosphorus as phosphate, potassium, sodium), enzyme activity (alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, gamma glutamyl transferase), glucose, total protein, and urea.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: Plasma was analyzed for vitamin E
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Organ Weights: Adrenal glands, brain, cecum, heart, kidneys, liver, ovaries/testes, spleen, and thymus were weighed. Mesenteric lymph nodes were weighed only in the tissue level and tissue level reversal groups. A complete and representative set of tissue samples was retained in 10% neutral-buffered formalin from all rats in the main and reversal groups.
Statistics:
Average data are presented as mean +/- the standard error of the mean (SEM). Also continuous variable data from untreated control and treated groups were tested for normality using the Kolmogorov-Smirnov (KS) test )p<0.05) and homogencity of variance using Bartlett's test (p<0.01). The Wilcoxin Mann-Whitney test was used if transformed data produced a nonsignificant Bartlett's test but a significant KS test. If the converse was observed, an appropriate t-test was used based on whether a pooled variance was suitable or not. If no suitable transformation could be made, one of the preceding tests was selected as the most appropriate based on the nature and distribution of the data. In all test comparisons, a probability of p S 0.05 in a 2-sided test was considered to be statistically significant. Individual animal data identified as statistical outliers were removed from the analysis. Data from the histopathological examination were tested for an increase in incidence between treated and untreated control animals using Fisher’s exact test. A probability level of p S 0.05 in a 1-sided test was considered to be statistically significant.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No adverse effects of treatment were observed on the physical appearance or behaviour of the rats during the study
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Overall body weight gain was not affected by treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
At the highest dose both male and female food consumption tended to show small but statistically significant increase. Generally the increased food consumption did not exceed control values by more than 10% and disappeared during the reversal period. The incremental food consumption was assumed to reflect the caloric loss for bulk substitution of mineral hydrocarbons for feed in the test diets.
Food efficiency:
not examined
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
Description (incidence and severity):
Dose-related and statistically significant clinical chemistry data for enzyme activity showed at doses 2000 ppm and 20000 ppm.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Adrenals, brain, cecum full and empty, heart, ovaries, testes and thymus were not affected. Statistically significant increases of organ weights were observed for kidney, liver, mesenteric lymph node, and spleen. Generally, liver weights were significantly increased at doses 2,000 ppm and higher for lower viscosity oils (N10A, N15H, and P15H). The increase in liver weight tended to occur in female groups than in males. Mesenteric lymph node weights were increased more tan 2.5 fold in rats treated with 20,000 ppm N10A,N15H, P15H, LMPW, MP, and IMPW. There was little report of reversal of the weight increase after 28 days and there was a slight increase for some treatment groups. Spleen and kidney weights were significantly higher than controls for females and males treated with 20,000 ppm (N10A, N15H, P15H, N70A, N70H, (and P70H, LMPW, MP and IMPW spleen only). The response in males was less than in females.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The effects were qualitatively similar to one another but differed in magnitude depending on the nature of the test material. The effects were dose-related, were of greater magnitude in females than in males, and were greater with the lower molecular weight test materials. Macrophage accumulation appeared to progress to histiocytosis in mesenteric lymph nodes and microgranuloma formation in liver.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Other: - A statistically significant increase in white blood cells was observed at high doses in some female treatment groups. An increase of about 20-25% for total white blood count occurred in females treated with 20,000 ppm N10A, N15H, P15H, MP and HSW and with 2000 ppm LMPW. A larger increase was observed for 20,000 ppm LMPW, a 48% increase over untreated controls.
Vitamin E: After the 28-day reversal, all serum vitamin E concentrations were not significantly different from untreated controls for all treatment groups except males treated with N10A, and N70H, where values remained decreased.
Key result
Dose descriptor:
NOAEL
Remarks:
P100H (C28-45)
Effect level:
>= 20 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Absence of treatment related effects
Remarks on result:
other: Equivalent to ≥1900 mg/kg bw/day
Key result
Dose descriptor:
NOEL
Remarks:
N10A (C15 -30), P15H (C18-30), N70A (C21-35), N70H (C22-37)
Effect level:
ca. 20 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: minimal histiocytosis at 200 ppm and above; minimal histopathological changes in mesenteric lymph nodes
Remarks on result:
other: Equivalent to 2 mg/kg bw/day
Key result
Dose descriptor:
LOEL
Remarks:
N15H C(17-30)
Effect level:
ca. 20 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: minimal histiocytosis at all dose levels; minimal histopathological changes in mesenteric lymph nodes.
Remarks on result:
other: Equivalent to 2 mg/kg bw/day
Critical effects observed:
not specified
Conclusions:
Under the study conditions, the NOEL values of 2 mg/kg bw/day (N10A, P15H, N70A, N70H), ≥1900 mg/kg bw/day (P100H) and LOEL value of 2 mg/kg bw/day (for N15H) were determined.
Executive summary:

A subchronic dietary toxicity study was conducted to determine the repeated dose toxicity of highly refined white mineral oils in rats, according to equivalent or similar to OECD Guideline 408. In a study, six highly refined base oils (P100H (C28-45), N10A (C15-30), P15H (C18-30), N70A (C21-35), N70H (C22-37), N15H (C17-30)) were administered [20 rats/sex/dose for treated group (main group), 60 rats/sex/dose for untreated control group and a reversal group (10 rats/sex for treated groups and 20 rats/sex for untreated group)] to male and female F-344 rats at dose levels 0, 20, 200, 2,000, and 20,000 ppm for 90 days [i.e., ca. 0, 1.7, 17, 173, 1815 mg/kg bw/day (males) and 0, 2, 19, 190, 1951 mg/kg bw/day (females)]. No adverse effects of treatment were observed on the physical appearance or behaviour of the rats during the study. Overall body weight gain was not affected by treatment. At the highest dose both male and female food consumption tended to show small but statistically significant increase. Generally the increased food consumption did not exceed control values by more than 10% and disappeared during the reversal period. The incremental food consumption was assumed to reflect the caloric loss for bulk substitution of mineral hydrocarbons for feed in the test diets. Dose-related and statistically significant clinical chemistry data for enzyme activity showed at doses 2000 ppm and 20000 ppm. Adrenals, brain, cecum full and empty, heart, ovaries, testes and thymus were not affected. Statistically significant increases of organ weights were observed for kidney, liver, mesenteric lymph node, and spleen. Generally, liver weights were significantly increased at doses 2,000 ppm and higher for lower viscosity oils (N10A, N15H, and P15H). The increase in liver weight tended to occur in female groups than in males. Mesenteric lymph node weights were increased more tan 2.5 fold in rats treated with 20,000 ppm N10A, N15H, P15H. There was little report of reversal of the weight increase after 28 days and there was a slight increase for some treatment groups. Spleen and kidney weights were significantly higher than controls for females and males treated with 20,000 ppm (N10A, N15H, P15H, N70A). The response in males was less than in females. The effects were qualitatively similar to one another but differed in magnitude depending on the nature of the test material. The effects were dose-related, were of greater magnitude in females than in males, and were greater with the lower molecular weight test materials. Macrophage accumulation appeared to progress to histiocytosis in mesenteric lymph nodes and microgranuloma formation in liver. A statistically significant increase in white blood cells was observed at high doses in some female treatment groups. An increase of about 20-25% for total white blood count occurred in females treated with 20,000 ppm N10A, N15H, P15H. After the 28-day reversal, all serum vitamin E concentrations were not significantly different from untreated controls for all treatment groups except males treated with N10A, and N70H, where values remained decreased. High-dose dietary exposure produced a syndrome of effects in liver and mesenteric lymph nodes with response greater in females than in male F-344 rats. Effects include increased organ weights, evidence for the presence of nonpolar hydrocarbons, and other observations suggestive of an inflammatory response. A NOEL of 20 ppm (i.e., ca. 2 mg/kg bw/day) for 4 of 6 oils (N10A (C15 -30), P15H (C18-30), N70A (C21-35), N70H (C22-37) was calculated, a LOEL of 20 ppm (i.e., ca. 2 mg/kg bw/day) was established for N15H (C17-30); and a NOEL of ≥20,000 ppm (i.e., ca. ≥1900 mg/kg bw/day) was calculated for P100H (C28 -45); based on histiocytosis. Under the study conditions, the NOEL values of 2 mg/kg bw/day (N10A, P15H, N70A, N70H), ≥1900 mg/kg bw/day (P100H) and LOEL value of 2 mg/kg bw/day (for N15H) were determined (Smith, 1996).

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Deviations:
not specified
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: CDF(F-344)/CrlBR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina
- Age at study initiation: Approximately 6 weeks old
- Weight at study initiation: Not reported
- Housing: Individually housed in suspended stainless steel cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24
- Humidity (%): 40% to 70%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light

IN-LIFE DATES: Not reported
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Certified rodent diet #5002 (meal)
- Storage temperature of food: Not reported
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
24 months
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations: 0, 60, 120, 240, or 1200 mg/kg bw/day
Basis: nominal in diet
No. of animals per sex per dose:
Fifty animals per sex per dose were used in the main study, twenty animals per sex per dose were used in the recovery groups, and twenty-five to forty-seven females per dose group were used for mineral hydrocarbon analysis and sacrificed at different times
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Not reported
- Rationale for animal assignment (if not random): Not reported
- Rationale for selecting satellite groups: Not reported
- Post-exposure recovery period in satellite groups: 12 months
- Section schedule rationale (if not random): Not reported
Observations and examinations performed and frequency:
Treatment of livers: At the end of the experiment the animals were killed by chloroform and their livers removed as free from blood as possible.
Treatment of faeces: The faeces were stored in alcohol.
Other examinations:
The faeces were collected throughout the experiment at weekly intervals and stored in alcohol.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on clinical signs or mortality.
Mortality:
no mortality observed
Description (incidence):
There were no treatment-related effects on clinical signs or mortality.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight was higher in the high-dose group compared to the controls, but this was a result of increased food consumption.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There were no toxicologically significant effects on food consumption, but the high-dose group consumed more food than the controls. Compound intake was not reported.
Food efficiency:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on food efficiency.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on ophthalmology.
Haematological findings:
no effects observed
Description (incidence and severity):
Although there were sporadic differences that were statistically significant, they were not consistent over time, or dose-dependent.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Although there were sporadic differences that were statistically significant, they were not consistent over time, or dose-dependent.
Urinalysis findings:
no effects observed
Description (incidence and severity):
Although there were sporadic differences that were statistically significant, they were not consistent over time, or dose-dependent
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males in the 1200 mg/kg/day group had increased mesenteric lymph node weights (absolute, relative to body weight and relative to brain weight). This effect was observed at 12 and at 24 months but did not occur in the recovery group animals. In females absolute mesenteric lymph node weights were increased in all dose groups (13, 15, 20 & 20% for the 60, 120, 240 & 1200 mg/kg/day groups, respectively). Increases in mesenteric lymph node/body weight and mesenteric lymph node/brain weight ratios were also slightly increased in all female dose groups at 24 months. There was also a significant increase in absolute mesenteric lymph node weights for 240 (55%) and 1200 mg/kg/day females (36%) in the recovery study.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on gross pathology.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
After 12 months of treatment there were no treatment-related histopathological findings in the liver at any dose level. At 24 months, however, there was an increased incidence of angiectasis and cystic degeneration in the males at all dose levels. Similar findings were also observed in control groups and treated females, but at a very low incidence. Although the incidence of these liver lesions was increased in the males, it was not in a dose-related manner. It was also noted by the authors of the report that this lesion is a common finding in F-344 rats, particularly in males. The authors therefore considered these findings to be of questionable relevance, especially in view of the minimal severity and lack of dose response. This lesion was not observed in the recovery group animals. Histiocytosis was observed in the mesenteric lymph nodes at all dose levels and also in the controls at 24 months, but the severity was slightly greater in the treated animals than the controls. It appeared that the increase in severity above controls was dose-related in the males, but not in the females. For the males, on a scale of 1 to 4 for increasing severity, the responses were 1.2, 1.6, 1.8, 2.3 and 2.1 for the 0. 60, 120, 240 and 1200 mg/kg/day groups, respectively. In the females the control value was 1.6 and all other dose groups ranged from 2.4 to 2.6 but not in a dose-related fashion.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
There was no increase in neoplastic findings.
Other effects:
no effects observed
Description (incidence and severity):
The level of mineral hydrocarbons in the liver was significantly increased at all doses, but there was no dose-response and levels were not significantly increased after the 12-month recovery period.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Critical effects observed:
not specified
Conclusions:
Under the study conditions, the NOAEL for these studies was considered to be 1,200 mg/kg bw/day.
Executive summary:

Two-year dietary studies were conducted to determine the chronic toxicity and the carcinogenicity of P70(H) and P100(H) white mineral oils in Fischer-344 rats (F-344), according to the OECD Guideline 453, in compliance with GLP. Additional endpoints evaluated were: (1) extent of mineral hydrocarbon deposition in liver, kidneys, mesenteric lymph nodes, and spleen of female rats at 3, 6, 12, 18 and 24 months, and (2) reversibility of effects following cessation of exposure. Dietary concentration of P70(H) and P100(H) were 60, 120, 240, and 1,200 mg/kg bw/day. No treatment-related mortality, neoplastic lesions, or changes in clinical health, hematology, serum chemistry, or urine chemistry were evident in any group administered either white oil. Statistically significant higher food consumption was noted in the 1,200 mg/kg group males and females exposed to either white oil and statistically significant higher body weights were noted in the 1,200-mg/kg males during the latter portion of the P100(H) study. Higher mesenteric lymph node weights were accompanied by increased severity of infiltrating histiocytes. This occurred to a greater extent with the P70(H) than the P100(H) oil. No other histopathology of significance was observed. Mineral hydrocarbons were detected in the liver following exposure to either oil. Maximal concentrations of mineral hydrocarbons in the liver were similar with both oils but occurred more rapidly with the P70(H) oil. Liver mineral hydrocarbon content returned to near-background levels during the reversibility phase. In conclusion, lifetime exposer of F344 rats to P70(H) (C27-34) and P100(H) C(28 -45) white oils resulted in only minimal findings and with no consequence to clinical health. Under the study conditions, the NOAEL for these studies was considered to be 1,200 mg/kg bw/day (Trimmer, 2004).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1926
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
The general plan of the experiment was to feed two groups of rats on a complete artificial diet, while one group received in addition a small quantity of squalene each day for 42 days
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
not specified
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Diet: complete artificial diet that described by Drummond and Coward [1920].
Route of administration:
other: dropped directly into their mouths from a micro-burette
Vehicle:
not specified
Duration of treatment / exposure:
dropped directly into their mouths from a micro-burette
Frequency of treatment:
Daily
Remarks:
660 mg actual ingested
No. of animals per sex per dose:
Test group: 10 rats
Negative control: 9 rats
Control animals:
yes, concurrent no treatment
Observations and examinations performed and frequency:
Treatment of livers: At the end of the experiment the animals were killed by chloroform and their livers removed as free from blood as possible.
Treatment of faeces: The faeces were stored in alcohol.
Other examinations:
The faeces were collected throughout the experiment at weekly intervals and stored in alcohol.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
It is clear from the weight of unsaponifiable matter from the faeces of the latter animals, and from its iodine value, that much of the squalene administered has been excreted.
Key result
Dose descriptor:
NOAEL
Effect level:
> 3 300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: body weight; amounts of cholesterol
Remarks on result:
other: considering 200 g bw and 660 mg per animal dose
Critical effects observed:
not specified

It was assumed that that fraction of unsaponifiable matter which was not squalene had an iodine value which was the same as that of the material from the control group, namely 80.4%, then it may be calculated from the iodine value of the whole of this unsaponifiable matter (316), that the amount of squalene present was 81.1 %, i.e. 16.79 g (squalene has iodine value 371). This figure could only be approximate, but some rough check on it was obtained from the fact that, if the weight of material not sterol in the faeces of the control group substracted from the corresponding figure for the faeces of the animals receiving squalene, it may arrive at the figure 17.60 g. These figures 16.79 and 17.60 g can only be approximate, but they would seem to show that about 17 g of the 27.7 g of squalene administered had been excreted. Thus about 10 g (approximately 38.6%) had been absorbed during the course of 42 days and this absorption had resulted in a marked increase in the weight of unsaponifiable matter in the livers and also in the bodies of these animals, namely, from 0.3680 to 1.0774 g in the case of the livers and from 1.8620 to 2.9114 g in that of the bodies. There was also a large increase in the amounts of cholesterol, from 0.2576 to 0.6189 g in the livers and from 1.3000 to 2.1620 g in the bodies.

No obvious ill effects followed the administration of squalene and post mortem examination showed that the organs of the animals were very healthy. There appeared to be a greater deposition of fat in the animals which received squalene and on the whole they seemed to grow at a somewhat greater rate than those of the control groups, possibly because the laxative action of the squalene caused a greater food consumption.

Conclusions:
Under the study conditions, the NOAEL of squalene was determined to be 3300 mg/kg bw/day in rats.
Executive summary:

A study was conducted to determine effect of co-administration of squalene in subchronic repeated dose study by oral administration. The general plan of the experiment was to feed two groups of rats on a complete artificial diet, while one group received in addition a small quantity of squalene each day. Immediately before feeding-time the animals were given approximately 660 mg of squalene dropped directly into their mouths from a micro-burette. Records were taken of the amount of squalene given each day. The faeces were collected throughout the experiment at weekly intervals and stored in alcohol. At the end of the experiment the animals were killed by chloroform and their livers removed as free from blood as possible. No obvious ill effects followed the administration of squalene and post mortem examination showed that the organs of the animals were very healthy. There appeared to be a greater deposition of fat in the animals which received squalene and on the whole they seemed to grow at a somewhat greater rate than those of the control groups, possibly because the laxative action of the squalene caused a greater food consumption. The experiment showed that when squalene was administered to the rat, it was in part absorbed and as a result of absorption there was a marked increase in the amounts of unsaponifiable matter and of cholesterol in the body and liver of the animal. No effects up to dose of 3300 mg/kg bw/day (considering 200g average animal body weight and 660 mg per animal dose) squalene in any animals were reported. Under the study conditions, the NOAEL of squalene was determined to be 3300 mg/kg bw/day in rats (Channon, 1926).

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2003
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Reason / purpose for cross-reference:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Diet containing 5% of the constituent β–sitosterol from tall oil and from cottonseed oil (equivalent to 2,571 mg/kg bw/day) was fed to 10 rats each for periods of 8, 18 and 22 months respectively and observed for toxicological effects.
GLP compliance:
not specified
Species:
rat
Strain:
not specified
Sex:
not specified
Details on test animals or test system and environmental conditions:
No data
Route of administration:
oral: feed
Vehicle:
not specified
Details on oral exposure:
No data
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data
Duration of treatment / exposure:
8, 18 and 22 months
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
5% (approx. equivalent to 2,571 mg/kg bw/day assuming 18 g as default food consumption/day; 0.35 kg as average adult rat body weight)
Basis:
nominal in diet
No. of animals per sex per dose:
10
Details on study design:
No data
Positive control:
No data
Observations and examinations performed and frequency:
BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes

CLINICAL CHEMISTRY: Yes


Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
None
Statistics:
No data
Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Details on results:
BODY WEIGHT AND WEIGHT GAIN: There was no detectable alteration in growth.


HAEMATOLOGY: There was no detectable alteration in blood cell counts

CLINICAL CHEMISTRY: There was no detectable alteration in blood urea nitrogen and serum proteins.


GROSS PATHOLOGY & HISTOPATHOLOGY: NON-NEOPLASTIC: There was no detectable alteration in gross or microscopic appearance of any organ or tissue
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 5 other: % in diet (i.e., ca. 2571 mg/kg bw/day)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: no treatment-related effects
Key result
Critical effects observed:
no

An additional repeated dose oral chronic toxicity study of Shipley et al., 1958 cited in the Scientific Committee on Food (SCF) opinion, indicates similar results:

Study details: A total number of 11 dogs were fed a diet with “β-sitosterols” at doses of 500 and 1,000 mg/kg bw for periods of 8 and 22 months. Body weight, haematological parameters, serum composition and the results of gross and microscopic pathological examination were not significantly different from those of two control dogs.

Conclusions:
Under the conditions of the study, the NOAEL of the substance in rats cwas determined to be 5% (approx. equivalent to 2,571 mg/kg bw/day) in diet.
Executive summary:

An oral chronic toxicity study was conducted in rats to investigate the dietary effects of the constituent β-sitosterol. Groups of 10 rats each were fed a diet with 5% of the substance (approx. equivalent to 2,571 mg/kg bw/day assuming 18 g as default food consumption/day; 0.35 kg as average adult rat body weight) from tall oil and from cottonseed oil for periods of 8, 18 and 22 months, respectively. There was no indication of any systemic toxicity (including body weight gain, hematology, clinical chemistry, gross and histopathology). Under the conditions of the study, the NOAEL of the substance in rats was determined to be 5% (approx. equivalent to 2,571 mg/kg bw/day) in diet based on lack of effects in the parameters observed (SCF, 2003).

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
(no. of animals/dose should be 10 while 3 were used in the study, not all parameters examined)
GLP compliance:
yes
Remarks:
Lab name not reported in the ANZFA risk analysis report
Species:
rat
Strain:
other: Alpk:APfSD
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Zeneca Pharmaceuticals, Alderley Park
- Acclimation period: 1 to 2 weeks

Route of administration:
oral: feed
Vehicle:
not specified
Details on oral exposure:
No data
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0.16, 1.57, 3.19 and 8.09% sterols w/w (equivalent to 77, 781, 1551 and 3910 mg sterol/kg bw/day in males, and 0, 87, 865, 1770 and 4204 mg sterol/kg bw/day in females)
Basis:
nominal in diet
No. of animals per sex per dose:
3/sex/dose
Control animals:
yes, concurrent no treatment
Details on study design:
No data
Positive control:
No data
Observations and examinations performed and frequency:
CLINICAL CONDITION AND BEHAVIOUR: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Before feeding (Day 1) and then weekly


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Not reported
- Food consumption: Determined weekly

FOOD EFFICIENCY:
- Food utilisation expressed as increase in bodyweight/100g food consumed for each animal

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before initiation of study and at Week 13
- Dose groups that were examined: All animals at study initiation and control and high dose groups at Week 13

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At study termination


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At study termination


OTHER: Serum 5’-nucleotidase was also determined at study termination.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Organ weights were also recorded.
Other examinations:
None
Statistics:
- Data were analysed by appropriate statistical techniques.
- 2-tailed student t-test was used to compare platelet counts in males treated at 5% sterols with controls.
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):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
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:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY: Three male animals from the 0.1% dosage group were sacrificed in extremis as a consequence of damage to the snout unrelated to the treatment regime. No deaths were associated with test material treatment. There were significant clinical observations with sterol treatment observed at Week 14 in male rats.


BODY WEIGHT AND WEIGHT GAIN: Bodyweights of either sex did not differ between dose levels.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No treatment related adverse effects on food consumption and food utilization was observed.
Treated male rats had group mean food consumption higher than in controls but this was toxicologically insignificant.

FOOD EFFICIENCY: No differences in food utilisation.

HAEMATOLOGY:
- Depression of mean platelet counts in females at all sterol diet levels and in males at 1 and 2% dietary sterols.
- Blood cell counts were slightly but statistically significantly increased in males at 5% sterol in diet (total white blood cell, neutrophil and lymphocyte count) and decreased at 2 and 5% (eosinophil count).
- In females, at all doses there was significant decrease in prothrombin time and in males at 2 and 5% activated partial prothrombin time was higher.

CLINICAL CHEMISTRY: The following significant changes were observed:
Increased plasma albumin (in females at 5% sterols)
Increased LDL (dose related in males but significant at 2 and 5%)
Increased cholesterol and HDL (in males and females at 1 and 2% but not 5%)
Increased alkaline phosphatase (in males at 5% and females at 1, 2 and 5%)
Increased alanine aminotransferase (in males and females at 2 and 5% and in males at 1%)
Increased aspartate aminotransferase, creatine kinase and lactate dehydrogenase (in females at 5%)
Increased phosphorus (in males at 2% but not 5%)
Increased magnesium (in females at 2 and 5%).

The extent of these haematological and clinical chemistry changes, although in general statistically significant, were considered of little biological or toxicological importance.

ORGAN WEIGHTS: No effects were observed

GROSS PATHOLOGY: No effects were observed

HISTOPATHOLOGY: NON-NEOPLASTIC: No effects were observed


Key result
Dose descriptor:
NOEL
Effect level:
3 911 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no treatment-related effects
Key result
Dose descriptor:
NOEL
Effect level:
4 204 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no treatment-related effects
Key result
Critical effects observed:
no

None

Conclusions:
Under the conditions of this study, the NOEL of the substance in rat was determined to be 5.0% (equivalent to 3911 and 4204 mg sterol/kg bw/day in males and females respectively) in diet based on lack of effects at the highest tested dose.
Executive summary:

A repeated dose subchronic oral toxicity study was conducted in rats to investigate the dietary effects of the constituent plant sterols. The method followed in the study was equivalent to OECD Guideline No. 408. Diets containing 0, 0.1, 1, 2, 5% of the substance (equivalent to 0, 77, 781, 1551 and 3910 mg sterol/kg bw/day in males, and 0, 87, 865, 1770 and 4204 mg sterol/kg bw/day in females) were fed to 3 Alpk:APfSD rats/sex/dose for 91 d. There was no indication of any systemic toxicity (including body weight gain, feed consumption, hematology, clinical chemistry, organ weights, gross and histopathology). Under the conditions of this study, the NOEL of the substance in rat can be concluded as 5.0% (equivalent to 3911 and 4204 mg sterol/kg/day in males and females respectively) in diet based on lack of effects at the highest tested dose (ANZFA, 2001 (1)).

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
A 14 day repeated dose subacute oral toxicity study was conducted in rats to determine the dietary effects of plant sterols at 1, 2 and 5 % w/w (approx. equivalent to 514, 1,028 and 2,571 mg/kg bw/day assuming 18 g as default food consumption/day; 0.35kg as average adult rat body weight). Mortality, clinical signs, body weight gain, water and food intake were measured during the course of the study. No post mortem evaluations were performed at termination of the study.
GLP compliance:
yes
Remarks:
Lab name not reported in ANZFA risk analysis report
Species:
rat
Strain:
other: Alpk:APfSD
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Zeneca Pharmaceuticals, Alderley Park
- Acclimation period: 7 d

Route of administration:
oral: feed
Vehicle:
not specified
Details on oral exposure:
No data
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data
Duration of treatment / exposure:
14 d
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
1, 2 and 5 % w/w (approx. equivalent to 514, 1,028 and 2,571 mg/kg bw/day assuming 18 g as default food consumption/day; 0.35 kg as average adult rat body weight)
Basis:
nominal in diet
No. of animals per sex per dose:
3/sex/dose
Control animals:
yes, concurrent no treatment
Details on study design:
No data
Positive control:
No data
Observations and examinations performed and frequency:
OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before feeding on Day 1 and on Days 4, 8, 11 and 15

BODY WEIGHT: Yes
- Time schedule for examinations: Before feeding on Day 1 and on Days 4, 8, 11 and 15


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption: Determined weekly

FOOD EFFICIENCY:
- Food utilisation expressed as increase in bodyweight/100g food consumed for each animal

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Weekly, expressed as weekly mean per animal

Sacrifice and pathology:
GROSS PATHOLOGY: No
HISTOPATHOLOGY: No
Other examinations:
No data
Statistics:
No data
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):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY: No animals died during the study and no clinical signs were observed

BODY WEIGHT AND WEIGHT GAIN: Bodyweights of either sex did not differ between dose levels

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No differences in food consumption


FOOD EFFICIENCY: No differences in food utilisation.



Key result
Dose descriptor:
NOEL
Effect level:
ca. 5 other: % in diet (i.e., ca. 2571 mg/kg bw/day)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment-related effects
Key result
Critical effects observed:
no

None

Conclusions:
Under the conditions of this study, the NOEL of the substance in rats was determined to be 5% (equivalent to 2,571 mg sterol/kg bw/day) in diet.
Executive summary:

A repeated dose oral subacute toxicity study was conducted in rats to determine the dietary effects of the constituent plant sterols. Diets containing 0, 1, 2 and 5% w/w level of the substance (approx. equivalent to 0.0, 514, 1,028 and 2,571mg/kg bw/day assuming 18 g as default food consumption/day; 0.35 kg as average adult rat body weight) were fed to 3 Alpk:APfSD rats/sex/dose for 14 d. No effects were observed on mortality, clinical signs, body weight gain, feed utilization and feed efficiency. Under the conditions of this study, the NOEL of the substance in rat was determined to be 5% (equivalent to 2,571 mg sterol/kg bw/day in males and females) in diet based on lack of effects at the highest tested dose (ANZFA, 2001 (2)).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2002
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Reason / purpose for cross-reference:
reference to other study
Principles of method if other than guideline:
A sub-chronic study was conducted in groups of male/female Fischer 344 rats to investigate the systemic toxicity of repeated administration of d-alpha tocopheryl acetate at 125, 500, or 2000 mg/kg/day of d-alpha-tocopheryl acetate in corn oil by gavage at a dose of 3.5 mL/kg for 90 d. Parameters such as clinical signs, body weight, feed consumption, organ weights, haematology and histopathological changes were observed at regular intervals.
GLP compliance:
not specified
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
Not reported
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
3.5 mL/kg of 125, 500, or 2000 mg/kg/day of d-alpha-tocopheryl acetate in corn oil was administered by gavage
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Not applicable
Duration of treatment / exposure:
90 d
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
3.5 mL/kg of 125, 500, or 2000 mg/kg/day d-alpha-tocopheryl acetate in corn oil
Basis:
actual ingested
No. of animals per sex per dose:
60/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Not reported
Positive control:
Not reported
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT and FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After sacrifice of 10 animals on Days 5 and 45, and all surviving animals were killed at study termination
- How many animals: Ten males and 10 females on Days 5 and 45 and remaining on study termination

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After sacrifice of 10 animals on Days 5 and 45, and all surviving animals were killed at study termination
- How many animals: Ten males and 10 females on Days 5 and 45 and remaining on study termination

OTHERS
- Levels of thyroid stimulating hormones

Sacrifice and pathology:
HISTOPATHOLOGY: Yes
- In the control and high-dose groups, all tissues were examined and in the low- and mid-dose groups, those tissues designated as targets for vitamin E toxicity were examined microscopically.
Other examinations:
Liver weights
Statistics:
Not reported
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Mortality, signs of toxicity, including diarrhea, tachypnea, bleeding through the nose, dark feces, and a red crust around the eyes (often 1 d before death), were observed in males of the high-dose group.
Mortality:
mortality observed, treatment-related
Description (incidence):
Mortality, signs of toxicity, including diarrhea, tachypnea, bleeding through the nose, dark feces, and a red crust around the eyes (often 1 d before death), were observed in males of the high-dose group.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weights were similar to that of the vehicle control group
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Feed consumption was similar to that of the vehicle control group
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Significant dose-related changes in several hematological parameters were only observed for males of the mid- and high-dose groups
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No treatment-related trends were observed for clinical chemistry values
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Relative liver-to-body weights were significantly increased in females of the 500 and 2000 mg/kg/day groups
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
See above for details on results.
- Haematological effects: The effects observed in males were attributed to a response to blood loss caused by hemorrhagic diathesis. No treatment-related hematological trends were observed for females.
- Significant increases in thyroid-stimulating hormone were observed in animals of all test groups at Day 90.
- Microscopical examinations: Treatment-related pulmonary adenomatous hyperplasia and chronic interstitial inflammation, characterized by increased cellularity, vascular congestion, thickened alveolar walls, and the presence of foamy macrophages, occurred in animals of all test groups. The incidence and severity increased in a dose-dependent manner. Increased extramedullary erythropoiesis was observed in four males of the high-dose group.
Key result
Dose descriptor:
LOAEL
Effect level:
125 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
125 mg/kg bw/day (nominal)
System:
respiratory system: lower respiratory tract
Organ:
lungs
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

None

Conclusions:
Under the conditions of the study, the LOAEL of the substance was concluded to be 125 mg/kg/day in corn oil.
Executive summary:

A 90 d sub-chronic study was conducted in groups of male/female Fischer 344 rats to investigate the systemic toxicity of repeated administration of the constituent d-alpha tocopheryl acetate. Groups of 30 male/female Fischer 344 rats were given 125, 500, or 2000 mg/kg/day of the substance in corn oil by gavage at a dose of 3.5 mLkg for 90 d, and two control groups were given 3.5 mL/kg vehicle. Body weights and feed consumption were measured and clinical observations were made weekly. Hematology and clinical chemistry parameters were examined for the animals killed at Days 5 and 45 and for all animals at termination of study. In the control and high-dose groups, all tissues were examined and in the low and mid-dose groups, those tissues designated as targets for vitamin E toxicity were examined microscopically. The mean body weights and feed consumption were simialr to the vehicle control group. However, mortality, signs of toxicity including diarrhea, tachypnea, bleeding through the nose, dark feces, and a red crust around the eyes, were observed in males of the high-dose group. Relative liver-to-body weights were significantly increased in females of the 500 and 2000 mg/kg/day groups. After 90 d, significant dose-related changes in hematological parameters were observed for males of the mid- and high-dose groups. This was attributed to a response to blood loss caused by hemorrhagic diathesis. No treatment-related trends were observed for clinical chemistry values; significant increases in thyroid-stimulating hormone were observed in animals of all test groups at Day 90. At microscopic examination, treatment-related pulmonary adenomatous hyperplasia and chronic interstitial inflammation, characterized by increased cellularity, vascular congestion, thickened alveolar walls, and the presence of foamy macrophages, occurred in animals of all test groups. Increased extramedullary erythropoiesis was observed in four males of the high-dose group. Under the conditions of the study, the LOAEL of the substance was concluded to be 125 mg/kg/day in corn oil (Fiume, 2002 (2)).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1987
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Reason / purpose for cross-reference:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Groups of 30 Charles River CD rats of each sex were fed diets containing hte constituent total tocopherol at dietary concentrations of 0, 0.002, 0.2, or 2% (i.e., 1.14, 110.1 and 1085.5 mg/kg bw/day) for 90 d. Effects on haematological, clinical chemistry parameters at 42 and 84 d in high dose and control groups; organ weights for liver, spleen, brain, pituitary, kidneys, gonads, adrenals, and thyroids, and a histopathological examination after terminal necropsy were observed.


GLP compliance:
not specified
Species:
rat
Strain:
other: Charles River CD
Sex:
male/female
Details on test animals or test system and environmental conditions:
No details available in the JECFA evaluation report
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
No details available in the JECFA evaluation report
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Not applicable
Duration of treatment / exposure:
90 d
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0, 0.002, 0.2, or 2% (i.e. approx. 1.14, 110.1 and 1085.5 mg/kg bw/day actual ingested doses)
Basis:
nominal in diet
No. of animals per sex per dose:
30 rats/dose
Control animals:
yes, plain diet
Details on study design:
No details available in the JECFA evaluation report
Positive control:
No details available in the JECFA evaluation report
Observations and examinations performed and frequency:
Haematological and clinical chemical examinations were made on 15 rats of each sex in the control and high-dose groups at 42 and 84 d.


Sacrifice and pathology:
At terminal necropsy, organ weights were determined for liver, spleen, brain, pituitary, kidneys, gonads, adrenals, and thyroids, and a histopathological examination was performed
Other examinations:
No details available in the JECFA evaluation report
Statistics:
No details available in the JECFA evaluation report
Clinical signs:
not specified
Mortality:
not specified
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 specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Details on results:
Not reported

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 2 other: % in diet (i.e., ca. 1086 mg/kg bw/day)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment-related effects
Key result
Critical effects observed:
no

None

Conclusions:
Under the test conditions, the NOAEL of the substance in rats was determined to be 1086 mg/kg bw/day (the highest tested dose).
Executive summary:

A study was conducted to evaluate the sub-chronic repeated dose toxicity of the constituent d-alpha-tocopherol (polyethylene glycol) 1000 succinate (TPGS) in Charles River CD rats. Groups of 30 Charles River CD rats of each sex were fed diets containing 0, 0.002, 0.2, or 2% (i.e., 1, 110 and 1086 mg/kg bw/day) of the substance for 90 d. Haematological and clinical chemical examinations were made on 15 rats of each sex in the control and high-dose groups at 42 and 84 d. At terminal necropsy, organ weights were determined for liver, spleen, brain, pituitary, kidneys, gonads, adrenals, and thyroids, and a histopathological examination was performed. The substance at the doses studied had no effect on body-weight gain, food consumption, haematology, organ weights, serum chemistry, or histopathology. Under the test conditions, the NOAEL of the substance in rats was determined to be 1086 mg/kg bw/day (the highest tested dose), based on lack of significant treatment related adverse effects in rats (JECFA, 1987 (2)).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1987
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Reason / purpose for cross-reference:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Five groups of weanling rats were fed 875, 1750, 3500 and 35000 mg d-alpha-tocopheryl acetate/kg/day through diet for 13 weeks (i.e. approx. 45, 90, 180 and 1800 mg/kg bw/day, based on 18 g as as average food consumption of a 0.35 kg body weight rat). Its impact on food consumption, haematology, serum chemistry, urinary chemistry were monitored at regular intervals.
GLP compliance:
not specified
Species:
rat
Strain:
not specified
Sex:
male/female
Details on test animals or test system and environmental conditions:
No details available in the JECFA evaluation report
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
No details available in the JECFA evaluation report
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Not applicable
Duration of treatment / exposure:
90 d
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
1.8 (control) 45, 90, 180, 1800 mg/kg bw/day
Basis:
nominal in diet
No. of animals per sex per dose:
No details available
Control animals:
other: yes, with 1.8 mg/kg bw of test material in diet
Details on study design:
No details available in the JECFA evaluation report
Positive control:
No details available in the JECFA evaluation report
Observations and examinations performed and frequency:
Parameters like food consumption, haematology, serum chemistry and urinary chemistry were observed

Sacrifice and pathology:
No details available
Other examinations:
No details available in the JECFA evaluation report
Statistics:
No details available in the JECFA evaluation report
Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
significantly lower feed and protein efficiencies
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
Haemoglobin, serum cholesterol, and urinary creatine and creatinine were unaffected by treatment
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum and liver vitamin E levels increased progressively with dose; but SGPT was elevated in the highest-dose group
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Details on results:
Not reported
Key result
Dose descriptor:
NOAEL
Effect level:
180 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: based on lower feed and protein efficiencies, increased serum and liver vitamin E levels elevated SGPT in the highest-dose group
Key result
Critical effects observed:
no

None

Conclusions:
Under the test conditions, the NOAEL of the substance in rats was determined to be 180 mg/kg bw/day.
Executive summary:

A study was conducted to evaluate the sub-chronic repeated dose toxicity of tocopheryl acetate in rats. Five groups of weanling rats were fed 875, 1750, 3500 and 35000 mg/kg/day through diet for 90 d (i.e. approx. 45, 90, 180 and 1800 mg/kg bw/day, based on 18 g as as average food consumption of a 0.35 kg body weight rat). After 8 weeks, rats in the highest-dose group had significantly lower feed and protein efficiencies. Serum and liver vitamin E levels increased progressively with dose. After 13 weeks, hemoglobin, serum cholesterol, and urinary creatine and creatinine were unaffected by treatment, but SGPT was elevated in the highest-dose group. Under the test conditions, the NOAEL of the substance was determined to be 180 mg/kg bw/day, based on effects observed at higher dose (JECFA, 1987(1)).

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
secondary literature
Reason / purpose for cross-reference:
reference to other study
Principles of method if other than guideline:
Groups of 60 male/female Charles River CD rats, were fed a diet supplemented with dl-alpha-tocopheryl acetate at dose levels of 500, 1000, or 2000 mg/kg bw/day for 104 weeks. The systemic toxicity was evaluated by monitoring parameters like mortality, body weight gain, food consumption, haematological findings, urinalysis and histopatholgical changes at regular intervals.
GLP compliance:
not specified
Species:
rat
Strain:
other: Charles River CD rats
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 134 g (males) or 130 g (females)
- Water: Vitamin K1 was administered in the drinking water at 7 mg/L to counteract an observed haemostatic failure
Route of administration:
oral: feed
Details on route of administration:
0, 500, 1000, or 2000 mg/kg bw/day
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Mixing appropriate amounts with (Type of food):
(a) Test group: The basal diet was supplemented with dl-alpha-tocopheryl acetate at levels calculated to give a dose of 500, 1000, or 2000 mg/kg bw/day and 5 mg/kg of vitamin K1
(b) Control group: Received unsupplemented basal diet stated to contain 39 mg vitamin E/kg bw, 10 mg vitamin K3/kg bw. and 5 mg vitamin K1/kg bw



Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Not applicable
Duration of treatment / exposure:
104 wks
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0, 500, 1000, or 2000 mg/kg bw/day
Basis:
nominal in diet
No. of animals per sex per dose:
60 animals/sex/dose
Control animals:
yes, plain diet
Details on study design:
No details available
Positive control:
Not reported
Observations and examinations performed and frequency:
MORTALITY: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded weekly.

FOOD CONSUMPTION (if feeding study): Yes
- Time schedule for examinations: Recorded weekly.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 4, 8, 13, 26, 52, 78, and 95 weeks of treatment
- How many animals: 10 male and 10 female animals of the control and highest-dose groups
- Parameters examined: RBC and total leucocyte counts, haematocrit, haemoglobin, differential leucocytes, and prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After 4, 8, 13, 26, 52, 78, and 95 weeks of treatment
- How many animals: 5 male and 5 female animals of the control and highest-dose groups
- Parameters examined: urea, glucose, bilirubin, total protein, electrophoretic protein fractionation, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, sodium, and potassium

URINALYSIS: Yes
- Time schedule for collection of urine: After 4, 8, 13, 26, 52, 78, and 95 weeks of treatment
- How many animals: 10 male and 10 female animals of the control and highest-dose groups
- Parameters examined: SG, reducing substances, glucose, ketones, protein, sedimentary cell counts, and blood and bile pigments

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
- 10 rats/sex/dose were killed, necropsied, and examined histologically after 52 weeks of treatment. The remaining animals were examined after termination at 104 weeks.
-Detailed histopathological examination was performed on control and high-dose groups; in other dose groups, histopathology was restricted to the liver and any macroscopically-abnormal tissue.
Other examinations:
Organ weight:
- Rats which died on test or which were killed at termination were necropsied and absolute and relative organ weights were determined for adrenals, brain, heart, liver, lungs, testes or ovaries, pituitary, spleen, thyroid, and prostate or uterus
Statistics:
Not reported
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
similar to or slightly greater than controls
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
similar to or slightly greater than controls
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
transient, slight lowering of the haematocrit and haemoglobin at week 8 in both males and females in the highest-dose group.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Elevated serum alkaline phosphatase and alanine aminotransferase was observed in high dose-group.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
agglomerations of vacuolated ("foamy") macrophages in the centriacini of 17% of treated males and 77% of treated females
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
tumour incidence did not reveal any neoplastic effects of treatment
Details on results:
CLINICAL SIGNS AND MORTALITY:
- 10% mortality observed during the first 26 weeks in high dose group was balanced by a similar number of deaths in control males between weeks 26 and 52. At termination, there were no significant differences in mortality between any treatment groups of either sex and their respective controls.

FOOD CONSUMPTION AND BODY WEIGHT AND WEIGHT GAIN: Were similar to or slightly greater than controls

HAEMATOLOGY: Only, transient, slight lowering of the haematocrit and haemoglobin was observed at week 8 in both sexes in the highest-dose group.

CLINICAL CHEMISTRY:
- Serum alkaline phosphatase was occasionally significantly elevated (p < 0.05) in the high-dose group, but the differences were neither consistent nor progressive with time; no such changes were seen at lower-dose levels.
- A dose-related elevation of alanine aminotransferase was observed in treated males at week 4, persisting to week 26, but later it lost statistical significance.
- Aspartate aminotransferase and all other blood chemical parameters were unaffected.

URINALYSIS: No significant changes were seen on urinalysis.

ORGAN WEIGHTS: In females (not males) of high dose group, a slightly elevated absolute liver weight was seen at the interim sacrifice, but the relative liver weight was not increased and no significant differences in relative or absolute organ weights were seen at termination of the study.

GROSS PATHOLOGY: No macroscopic changes related to treatment were observed.

HISTOPATHOLOGY: NON-NEOPLASTIC: No treatment related changes was revealed in the liver, except for the agglomerations of vacuolated ("foamy") macrophages in the centriacini of 17% of treated males and 77% of treated females across the study as a whole, distributed among the treated rats without dose-relation. It was not found in the control group.

HISTOPATHOLOGY: NEOPLASTIC (if applicable): The aggregate tumour incidence did not reveal any neoplastic effects of treatment. In both sexes, there were indications of an inverse relationship between dosage and incidence of mammary fibro-adenomas, but this effect was statistically significant
only in females.

Key result
Dose descriptor:
LOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: haematological effects (i.e., transient, slight lowering of the haematocrit and haemoglobin at week 8 in both sexes) and presence of vacuolated lipid staining of macrophages in the liver
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
System:
haematopoietic
Organ:
blood
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Occasional difficulties in arresting bleeding were observed when blood samples were collected after 8 weeks and frank haemorrhages were observed in males only from week 15 (high-dose), week 16 (intermediate-dose), or week 18 (low-dose). Haemorrhages occurred variously in the gut, urinary tract, orbit and meninges and from minor injuries to the claws or vibrissal pits. Vitamin K supplementation was effective in bringing about recovery within one to three days.

Conclusions:
Under the test conditions, the LOAEL of the substance was determined to be 500 mg/kg bw/day, based on the haematological effects (i.e., transient, slight lowering of the haematocrit and haemoglobin at week 8 in both sexes) and presence of vacuolated lipid staining of macrophages in the liver.
Executive summary:

The dietary effects of dl-alpha-tocopheryl acetate were investigated in a repeated dose chronic toxicity study. Groups of 60 male/female Charles River CD rats, were fed a diet supplemented with the substance at dose levels of 500, 1000, or 2000 mg/kg bw/day for 104 weeks. The systemic toxicity was evaluated by monitoring parameters like mortality, body weight gain, food consumption, haematological findings, urinalysis and histopatholgical changes at regular intervals. There were no significant differences in mortality between any treatment groups of either sex and their respective controls. Food consumption and body weight and weight gain were similar to or slightly greater than controls. Serum alkaline phosphatase was occasionally significantly elevated (p < 0.05) in the high-dose group, but the differences were neither consistent nor progressive with time; a dose-related elevation of alanine aminotransferase was also observed in treated males at week 4, but it persisted (with statistical significance) only up to week 26. Transient, slight lowering of the haematocrit and haemoglobin was observed at week 8 in both sexes in the highest-dose group. No significant treatment related effects were observed except for except for haematological effects (i.e., transient, slight lowering of the haematocrit and haemoglobin at week 8 in both sexes) in the higher dose group. In females (not males) of high dose group, a slightly elevated absolute liver weight was seen at the interim sacrifice, but the relative liver weight was not increased and no significant differences in relative or absolute organ weights were seen at termination of the study. No treatment related histopathological changes was revealed in the liver, except for the agglomerations of vacuolated ("foamy") macrophages in the centriacini of 17% of treated males and 77% of treated females across the study as a whole, distributed among the treated rats without dose-relation. In both sexes, there were indications of an inverse relationship between dosage and incidence of mammary fibro-adenomas, but this effect was statistically significant only in females. Under the test conditions, the LOAEL of the substance was determined to be 500 mg/kg bw/day, based on the haematological effects (i.e., transient, slight lowering of the haematocrit and haemoglobin at week 8 in both sexes) and presence of vacuolated lipid staining of macrophages in the liver (JECFA, 1987 (3)).

Endpoint:
repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2002
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Reason / purpose for cross-reference:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
A 60 d repeated dose study was conducted to investigate the dietary effects of 400 mg/kg bw/day the constiteunt vitamin E (assumed to be tocopherol) in Wistar rats.
GLP compliance:
not specified
Species:
rat
Strain:
Wistar
Sex:
male
Route of administration:
oral: feed
Details on route of administration:
400 mg/kg bw/day
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Not applicable
Duration of treatment / exposure:
60 d
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
400 mg/kg bw/day
Basis:
nominal in diet
No. of animals per sex per dose:
Six males
Control animals:
yes, plain diet
Details on results:
When compared to animals fed a high-fat diet, tocopherol prevented the increase in plasma lipid concentrations, decreased the low-density lipoprotein cholesterol to high-density lipoprotein cholesterol (LDLc/HDLc) ratio and lipid peroxide concentrations, and increased reduced glutathione.

Key result
Dose descriptor:
NOEL
Effect level:
400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No effects at the tested dose
Key result
Critical effects observed:
no

When compared to animals fed a high-fat diet, tocopherol prevented the increase in plasma lipid concentrations, decreased the low-density lipoprotein cholesterol to high-density lipoprotein cholesterol (LDLc/HDLc) ratio and lipid peroxide concentrations, and increased reduced glutathione.

 

Conclusions:
Under the conditions of the study, the NOEL of the substance in rats was determined to be 400 mg/kg bw/day.
Executive summary:

A study was conducted to investigate the dietary effects of the constituent vitamin E (assumed to be tocopherol) in Wistar rats. Single group of six adult male Wistar rats was given 400 mg/kg/day of the substance in feed for 60 d, and a control group was fed basal chow. The substance was fount to be not toxic. When compared to animals fed a high-fat diet, tocopherol prevented the increase in plasma lipid concentrations, decreased the low-density lipoprotein cholesterol to high-density lipoprotein cholesterol (LDLc/HDLc) ratio and lipid peroxide concentrations, and increased reduced glutathione. Under the conditions of the study, the NOEL the substance in rats was determied to be 400 mg/kg bw/day (Fiume, 2002 (1)).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
7.7 mg/kg bw/day
Study duration:
chronic
Experimental exposure time per week (hours/week):
168
Species:
other: humans
Quality of whole database:
all studies with constituents were taken from peer reviewed or regulatory opinions; NOAEL based on the EFSA re-evaluated upper limit for tocopherol

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Reason / purpose for cross-reference:
data waiving: supporting information
Reason / purpose for cross-reference:
data waiving: supporting information
Reason / purpose for cross-reference:
data waiving: supporting information
Critical effects observed:
not specified
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

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Reason / purpose for cross-reference:
data waiving: supporting information
Reason / purpose for cross-reference:
data waiving: supporting information
Reason / purpose for cross-reference:
data waiving: supporting information
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In the absence of repeated dose toxicity study with the test substance,‘squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation’ the endpoint has been assessed based on animal and human studies for substances representative of the main constituents, which can be categorised as glycerides, fatty acids or fatty acid methyl esters (which will eventually hydrolyse to fatty acids (Mattson and Volpenhein, 1972)) and unsaponifiable matters (including tocopherols, sterols, squalene and hydrocarbons). As a large number of studies have been conducted on the individual constituents, particularly in the context of nutritional research, for practical reasons, only a limited number of studies are reported here.

Oral:

Glycerides and fatty acids:

At doses ranging from 7.5 to 18.5% in the diet, no significant toxicity was seen for various constituent glycerides and fatty acids with chain lengths varying between C8-18 or C16-18, including C18-unsatd. and C18-unsatd. hydroxy (Morin, 1967; Harkins and Sarrett, 1968; Nolen, 1981; Manorama and Rukmini, 1991; Coquet et al., 1977; Speijers et al., 2009; Irwin, 1992; HERA, 2002). The highest oral NOAEL could therefore be considered to be 18.5% in diet, i.e., approximately 9,250 mg/kg bw/day. In certain studies (also others not reported here), differences may be observed compared to controls on bodyweight gain, food consumption and certain measured parameters depending on the chain length distribution of the fatty acids (associated to the glycerides or free) and their degree of unsaturation. However, research indicates that when consumed at nutritionally relevant concentrations, there are no adverse effects on health and longevity. It is worth noting that, due to their innocuous nature, fats and oils are commonly used as controls and as vehicles in animal toxicity studies. For example, OECD Guideline 408 (repeated dose 90-day oral toxicity study in rodents) recommends the use of “a solution/emulsion in oil (e.g. corn oil)” as a vehicle where an aqueous vehicle is not suitable (OECD, 1993). Several fatty acids (stearic acid; oleic acid and sodium palmitate) are Generally Recognised as Safe (GRAS) by the U.S. Food and Drug Administration (US FDA). Also, fatty acids as a group are permitted as direct food additives (HERA, 2002).

Unsaponifiable matter:

Tocopherols:

Tocopherols have been tested in numerous repeated dose oral toxicity studies. Overall, animals appear to tolerate high levels (i.e. at least two orders of magnitude above nutritional Vitamin E levels, e.g. 1,000 - 2,000 IU/kg diet) without adverse effects. At very high doses, signs indicative of antagonism with the function of other fat-soluble vitamins occur (Tomassi and Silano, 1986; Fiume, 2002; EFSA, 2008). A NOAEL of 125 mg/kg bw/day was reported for a 64-week feeding study in rat with tocopheryl acetate (Tomassi and Silano, 1986).

The tocopherols and their stereoisomers have also been reviewed and evaluated by a number of regulatory authorities along with derivation of the threshold/safe upper limits: 

1)   A group ADI of 0.15–2 mg/kg bw/day for dl-α-tocopherol and d-α-tocopherol was allocated by Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 1987. This is based on clinical studies in humans and takes into account the fact that α-tocopherol is an essential nutrient (JECFA, 1987).

2)   The Antioxidant Panel of the Food and Nutrition Board (FNB) at the Institute of Medicine of the US National Academy of Sciences set the tolerable upper intake level (UL) for adults at 1000 mg (2,325 μmol)/day of any supplemental form of alpha-tocopherol. This was based on a LOAEL of 500 mg/kg bw/day for dl-α-tocopherol (based on reduced blood clotting in thestudy by Wheldon et al. (1983)), divided by an uncertainty factor of 36 (LOAEL to NOAEL = 2; subchronic to chronic intake = 2; intra-species variation = 3; inter-species variation = 3) and assuming a human body weight of 68.5 kg. The same UL for pregnant and lactating women was recommended. For infants (0–12 months), a UL was not determined, as it was considered that the only source of intake should be from food or formula. The recommended ULs for ages 1–3, 4–8, 9–13 and 14–18 years were 200, 300, 600 and 800 mg/day of any isomeric form of α-tocopherol, respectively (IOM, 2000).

3)   The EU Scientific Committee on Food (SCF) has not derived an ADI for vitamin E or any of the tocopherols, but based on a placebo controlled, dose-response supplementation study in 88 healthy humans (Meydani et al., 1998), set the NOAEL at 540 mg alpha-tocopherol equivalents, the highest dose used in the study. Considering an uncertainty factor of 2 to cover for interindividual differences in sensitivity, the SCF established an UL of 270 mg alpha-tocopherol equivalents for adults, which was rounded to 300 mg alpha-tocopherol equivalents, which is equivalent to 5 mg/kg bw/day in a 60 kg adult. The ULs were scaled for children in the age ranges 1–3, 4–6, 7–10, 11–14 and 15–17 years to give ULs of 100, 120, 160, 220 and 260 mg/day, respectively (SCF, 2003).

4)   The UK’s Expert Group on Vitamins and Minerals (EVM) established a NOAEL for humans (age not defined) of 540–970 mg d-α-tocopherol equivalents per day (EVM, 2003). An uncertainty factor to account for inter-individual differences was not considered necessary, as human study results (such as those of Meydani et al. (1998)) supported a NOAEL of 540 mg d-α-tocopherol equivalents per day; therefore, the safe upper limit was established as 540 mg d-α-tocopherol equivalents for supplemental vitamin E, which is equivalent to 90 mg/kg bw/day in a 60 kg adult (EVM, 2003).

5)   The EFSA Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food (AFC) reviewed and concluded that mixed tocopherols (d-α-tocopherol, d-β-tocopherol, d-γ- tocopherol and d-δ-tocopherol) are not a safety concern at the proposed levels of use of 300 mg/day or 5 mg/kg bw/day for 60 kg adults (EFSA, 2008).

 

Sterols and sterol esters:

In animal studies, sterols and sterol esters demonstrated low repeated dose oral toxicity. The lowest NOAEL found was equivalent to ca. 2,500 mg sterols/kg bw/day in a 22-month study with rats (SCF, 2003). Healthy human volunteer studies conducted with phytosterols at 13-47 mg sterols/kg bw/day in diet, revealed no significant systemic toxicity other than minor adverse effects such as moderate constipation, mildly increased body weights, deficiencies of carotenoid and fat soluble (Vit E and K1) vitamin levels etc. These deficiencies of the carotenoid and vitamin levels were not reported to be associated with any symptoms and the highest tested dose was well tolerated. Furthermore, phytosterols are also well known to reduce the serum cholesterol levels and their efficacy depends on the dose, the existing serum cholesterol level and the nature of the phytosterols ingested. Apart from carotenoid-lowering effects, no other adverse effects were observed in humans with repeated intake as high as 8 g/day, although a maximum uptake of 3 g/day (i.e. 50 mg/kg bw/day for a 60 kg adult) is recommended, with no added benefits observed above that level (SCF, 2002).

Squalene:

A study was conducted to determine the effect of co-administration of squalene in sub-chronic repeated dose study by oral administration. The general plan of the experiment was to feed two groups of rats on a complete artificial diet, while one group received in addition a small quantity of squalene each day. Immediately before feeding-time the animals were given approximately 660 mg of squalene dropped directly into their mouths from a micro-burette. Records were taken of the amount of squalene given each day. The faeces were collected throughout the experiment at weekly intervals and stored in alcohol. At the end of the experiment the animals were killed by chloroform and their livers removed as free from blood as possible. No obvious ill effects followed the administration of squalene and postmortem examination showed that the organs of the animals were very healthy. There appeared to be a greater deposition of fat in the animals which received squalene and on the whole they seemed to grow at a somewhat greater rate than those of the control groups, possibly because the laxative action of the squalene caused a greater food consumption. The experiment showed that when squalene was administered to the rat, it was in part absorbed and as a result of absorption there was a marked increase in the amounts of unsaponifiable matter and of cholesterol in the body and liver of the animal. No effects up to dose of 3300 mg/kg bw/day (considering 200 g average animal body weight and 660 mg per animal dose) squalene in any animals were reported. Under the study conditions, the NOAEL of squalene was determined to be 3300 mg/kg bw/day in rats (Channon, 1926).

Apart from the presented study and the epidemiological evidence deriving from the presence of squalene as a component of common edible oils and fat constantly present in human diet, several studies have been performed in the framework of use of squalene as adjuvant in recently developed influenza vaccines. The evaluation report for Humenza (vaccine against H1N1 virus containing squalene-based adjuvant) submitted to the European Medicines Agency (EMA) in 2010, reported two repeat dose toxicity studies, one in rodents (in rats) and one in non-rodents (in rabbits) for assessing systemic toxicity and local tolerance. In the rat repeat dose and in the reproductive toxicity studies, several dose levels of the vaccine were assessed (from 1.25% to 10% squalene, including the human dose 2.5% squalene). In the rabbit repeat dose toxicity study, the human dose of the squalene-based adjuvant used in the vaccine was evaluated. No specific safety concerns were raised. Similar toxicology studies were submitted by Novartis, which were designed to meet the US FDA and EMEA requirements complying with applicable international guidelines for the nonclinical assessment of vaccines and adjuvants. The pivotal toxicology studies performed with the adjuvant alone included the evaluation of local tolerability, repeat-dose toxicity (one clinical dose administered to rabbits once daily for 14 days), genotoxicity, sensitisation, and embryofetal and developmental toxicity. These studies did not indicate any potential for systemic toxicity or local reactogenicity. In repeat-dose rabbit studies, clinical pathology findings of increased fibrinogen, and minor inflammatory and degenerative changes at the injection site, which were consistent with the effects of intramuscular (i.m.) injections of an immunological adjuvant. These reactions were readily reversible within days to 1–2 weeks. Furthermore, squalene is a common component of the human diet through olive oils and other oils and fats and it is assumed in a quantity between 30 and 200 mg/day, without adverse effect in a whole human life. It is also used since decades in cosmetic formulations, creams and similar, where a daily application is performed during a lifetime by humans. No systemic effects have been reported, about eventual systemic toxicity.

Hydrocarbons:

In a subchronic dietary toxicity study, six highly refined base oils were administered to male and female F-344 rats at dose levels 20, 200, 2,000, and 20,000 ppm of for 90-days. High-dose dietary exposure produced a syndrome of effects in liver and mesenteric lymph nodes with response greater in females than in male F-344 rats. Effects include increased organ weights, evidence for the presence of nonpolar hydrocarbons, and other observations suggestive of an inflammatory response. A NOEL of 20 ppm or 2 mg/kg bw/day for 4 of 6 oils (N10A, P15H, N70A, N70H; carbon number ranging from 15 to 37) was calculated, a NOEL was not established for N15H (carbon number 17-30); and a NOEL of greater than or equal to 20,000 ppm or 1900 mg/kg bw/day was calculated for P100H (carbon number 28-45); based on histiocytosis. In another sub-chronic dietary toxicity study for P15(H) white oil (carbon number 18-30), the NOAEL in SD female rats and LOAEL in F344 female rats were determined to be ≥1624 mg/kg bw/day (based on absence of adverse effects) and 161 mg/kg bw/day (based on the histopathology in the liver and mesenteric lymph nodes accompanied by changes in the weight of those organs) respectively (Firriolo, 1995). As these effects are now shown to be strain-specific, the adversity of the mesenteric lymph nodes (MLN) histiocytosis is questionable. In two-year dietary studies conducted to determine the chronic toxicity and the carcinogenicity of P70(H) (carbon number 27-43) and P100(H) (carbon number 28-45) white mineral oils in Fischer-344 rats (F-344), the NOAEL for these studies was considered to be 1200 mg/kg bw/day (highest tested dose), based on the absence of carcinogenicity and chronic toxicity (Trimmer, 2004).

Based on the available weight of evidence information on the constituents, the test substance ‘squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation’ is overall considered to have low systemic toxicity.

Inhalation:

There is no information on the repeated dose inhalation toxicity of the test substance. However, exposure via the inhalation route is not expected considering the intermediate use and low vapour pressure (0.00108 Pa at 20°C) of the substance. In addition, studies conducted via the oral route with the constituents or its representative substances indicated a lack of significant toxicity of the test substance. Therefore, testing via inhalation route is unlikely to result in any additional hazard identification and hence further testing involving vertebrate animals may be omitted, in accordance with Annex XI (1.2) of the REACH regulation. The risk assessment for the likelihood of any potential inhalation exposure in workers has been conducted based on the conservative NOAEL identified from the studies with the constituents and by using appropriate route-to-route extrapolation assessment factors as per the ECHA Guidance R.8.

Dermal:

There is no information on the repeated dose dermal toxicity of the test substance. However, given the intermediate use of the test substance where it completely gets converted to crude squalene, there is only a low dermal exposure potential of the test substance itself in workers, during its handling. However, as discussed in the toxicokinetic section, dermal absorption of the test substance is not expected to be higher than via the oral route. Considering the intermediate use and physico-chemical properties of the test substance and/or its constituents, the absorption potential via the dermal route is not expected to be higher than oral route. Therefore, testing via the dermal route is unlikely to result in any additional hazard identification and further testing involving vertebrate animals may be omitted, in accordance with Annex XI (1.2) of the REACH regulation. The risk assessment for the likelihood of any potential dermal exposure in workers has been conducted based on the conservative NOAEL identified from the studies with the constituents and by using appropriate route-to-route extrapolation assessment factors as per the ECHA Guidance R.8.

Justification for classification or non-classification

Based on the available weight of evidence information on the constituents, the test substance ‘squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation’ does not warrant classification for repeated dose toxicity according to EU CLP criteria (EC 1272/2008).