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The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Description of key information

The repeated dose toxicity potential of ‘Soaps, stocks, vegetable oil, acidulated’ can be deduced based on information available for its individual constituents. Studies conducted with the two main constituents, glycerides and fatty acids, indicate overall low toxicity. The highest free-standing NOAEL from a chronic repeated dose oral toxicity was equivalent to18.5% in diet, i.e., equivalent to 9,250 mg/kg bw/day. The sterols, sterol esters and squalene are unlikely to contribute significantly to toxicity as they were found to be poorly absorbed through oral as well as dermal routes. 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.

Considering the available dermal studies with some of the constituents together with their absorption potential, toxicity of the individual constituents via the dermal route is not expected to be higher than oral route. Exposure via the inhalation route is not expected considering the low vapour pressure of the substance. Further, the inhalation exposure potential in workers where the substance is handled in aerosolized or spray form is considered to be low due to implementation of strict risk management measures at workplace (See sections 9 and 10 of the CSR). Therefore, repeated dose inhalation toxicity is not expected to pose an issue for human health and no further testing is required for this endpoint, in accordance with Annex VIII, column 2 of the REACH regulation.

Alternative text:

Chronic and sub-chronic dietary studies were conducted in rodents with various glycerides and fatty acids which are representative of the REVODS category members and covering the fatty acid chain length of C8 to C18. In these studies, no treatment-related effects were seen in the test animals due to supplementation of the diet with these substances and consequently, the no observed effect levels for these studies were the respective highest tested dose levels. For perspective, the highest free standing NOAEL from the long term studies is 18.5% in diet from the 47-week study with coconut oil, equivalent to 9250 mg/kg bw/day (Harkins, 1968). This NOAEL is supported by free standing NOAELs from other chronic and sub-chronic studies (Morin, 1967; Nolen, 1981; Speijers, 2009; Coquet, 1977 and Manorama and Rukmini, 1991).

The tocopherols, in general, contribute less to the composition of deodorizer distillates than the phytosterols and sterol esters. The sterols and sterol esters are generally poorly absorbed from the gastro-intestinal tract and slowly absorbed through the skin. As a result, inspite of their relative greater extent of absorption through oral well as dermal routes, overall contribution of the tocopherols to the systemic dose of the deodorizer distillates could be considered to be low. On the other hand, major constituents of the deodorizer distillates are glycerides and fatty acids of C8 to C18 chain length. Systemic absorption of the shorter chain fatty acids is greater than the longer chain counterparts. Therefore, C8-C12 fatty acids could be considered to show higher degree of systemic absorption compared to the longer chains. With this in mind, coconut oil (rich in C12 and C14 fatty acids) as a representative of the constituent glycerides, C8 -18 and C18 -unsatd.’ would be appropriate and studies conducted with coconut oil as test substance can be regarded as major contributor in the evaluation of repeated dose exposure.

Considering the available dermal studies with some of the constituents together with their absorption potential, toxicity of the individual constituents via the dermal route is not expected to be higher than oral route. Exposure via the inhalation route is not expected considering the low vapour pressure of the substance. Further, the inhalation exposure potential in workers where the substance is handled in aerosolized or spray form is considered to be low due to implementation of strict risk management measures at workplace (See sections 9 and 10 of the CSR). Therefore, repeated dose inhalation toxicity is not expected to pose an issue for human health and no further testing is required for this endpoint, in accordance with Annex VIII, column 2 of the REACH regulation.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
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
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:
13 200 mg/kg bw/day (nominal)
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:
14 600 mg/kg bw/day (nominal)
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
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
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
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
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
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)
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
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
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:
repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
weight of evidence
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
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
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
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:
8 866 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no toxicologically significant effects
Key result
Dose descriptor:
NOAEL
Effect level:
10 242 mg/kg bw/day (actual dose received)
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
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:
repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
weight of evidence
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
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:
; 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
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
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
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:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
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:
2 571 mg/kg bw/day (nominal)
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
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
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:
> 2 571 mg/kg bw/day (nominal)
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
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
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:
1 086 mg/kg bw/day (actual dose received)
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
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: no treatment-related adverse effects
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 wasv 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
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
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
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
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
9 250 mg/kg bw/day
Study duration:
chronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
other justification
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Non-Human information

Oral:

There is no published information on the repeated dose oral toxicity of ‘Soaps, stocks, vegetable oil, acidulated’ in animals. However, 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 (see also Table above).

-      At doses ranging from 7.5 to 18.5% in 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).

-       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). Further, the US Institute of Medicine (IOM) established an UL of 1,000 mg/day (i.e., equivalent to 13.9 mg/kg bw/day) for any isomeric form of α-tocopherol (IOM, 2000). This was based on a Lowest Observed Adverse Effect Level (LOAEL) of 500 mg/kg bw/day for dl-α-tocopherol (based on reduced blood clotting in a chronic carcinogenicity study by Wheldon et al. (1983) in rats), 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 was recommended for pregnant and lactating women and the other 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 cited in ECHA, 2015).

-       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).

-       No data could be located on the repeated dose oral toxicity of squalene. This substance is however poorly absorbed through the gastrointestinal tract; therefore repeated dose toxicity is not expected under normal and foreseeable use conditions.

Inhalation:

Under normal atmospheric conditions, exposure to ‘Soaps, stocks, vegetable oil, acidulated’ via the inhalation route is limited considering its low vapour pressure (0.0328 Pa at 20°C). Further, the inhalation exposure potential in workers where the substance is handled in aerosolized or spray form is considered to be low due to implementation of strict risk management measures at workplace (See sections 9 and 10 of the CSR). In addition, studies conducted via the oral and dermal routes indicated a lack of significant toxicity of the test substance. Thus, repeated inhalation exposure is not expected to pose an issue for human health and no further testing is required for this endpoint, in accordance with Annex VIII, column 2 of the REACH regulation.

Dermal:

There is no information on the repeated dose dermal toxicity of ‘Soaps, stocks, vegetable oil, acidulated’ in animals. However, data is available for some of its constituents:

·        Glycerides with chain lengths bteween C8 - 18 including C18 -unsatd. present low systemic toxicity upon oral exposure where absorption is higher than via the dermal route (see section 5.1 of the CSR). Repeated dose dermal toxicity is therefore expected to be minimal. This is supported by very long history of safe use of these types of substances in nutritional (food and feed), cosmetic and/or industrial applications.

·        Significant systemic toxicity is also not expected from repeated dermal exposure to fatty acids. Studies with various chain length fatty acids are summarised in HERA (2002) and CIR (1982 and 1987) reports. For example, no adverse effects apart from changes to the superficial layer of the dermis were resulted from daily topical application of myristic acid (C14) to rabbit skin for 30 d (0.5 mL of a 30% preparation applied to the depilated skin of the flanks). A formulation of “bath soap and detergent” containing 10-25% sodium stearate (C18) was used to conduct a dermal toxicity study in rabbits. Formulations at a dose of 2,000 mg/kg bw were applied for 3 months to the skin by syringe daily, five days a week. No untoward reactions were observed.

Human information:

Oral, dermal and inhalation:

There is no information on the repeated dose dermal toxicity of ‘Soaps, stocks, vegetable oil, acidulated’ in humans. However, the following information is available on its constituents:

-         Repeated oral, inhalation or dermal exposure to glycerides or fatty acids is not expected to pose an issue for human health under normal and foreseeable handling and use conditions.

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

o   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).

o   The EU Scientific Committee on Food (SCF) has not derived an ADI for vitamin E or any of the tocopherols, but established a UL of 300 mg/day (i.e. equivalent to 0.19 mg alpha-tocopherol equivalents/kg bw/day assuming 60 kg as adult body weight) for vitamin E in adults (SCF, 2003, EFSA, 2008). 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).

o   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 9.0 mg/kg bw/day in a 60 kg adult (EVM, 2003).

o   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 for adults (EFSA, 2008).

-         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).

-         No specific information on the repeated dose toxicity of squalene to humans was found. This substance is however poorly absorbed through the gastrointestinal tract; therefore repeated dose toxicity is not expected under normal and foreseeable use conditions.

Justification for classification or non-classification

The repeated dose toxicity potential of ‘Soaps, stocks, vegetable oil, acidulated’ can be deduced based on information available on its individual constituents. Studies conducted on glycerides and fatty acids, the two main constituents, indicated low overall toxicity. The highest free-standing NOAEL from a chronic repeated dose oral toxicity was equivalent to18.5% in diet, i.e. ca. 9,250 mg/kg bw/day. The sterols, sterol esters and squalene are found to be poorly absorbed both orally and dermally, therefore unlikely to contribute significantly to toxicity. Tocopherols on the other hand are well absorbed and, although beneficial at nutritionally relevant doses, may antagonise the function of other fat-soluble vitamins at higher doses. Considering the available dermal studies for some of the constituents together with their low absorption potential, toxicity of the individual constituents via the dermal route is not expected to be higher than oral route. Exposure via the inhalation route is not expected considering the low vapour pressure of the substance. Further, the inhalation exposure potential in workers where the substance is handled in aerosolized or spray form is considered to be low due to implementation of strict risk management measures at workplace (See section 9 and 10 of the CSR).

Based on the above information, the substance does not warrant classification for repeated dose toxicity according to CLP Regulation EC/1272/2008.