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Description of key information

Repeated dose information was available for the structural analogues D- and D,L-alpha-tocopheryl acetate (vitamin E acetate). These vitamin E esters  will be rapidly hydrolysed to the free alpha-tocopherol forms under physiological conditions.  In summary, Vitamin E exerts low  toxicity in repeated dose studies. The NOAEL of 500 mg/kg bw/d for D-alpha-tocopheryl acetate was based on a well conducted 90-day study in rat comparable to OECD guideline 408 (Abdo, 1986). In a chronic rat study with doses of 500, 1000 or 2000 mg/kg bw/d D,L-alpha-tocopherol acetate the animals showed at all dose levels spontaneous haemorrhages in several organs which was correctable by administration of Vitamin K3.
The following repeated studies were also part of the assessment for this endpoint:
28-day studies (OECD 407) with D,L-alpha-tocopheryl acetate: gavage study in rats (Pfister, 1999; BASF AG, 1983)
28-day study with D,L-alpha-tocopheryl acetate: feed study in dogs (BASF AG, 1982)
2-year study (OECD 453): feeding study in rats with D,L-alpha-tocopheryl acetate supplemented with Vitamin K (Wheldon, 1978)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP near-guideline study, acceptable for assessment.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratory, Kingston, NY
- Age at study initiation: 25 days
- Housing: stainless steel cages
- Diet: ad libitum, modified AIN-76 diet
- Water: ad libitum
- Acclimation period: 11 days


Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
The vitamin (>99% pure and obtained from Eastman Kodak Company, Rochester, NY) was administered in corn oil by gavage at the rate of 3.5 ml/kg
body weight daily for up to 13 weeks.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
125, 500, 2000 mg/kg bw/d
Basis:

No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
Two control groups (10 rats/sex/group) were either administered corn oil by gavage (vehicle control group) or remained untreated (untreated control group).
Post-exposure period: none
Positive control:
None
Observations and examinations performed and frequency:
Individual blood samples (with or without heparin) for haematology and clinical chemistry determinations were collected by terminally bleeding ten males and ten females from each treatment group via the vena cava at days 5, 45 and 90 of dosing treatment.
Haematology determinations, which included mean corpuscular cell volume, haemoglobin, haematocrit, erythrocyte count and total and differential leucocyte counts, were made on a Baker 7000 cell counter (Baker Instruments, Allentown, PA) . Prothrombin time (PT) and activated partial thromboplastin time (APTT) were determined according to the Coag-a Mate operator's manual (General Diagnostics, Division of Warner-Lambert Co, Morris Plains, NJ) . Fibrinogen levels were determined according to the turbidimetric procedure of Exner using the Cetrifichem 400 (Exner, 1979). Serum samples were analysed for chloride, alkaline phosphatase, thyroxine (T4) and γ-glutamyl transpeptidase (GGT) using the Centrifichem 400 according to the procedures specified by the manufacturer (Baker Instruments, Allentown, PA). Triiodothyronine (T3) and thyroid-stimulating hormone (TSH) were determined by radioimmunoassay procedures (Kieffer, 1974) .
Sacrifice and pathology:
At the end of the treatment period, surviving animals were sacrificed and subjected to complete necropsy.
Organ weights and organ-to-body weight ratios were determined for brain, heart, kidneys, liver, spleen, thyroid and parathyroid (combined), thymus, pituitary gland, ovary, uterus, prostate, epididymis and testes.
In addition to these organs, the spinal cord, eyes, salivary gland, mammary gland, oesophagus, lungs with mainstem bronchi, trachea, stomach, small and large intestine, adrenals, pancreas, urinary bladder, mesenteric lymph node, bone marrow, sternum and sciatic nerve with skeletal muscle were
collected, fixed in 10% buffered formalin, embedded in paraffin, sectioned at 5 ,um and stained with haematoxylin and eosin. All tissues from the control and the high-dose groups were examined microscopically. For the lower-dose groups, only tissues that were designated as targets for vitamin E toxicity were examined (not further specified).
Statistics:
The significance of differences between Vitamin E-treated and control groups was assessed using the Dunnett's multiple comparison procedure. Dose-response trend was assessed by Jonckheere's test. Results were considered significant when the calculated P value was 0.05 or less.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
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:
effects observed, treatment-related
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Details on results:
Seven out of 10 high dose males died during weeks 9-11.
The test substance had no effect on food consumption or body weights.
The relative liver weight was significantly increased in high dose females.
Administration of 2000 mg/kg bw/d caused hematological changes: prolongation of both prothrombin and activated partial thromboblastin (APTT) times, reticulocytosis and a decrease in hematocrit values and hemoglobin concentrations was observed in males; APTT times were also increased in females. Hemorrhagic diathesis was observed in males and females of the high dose group; and increased medullary erythropoiesis was seen in the spleen of one high dose male.
A compound-related increase was also observed in the plasma levels of thyroid-stimulating hormone. However, without a corresponding increase in T3 and T4, and no evidence for increased energy metabolism ( food consumption and body weights were comparable to control groups), the toxcicological relevance was doubted.

The test substance at all dose levels tested caused interstitial inflammation and adenomatous hyperplasia of the lung. The lung lesions were observed in all vitamin E-treated groups, and the incidence and severity increased in a dose-dependent manner. These lesions were characterized by increased cellularity, vascular congestion, thickened alveolar walls and the presence of foamy macrophages (some of which had undergone cell death and degeneration) in the alveolar spaces. A lipid-like yellow pigmentation was often present within either the macrophages or alveoli.
None of these lesions, however, were considered to reflect systemic toxic effects of the test or reference articles. The changes in the lungs were considered to be a local reaction to the aspiration of a-tocopheryl acetate into the lungs that would not occur under conditions of normal use.

Therefore, for the NOAEL derivation the effects in the lungs were not considered.
Because at 500 mg/kg only the APTT value is increased, but not the PT and fibrinogen values, the NOAEL is set at 500 mg/kg.

Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subchronic
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

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

No studies specifically conducted with the free racemic Vitamin E form (D,L-alpha-tocopherol) have been reported. Therefore, experimental data for the Vitamin E esters D- and D,L-alpha-tocopheryl acetate is used which will be rapidly hydrolysed to the free Vitamin E alcohol (D- or D,L-alpha tocopherol) under physiological conditions. Please see read-across justification section 13.

A reliable (GLP, OECD 407) 28 -day study with D,L-alpha tocopheryl acetate in rats is available (Pfister, 1999). The rats were treated by oral gavage at 180, 600 and 2000 mg/kg bw d. As no treatment-related findings were noted, the NOAEL in this study was set at 2000 mg/kg bw/d.

The low toxicity of Vitamin E acetate after repeated administration was supported by further 28 -day feeding studies in rats and dogs (BASF 1983 and 1982). The rats were treated with D,L-alpha tocopheryl acetate at dose levels ca. 139, 278, 556 and 1111 mg/kg bw/d and the dogs at dose levels of ca. 45, 90, 180, 360 mg/kg bw/d in the diet. There were no significant, substance-related differences between the groups with regard to body weight, food consumption, and clinical-chemical, hematological, and urine parameters. The NOAELs can be set at >= 1111 mg/kg bw/d in the rat study and >= 360 mg/kg bw/d in the dog study.

A 90-day repeated dose (oral gavage) study is available for D-alpha-tocopheryl acetate (Abdo et al. 1986). In this study, the rats were dosed at 125, 500 and 2000 mg/kg bw/d D-alpha tocopheryl acetate. The relative liver weight was significantly increased in high dose females. Administration of 2000 mg/kg bw/d caused hematological changes: prolongation of prothrombin and activated partial thromboblastin (APTT) times and an increase in fibrinogen value, reticulocytosis and a decrease in hematocrit values and hemoglobin concentrations was observed in males; APTT times were also increased in females. Hemorrhagic diathesis was observed in males and females of the high dose group; and increased medullary erythropoiesis was seen in the spleen of one high dose male.

The test substance at all dose levels tested caused interstitial inflammation and adenomatous hyperplasia of the lung. The lung lesions were observed in all vitamin E-treated groups, and the incidence and severity increased in a dose-dependent manner. These lesions were characterized by increased cellularity, vascular congestion, thickened alveolar walls and the presence of foamy macrophages (some of which had undergone cell death and degeneration) in the alveolar spaces. A lipid-like yellow pigmentation was often present within either the macrophages or alveoli. These effects were attributed to local aspiration of the test substance (as in the other oral gavage 90-day study this was not observed), which would not occur under normal circumstances. Furthermore, these effects were not seen in the chronic feed study (Wheldon, 1978). Therefore, for the NOAEL derivation the effects in the lungs were not considered. Because at 500 mg/kg only APTT values were increased in absence of an increase in PT and fibrinogen value, the NOAEL is set at 500 mg/kg bw/d.

In a long-term toxicity study rats were fed D,L-alpha-tocopheryl acetate at dietary concentrations providing dosages of 0, 500, 1000 and 2000 mg/kg bw/d for 104 weeks (Wheldon et al., 1983). Vitamin K supplementation suppressed the induced hypoprothrombinemia. Growth rate and survival were unaltered by treatment. The observation of agglomerations of vacuolated macrophages in the hepatic centriacini in all treatment groups, alongside changes in serum liver enzyme activity, suggested a limited hepatic response to vitamin E overload. A NOAEL could not be established, since hemorrhage effects were treated by supplementing Vitamin K after 26 weeks.

 

For the DNEL-derivation, a NOAEL of 500 mg/kg bw /d derived from the 90 -day repeated dose with D-alpha-tocopheryl acetate (Abdo,1986) study will be used.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Reliable study which followed current guideline requirements

Repeated dose toxicity: via oral route - systemic effects (target organ) cardiovascular / hematological: blood coagulation

Justification for classification or non-classification

According to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 classification is not warranted.