3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-benzopyran-6-yl acetate

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Remarks:

combined repeated dose and carcinogenicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 November 1974 - 2 December 1976

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study was conducted according to an early version of the method described in OECD guideline 453 and not under GLP conditions, as the study was initiated before introduction of the guideline and GLP. Report is very well-documented.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1978

Report date:

1978

Reference 2

Reference Type:

publication

Title:

D,L-alpha-tocopheryl acetate (vitamin E): A long-term toxicity and carcinogenicity study in rats

Author:

Whelton et al

Year:

1983

Bibliographic source:

Whelton 1983, Int J Nutr Res 53: 287-296

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Remarks:

study conducted before introduction of OECD guideline: No stability test of the test article in food was done prior to the study. Analysis of food and drinking water is not performed.

Principles of method if other than guideline:

Not relevant

GLP compliance:

no

Remarks:

Study was performed prior to introduction of GLP

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-benzopyran-6-yl acetate

EC Number:

231-710-0

EC Name:

3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-benzopyran-6-yl acetate

Cas Number:

7695-91-2

Molecular formula:

C31H52O3

IUPAC Name:

2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-3,4-dihydro-2H-1-benzopyran-6-yl acetate

Details on test material:

- Name of test material (as cited in study report): dl-alpha-tocopheryl acetate
- Physical state: Liquid (viscous oil)
- Storage condition of test material: In metal containers stored in dark unheated area

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River U.K. Ltd, Margate, Kent, England
- Weight at study initiation:
Males: 134 +/- 1 g
Females: 130 +/- 1 g
- Housing: Five rats in one polypropylene cage
- Diet (e.g. ad libitum): Ad libitum, complete rodent diet
- Water (e.g. ad libitum): Ad libitum, tap water
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2

IN-LIFE DATES: From: - To: 2 December 1976

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Dietary concentrations of dl-alpha-tocopheryl acetate were adjusted weekly, taking into account changing bodyweight and food intake, so as to preserve approximately constant dose levels in terms of quantity of drug per unit bodyweight. Homogeneity was achieved by blending each mixture for 20 minutes in a rotary double-cone mixer.
Vtiamin K1 is administered throughout weeks 24, 25 and 26 by adding 1 ml of Konakion (2% Vit K1) to three liters of water, and from week 27 until the end via the diet (5 mg/kg).

DIET PREPARATION
- Rate of preparation of diet (frequency): Each week
- Mixing appropriate amounts with (Type of food): Spratts Laboratory Diet No. 2

VEHICLE
- No vehicle used

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Not relevant

Duration of treatment / exposure:

104 weeks

Frequency of treatment:

Daily in diet

Doses / concentrations

Remarks:

Doses / Concentrations:
500, 1000, 2000 mg/kg/day
Basis:
nominal in diet

No. of animals per sex per dose:

60

Control animals:

yes, plain diet

Details on study design:

- Rationale for selecting satellite groups: No satellite groups

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked:
Ill-health, mortality
Adverse reaction to treatment in respect to behaviour, appearance and locomotion

BODY WEIGHT: Yes
- Time schedule for examinations: study start and weekly

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

WATER CONSUMPTION: Yes, by visual inspection of the water bottles (no results given)
- Time schedule for examinations: No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:
Control and 2000 mg/kg/day group: week 4, 8, 13, 26, 52 and 82
All groups: Week 103

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before treatment start and after 4, 8, 13, 26, 52, 78 and 95 weeks of treatment
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10 males, 10 females from control and 2000 mg/kg/day group
- Parameters checked:
RBC count
Haemoglobin concentration
Packed cell volume
Leucocyte count, total
Leucocyte count, differential
Prothrombin time
Mean cell volume and mean cell haemoglobin were calculated from RBC data

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before treatment start and after 4, 8, 13, 26, 52, 78 and 95 weeks of treatment
- Animals fasted: No data
- How many animals: 5 males, 5 females from control and 2000 mg/kg/day group
- Parameters checked:
Urea concentration
Glucose concentration
Alkaline phosphatase activity (SAP)
Glutamate-pyruvate transaminase activity (SGPT)
Glutamate-oxalacetate transaminase activity (SGOT)
Bilirubin concentration
Total protein concentration
Electrophoretic protein fractions
Sodium (Na) and Potassium (K) concentrations
URINALYSIS: Yes
- Time schedule for collection of urine: before commencement of treatment and after 4, 8, 13, 26, 52, 82 and 92 weeks of treatment
- Parameters checked:
Volume
pH
Specific gravity
Protein concentration
Total reducing substances
Glucose
Ketones
Bile pigments
Blood pigments
Microscopy (presence of cells)

Week 82 and 92: Urobilin

Sacrifice and pathology:

Termination after 52 and 104 weeks

GROSS PATHOLOGY: Yes, for:

Macroscopic examination:
External openings
Cranial cavity
Subcutaneous structures
Thoracic cavity
Abdominal cavity

Organ weight analysis:
Adrenals
Brain
Heart
Kidneys
Liver
Lungs
Ovaries
Pituitary
Prostate
Spleen
Testes
Thyroids
Uterus

HISTOPATHOLOGY: Yes, by microscopic examination
Tissues preserved histology:
Adrenals
All tissue masses and adjacent tissues
Any other macroscopic abnormality
Brain
Caecum
Duodenum
Eye and optic nerve
Heart
Ileum
Kidneys
Liver
Lungs
Lymph nodes (cervical and mesenteric)
Mammary gland ( posterior)
Oesophagus
Ovaries
Pancreas
Pituitary
Prostate
Salivary glands
Spleen
Stomach
Testes
Thymus (where present)
Thyroids
Trachea
Uterus
Urinary bladder
A bone marrow smear was also prepared

Only fixed:
Aorta
Second eye
Bone
Seminal vesicle
Colon
Skeletal muscle
Mammary gland (anterior)
Skin
Sciatic nerve
Tongue

Other examinations:

Not relevant

Statistics:

Inter-group differences in incidence (e.g. mortality or tumour distribution) were examined for significance by the Chi2 test.
Variations in numerical data were examined by Student's t-test (using a pooled variance when more than two groups were involved); unless there were indications (e.g. a significant difference in variance as revealed by Bartlett's test) that a non-parametric test would be more appropriate, in which case the Mann-Whitney test was employed.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

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

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
The only treatment-related sign, and the only sign to occur with any significant frequency, was persistent bleeding from minor trauma: the occurrence of haemorrhagic incidents was clearly circumscribed with respect to time, sex and groups affected

First 26 weeks: 11 males died, no females and no control animals. Among the treated males, the incidence showed no relation to treatment level, remaining at about the same level in each of the three groups. Within 24 hours of commencement of vitamin K1 supplementation, almost all cases of haemorrhage disappeared.
Between weeks 27 and 52, mortality was higher in control than in treated groups, which leaded to an equilibrium in deaths among treated and control group. Animals died mostly on heamorrhage in the first year.
During the second year mortality rates in the 1000 and 2000 mg/kg/day group were very similar to controls. In the 500 mg/kg/day group the number of terminal survivors was higher. Pituitary tumours were more frequently deemed to constitute the cause of deaths in the highest dose group than in other groups (not remarkable).

BODY WEIGHT AND WEIGHT GAIN
In the first year of the study, body weight gain of males was closely comparable, while females receiving 1000 and 2000 mg/kg/day showed slightly enhanced weight gains. 500 mg/kg/day females made marginally higher weight increments than did controls, so that group mean bodyweights at the end of the first year were perceptibly related to dose. This relationship was lost in the second year. In the third quarter of study, no treatment related effects were seen. The last six months of study, age-related changes obscured any trends relating to treatment.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
The food intake of rats receiving 2000 mg/kg/day and of females receiving 1000 mg/kg/day was significantly greater than the control consumption during the first half of the study. In the second year, however, the food intake of females receiving 500 mg/kg/day was significantly lower than that of the controls.

HAEMATOLOGY
No treatment-related effects on cellular components of blood were found.
Prolongation of the prothrombin time was evident in males of all three treated groups (after 4 weeks). At week 8 this was more evident. At week 13, the prothrombin times returned towards normality. Sampling at week 13 revealed that prothrombin times were longer for all three treatment groups as compared to controls. After 3 weeks of treatment with Vitamin K1, the parameter had resumed to normal levels.

CLINICAL CHEMISTRY
Dosage-related elevation of SGPT activity was evident in treated male rats, degree of change increasing as the study progressed through the first six months of treatment. In treated females, increased activity only became apparent after 13 weeks of treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
After 52 weeks:
Liver: Agglomerations of foamy macrophages within the centriacini were noted in several treated rats, distributed among the groups without dosage-relation. However, there was no comparable finding in any control animal, and by counting as positive any rat displaying foamy macrophages in two or more centriacini on the examined section, it was possible to arrive at the distribution given in Table 16 (see background material of this endpoint study record). These data suggest a positive correlation between incidence and period of treatment. In the sample killed at 52 weeks, it was clear that no dosage-relation existed, the lowest dose being apparently more effective than the high dosage in eliciting the foamy macrophage response.
After 104 weeks:
Further examples of centriacinar aggregations of foamy macrophages were found, again confined to rats from treated groups. Indices are recorded again in Table 16 (background material). In males the incidence was distinctly lower. Overall incidence in females was markedly higher as compared to males.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No treatment related effects in first year of study.
There was no evidence that the distribution of the various tumour types was disturbed by treatment with dl-alpha-tocopheryl acetate.

Effect levels

Dose descriptor:

NOAEL

Basis for effect level:

other: Treatment-related incidence of foamy macrophages in liver was observed, however not related to dosage

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

See attached background material.

Applicant's summary and conclusion

Conclusions:

It was concluded that massive dosages of dl-alpha-tocopheryl-acetate, although without tumorigenic effect, are capable of eliciting reaction in the liver, manifesting as the presence of foamy macrophages, a tendency towards higher relative liver weight, increased SGPT activity and lengthened prothrombin times, with irregular increases in SAP. The haemostatic lesion, but not the serum enzyme changes, is rapidly reversible by vitamin K1 supplementation. The only other evidence of response to dl-alpha-tocopheryl acetate consists of increased growth rate and food intake in females receiving 1000 or 2000 mg/kg/day.

Executive summary:

Dl-alpha-tocopheryl acetate was fed to groups each consisting of 60 male and 60 female rats at dietary concentrations providing dosages of 500, 1000 and 2000 mg/kg body weight/day. Diet without test article served as control. After 52 consecutive weeks of treatment, ten males and ten females from each group were killed for histological examination. After 104 weeks of treatment, all surviving animals were killed.

During the first 14 weeks of treatment, prothrombin times were consistently prolonged in males (but not females) at all dosages. Persistent bleeding from minor trauma became a frequent occurrence among these animals, until the vitamin K intake was supplemented from the beginning of Week 24. All groups (including controls) received the same level of supplementation, via the drinking water (for the first three weeks) or the diet (subsequently).

Haemorrhagic incidents then declined within a few days, and prothrombin times resumed normal values. There were no other signs of reaction to treatment. Ophthalmoscopy revealed no treatment-related changes.

Impaired haemostasis contributed to the mortality among males receiving dl-alpha-tocopheryl-acetate at 1000 or 2000 mg/kg/day prior to vitamin K supplementation; five males died (or were killed in extremis) in each of these dosage groups during this period. A further death occurred at the highest dosage during Week 26, but infection rather than impaired haemostasis was causative.

From Weeks 27 to 52, mortality was higher among control than treated males; the total mortality for both sexes displayed no relationship to dosage at the end of the 104 -week period.

Throughout the treatment period, rats of either sex given 2000 mg/kg/day and females given 1000 mg/kg/day consumed slightly but significantly more food than did controls. The body weight increments of females given the test article at 1000 or 2000 mg/kg/day were significantly increased above those of controls during Weeks 4 to16. During this period, males receiving 2000 mg/kg/day were not affected, and although between Weeks 63 and 68 this sub-group suffered a transient diminution in mean bodyweight, this was not considered to relate to treatment. Males receiving 1000 mg/kg/day and both sexes receiving 500 mg/kg/day were not affected at any time. Between Weeks 37 and 40, the bodyweight gains and food intake of both treated and untreated rats were depressed by climatic extremes, with subsequent recovery.

Apart from the lengthened prothrombin time noted above, repeated haematological examinations revealed no significant response to treatment with dl-alpha-tocopheryl acetate. Routine urinanalysis similarly yielded no evidence of effect, although blood was evident in the urine (either voided in vivo, or found in the bladder post mortem) of four males that died in Weeks 18 or 19 while receiving 1000 or 2000 mg/kg/day. Treated males, and later treated females, displayed elevations of serum glutamate-pyruvate transaminase activity (SGPT), unaccompanied by any disturbance of glutamateoxalacetate transaminase activity. All dosage groups were eventually affected, although dosage-relation was not maintained and the difference from control values did not always attain statistical significance. Since SGPT values in controls remained undisturbed throughout, it was concluded that the observed SGPT elevations related to administration of the test article rather than of vitamin K1. Serum alkaline phosphatase activity (SAP) was occasionally elevated in rats receiving dl-alpha-tocopheryl acetate, but bore no consistent relation to SGPT levels. There was no disturbance of any other parameter examined.

At necropsy after 52 weeks of treatment, no treatment-related macropathology was discovered. Organ weight analysis indicated only inter-group differences that were secondary to differences in bodyweight.

Necropsy of animals dying during the second half of the study or killed after 104 weeks revealed higher liver weights (when expressed as a percentage of bodyweight) in females receiving 1000 mg/kg/day. Microscopic evaluation of liver sections revealed foamy macrophages in centriacini in most (60% or more) treated females and some 15% of the treated males. In a higher proportion of cases, these macrophages yielded positive reactions to Oil-Red-O and to the Per-iodic Acid Schiff technique. The inter-group distribution of this change was clearly related to treatment with the test article but was not related to dosage.

There were small but consistently dosage-related reductions i n the incidence of mammary fibroadenomas in both sexes. With this possible exception, there was no evidence of any treatment related alteration of the tumour frequency in any tissue. Occasional cases of hepatic nodular hyperplasia were discovered in all groups, including controls, and their distribution was unrelated to dosage.

It was concluded that massive dosages of dl-alpha-tocoperyl-acetate, although without tumorigenic effect, are capable of eliciting reaction in the liver, manifesting as the presence of foamy macrophages, a tendency towards higher relative liver weight, increased SGPT activity and lengthened prothrombin times, with irregular increases in SAP. The haemostatic lesion, but not the serum enzyme changes, is rapidly reversible by vitamin K1 supplementation. The only other evidence of response to dl-alpha-tocopheryl acetate consists of increased growth rate and food intake in females receiving 1000 or 2000 mg/kg/day.