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    TRENBOLONE ACETATE

    1.  EXPLANATION

          Trenbolone acetate was previously evaluated at the twenty-sixth,
    twenty-seventh and thirty-second meetings of the Joint FAO/WHO Expert
    Committee on Food Additives (Annex 1, references 59, 62 and 80). At
    the thirty-second meeting the Committee established a temporary ADI of
    0 - 0.01 g/kg bw for trenbolone acetate (TBA) based on a
    no-hormonal-effect level of 2 g/kg bw/day.

          At the thirty-second meeting, the Committee requested the
    submission of (1) the final reports, with supporting data, for the
    tissue residue studies in which TBA was administered to heifers and
    TBA in combination with estradiol-17beta was administered to steers;
    (2) data on individual animals from the three hormonal studies in pigs
    that were reviewed at the thirty-second meeting; (3) results from a
    90-day study, in an appropriate species, with orally administered
    alpha-trenbolone hydroxide (alpha-TBOH).

          This monograph addendum summarizes the data that was submitted in
    response to this request, as well as genotoxicity data that have been
    published recently.

    2.  BIOLOGICAL DATA

    2.2  Toxicological studies

    2.2.2  Short-term studies

    2.2.2.1  Rat

          Groups of CD(UK) rats (10/sex/group) received suspensions of 0,
    10, 40, 360 or 3600 g 17-alpha-trenbolone (17alpha-TBOH) in
    methylcellulose/kg bw/day for 23 weeks. Another group (10/sex)
    received 40 g 17-beta-trenbolone 17-TBOH/kg bw/day as a reference
    compound. All animals were observed for clinical signs and mortality.
    Body weight, water and food consumption, food conversion, hematology,
    ophthalmoscopy, clinical chemistry, organ weights, macroscopy and
    histopathology were recorded.

          One high dosed male rat was found dead in the second week of the
    study, probably as a result of an intubation error. Salivation was
    observed in high dosed male rats. Food consumption was significantly
    increased in males at 3600 g/kg bw and platelet counts, PCV and Hb
    values were significantly decreased at all dose levels in male rats.
    MCV values and Trombotest time were significantly decreased at the
    highest dose only. In females a significant increase in Hb and RBC
    values and Trombotest time was observed at the highest dose only. 
    Calcium-ion concentrations appeared to be decreased, but this is most
    probably due to a relatively high control value. Sodium- and
    potassium-ion concentrations were significantly increased in males and
    cholesterol levels significantly decreased in high-dose male and
    female rats. Total protein levels were significantly decreased and
    alkaline phosphatase activity was increased in females at 360 and 3600
    g 17alpha-TBOH/kg bw/day. At the highest dose, prostate and seminal
    vesicle weight were significantly decreased in males and uterus weight
    was significantly decreased in females. Pituitary weight was
    significantly increased in males and significantly decreased in
    females at 3600 g/kg bw. According to the authors no
    treatment-related changes were observed at macroscopical and
    histopathological observation. Specific hormonal parameters were not
    measured in this study. The NOAEL in this study is 40 g/kg bw
    17alpha-TBOH (Dean, 1988; Hooks  et al., 1988).

    2.2.5  Special studies on macromolecular binding

          -TBOH (purity >97%) was irreversibly bound to DNA isolated from
    Salmonella typhimurium TA 100 after incubation of bacteria with [3H]
    -TBOH (Lutz  et al., 1988).

          Covalent binding of -TBOH (purity >97%) to calf thymus DNA was
    studied  in vitro after incubation with and without rat liver S9. The
    greatest DNA binding was found in the absence of the "activation"

    system. Addition of inactive S9 (without cofactors) reduced the DNA
    binding about 20-fold. Intermediate results were found with active S9
    (Lutz  et al., 1988).

          -TBOH (purity 99%) was administered p.o. and i.p. to female SD
    rats and male Wistar rats, respectively. After 8 hours (females) or l6
    hours (males), DNA was isolated from the livers and purified to
    constant specific radioactivity. The Covalent Binding Index (CBI)
    values ranged from 8 to 17. This is relatively low compared to
    aflatoxin B1 and dimethylnitrosamine with CBIs of 10000 and 6000
    respectively (Lutz  et al., 1988).

    2.2.6  Special studies on genotoxicity.

          The results of genotoxicity assays are summarized in table 1.

        Table 1:  Results of genotoxicity assays on TRA, alpha-TBOH and -TBOH

                                                                                               
                                        Concentration
    Test              Test              of substance
    system            object            tested              Purity   Results          Reference
                                                                                               

    Ames test         S.typhimurium     0-1000                                        Lutz
    (both with                          g/plate? -TBOH,                              et al.,
    and without                         333 g/plate                                  1988
    activation)       TA 100                                         "positive"(1)
                      TA 98                                          negative
                      TA 102                                         negative

    Cell              Syrian hamster    1.0-7.5 g ml       >99%     positive         Schiffmann
    transformation    embryo            -TBOH                                         et al.,
                      fibroblasts       1.0-7.5 g ml       >99%     positive         1988
    assay                               alpha-TBOH

    Cell              Mouse             1-10 g ml          >99%     negative (2)     Schiffmann
    transformation    C3H10T1/2         -TBOH                                         et al.,
                      cells                                                           1988
    assay

    Micro-            Syrian hamster    5x10-6-10-4M        both     positive         Shiffmann
    nuclei            embryo            - and               99%                       et al., 
    induction         fibroblasts       alpha-TBOH                                    1988

    Micro-            Mouse             5x10-6-10-4M        both     negative         Schiffmann
    nuclei            C3H10T1/2         - and               99%                       et al.,
    induction         cells             alpha-TBOH                                    1988
                                                                                               
    Table 1 (continued)

    (1)  evaluated as positive because of a consistent dose-related increase
         which never exceeded 1.3 x control, observed only without rat liver
         S9 fraction in TA 100;
    (2)  positive control yielded positive results.
    
    2.2.7  Special studies on no-hormonal effect levels.

    2.2.7.1  Pigs

          Sixty mature pigs (large white, 3-7/group) were administered
    doses of 17alpha-TBOH (0.1, 10, 100, 160, 240 or 360 g/kg bw/day) and
    17-TBOH (0.1, 1, 10, 16, 24 or 36 g/kg bw/day) in corn oil in
    gelatin capsules with the feed for 14 days following castration. A
    control group of 11 castrated pigs received corn oil only. Blood
    samples were taken on days 0, 7, 14, 2l and 28. LH was determined
    before castration and on days 14 and 28 (only for control animals and
    animals treated with 17-TBOH). All pigs were sacrificed at day 28.
    The pituitary, prostate and seminal vesicles were weighed and examined
    by gross pathology and histopathology.

          No effects have been found on body weight and organ weights. A
    decrease in difference in LH concentrations between day 14 and day 0
    was observed at 16, 24 and 36 g 17-TBOH/kg bw/day, a more pronounced
    significant difference was seen between the LH values of days 28 and
    14. At 160, 240 and 360 g 17alpha-TBOH/kg bw/day, the difference in
    LH values between day 14 and day 0 was decreased, although for the
    latter dose group this difference was not significant. Changes in the
    morphology of glandular epithelium of the prostate (an increase in the
    height and acinar size) was observed in the groups treated with 16, 24
    and 36 g -TBOH/kg bw/day (3/7, 2/7 and 6/7 pigs, respectively).

          As found in the previous evaluation, the no observed hormonal
    effect levels in this study were 10 g 17-TBOH/kg bw/day and 100 g
    17alpha-TBOH/kg bw/day (Roberts  et al., 1983).

          Groups of pigs (large white hybrid 5-6 months, 5/sex/group) were
    orally administered 0, 5.0, 7.5 or 10.0 g/kg bw/day TBA in corn oil
    in gelatine capsules with the food for 14 weeks. Observations included
    clinical signs, body weight, food consumption, testosterone,
    estradiol-17beta and progesterone assays (blood samples were obtained
    weekly), macroscopy and histopathology (testes, seminal vesicles,
    uterus, ovaries, mammary glands and liver).

          One female control group pig died at week 11 (as result of a
    myocardial rupture). Body weight and food consumption were not
    adversely affected by the TBA administration. In contrast to
    observations in other studies in male rats, an occasional increase in
    testosterone in males compared to the controls was observed. In males
    of the two highest dose groups, occasional statistically significant

    decreases in progesterone were observed. However, no dose-related
    effects were found. No statistically significant changes for estradiol
    were observed for males or females. Progesterone levels in females,
    controls as well as treated animals, were variable due to estrous
    cyclical variation. A dose-related decrease in thymus weight and
    increase in liver weight were found in males of the two highest dose
    groups. At 10 g TBA/kg bw/day epididymis weight was decreased in male
    pigs and spleen weight was increased in female pigs. Histological
    changes in the hepatocyte cytoplasm (described as a "partial ground
    glass" appearance) were observed in the livers of treated male pigs
    (4/5, 5/5 and 5/5 at 5, 7.5 or 10 g/kg bw/day, respectively).
    According to the authors this effect was not associated with any
    degenerative change, and was probably due to an adaptive response. The
    NOEL in this study was between 5 and 7.5 g TBA/kg bw/day (Cherry,
    1986; Roberts  et al., 1986).

          Groups of large white hybrid domestic pigs (Sus scrofa, 26 weeks
    old, 4/sex) were fed diets containing 0, 0.1, 2.0 or 20 ppm (equal to
    2-3, 40-100 or 400-600 g/kg bw/day) TBA for fourteen weeks (spanning
    the period of puberty). Observations included clinical signs,
    mortality, body weight, food consumption, ophthalmoscopy, hematology,
    clinical chemistry, steroid hormone assays, organ weight (mean and
    relative data are only given for males and females together),
    macroscopy, examination of bone marrow smears and histopathology.

          No effects were seen on most parameters. One pig in the
    highest-dose group was sacrificed after developing a partial paralysis
    of the hindquarters during week 10. Significant dose-related
    hematological effects were found (at week 12) in females receiving 2.0
    or 20 ppm TBA.

          Cholesterol levels were significantly increased in males and
    females at 2.0 and 20 ppm (at weeks 6 and 12). Serum levels of urea
    and ASAT were increased at the highest dose only. Testosterone and
    estradiol levels were significantly decreased in males at 2 and 20 ppm
    TBA and a slight, not significant decrease was found at 0.1 ppm,
    although the predosing estradiol values in this group were relatively
    low. Serum progesterone levels were significantly reduced at the two
    highest doses in females. Seminal vesicle weight and pituitary weight
    were increased at the highest dose only. At 2 and 20 ppm TBA,
    dose-related changes were observed in liver and kidney weight
    (increase), uterus weight (decrease) and testes weight (decrease). A
    marginal effect on testicular weight was observed at the lowest dose
    of 0.1 ppm.

          Dose-related abnormalities were observed in the liver
    (enlargement of hepatocytes, with associated "ground glass" appearance
    of the cytoplasm), testes (interstitial cell atrophy), ovaries

    (suppressed or abnormal cyclical activity, characterized by the
    absence of maturing follicles and/or mature or early regressing
    corpora lutea) and in the uterus (absence of glandular development of
    the endometrium) at 2 and 20 ppm TBA.

          A marginal no-hormonal effect level of 0.1 ppm TBA (equal to a
    range of 2-3 g/kg bw/day) is indicated (Ross  et al., 1980).

    3.  COMMENTS

          In Syrian hamster embryo fibroblasts, morphological
    transformation was induced by both alpha-TBOH and -TBOH. The
    neoplastic potential of the transformed cells was examined by
    injecting them subcutaneously into nude mice; fibrosarcomas developed
    at the sites of injection of the -TBOH- but not the
    alpha-TBOH-transformed cells. No indication of cell transformation was
    obtained in mouse C3H2OT1/2 cells. In Syrian hamster embryo cells but
    not in C3H10T1/2 cells, induction of micronuclei was observed with
    both alpha-TBOH and -TBOH. On the basis of regression analysis,
    -TBOH showed a weak increase in revertant count in  Salmonella
     typhimurium (in the absence of metabolic activation), which would
    generally not be regarded as a positive result.  In vitro covalent
    binding of -TBOH was observed to DNA from  S. typhimurium and to
    calf thymus DNA. In the latter assay. addition of inactive rat
    microsomal protein reduced the binding about 20-fold.

          However, taking into account the results both of the long-term
    feeding studies in rats and mice, and of the comprehensive battery of
    short-term tests, it was concluded that it was unlikely that TBA was
    genotoxic.

          In accordance with the decisions taken at the thirty-second
    meeting, it was decided to base the evaluation of TBA and its
    metabolites on their no-hormonal-effect level.

          The results of the 90-day study with alpha-TBOH in rats were
    reviewed but found unsuitable for establishing a no-hormonal-effect
    level for the alpha-epimar.

          The results of three hormonal studies in pigs carried out with
    TBA, alpha-TBOH or -TBOH were re-evaluated. The previous
    no-hormonal-effect levels of 10 g/kg bw/day for -TBOH and 100 g/kg
    bw/day for alpha-TBOH, determined in castrated pigs, were confirmed.

          In a 14-week study in male and female pigs given TBA in capsules,
    the no-hormonal-effect level in male pigs was between 5 and 7.5 g/kg
    bw/day based on changes in epididymus weight and in plasma
    progesterone concentration. In a second 14-week dietary study using
    TBA in growing pigs, the most sensitive effects observed were changes
    in serum testosterone and estradiol concentrations and testes weight
    in male pigs. These effects were dose-related and significant at
    higher dose levels but marginal at 0.1 ppm (equal to a dose of TBA
    ranging from 2 to 3 g/kg bw/day).

          A safety factor of 100 was applied to the marginal-effect level
    of 2 g/kg bw/day for TBA in the 14-week study in pigs, giving an ADI
    of 0-0.02  g/kg bw for TBA. A no-hormonal-effect level of 2 g/kg
    bw/day for TBA was supported by the no-hormonal-effect level of 2
    g/kg bw/day for TBA was supported by the no-hormonal-effect level of
    2 g/kg bw/day for -TBOH.

    4.  EVALUATION

          Level causing no hormonal effect

          Pig: marginal no effect level for TBA: 0.1 ppm in the diet, equal
          to 2 g/kg bw/day.

          Monkey: 17 -TBOH: 2 g/kg bw/day.

          Estimate of acceptable daily intake

          O - 0.02 g/kg bw TBA.

    5.  REFERENCES

    CHERRY, C.P. (1986). Photomicrography addendum to histopathology
    report No. RSL/663. Oral administration of trenbolone acetate to
    growing pigs over a 14-week period. Unpublished report No. RSL
    663/86476 from Huntingdon Research Centre Ltd., Huntingdon,
    Cambridgeshire, England. Submitted to WHO by UCLAF, 75020 Paris,
    France.

    DEAN, G.A. (1988). 17 alpha trenbolone toxicity to rats by repeated
    oral gavage for 132 weeks. Draft results. Unpublished report No.
    RSL/756 from Huntingdon Research Centre Ltd., Cambridgeshire, England.
    Submitted to WHO by Roussel UCLAF, 75020 Paris, France.

    HOOKS, W.N., BOWMAN, J.C., RAO, R.S., GIBSON, W.A. & GOPINATH, C.
    (1988).  17 alpha trenbolone toxicity to rats by repeated oral
    administration for 13 weeks (final report). Unpublished report No. RSL
    756/881104 from Huntingdon Research Centre, Ltd., Cambridgeshire,
    England. Submitted to WHO by Roussel UCLAF, 75020 Paris, France.

    LUTZ, W.K., DEUBER, R., CAVIEZEL, M., SAGELSDORFF, P., FRIEDERICH, U.,
    & SCHLATTER, C. (1988). Trenbolone growth promotant: covalent DNA
    binding in rat liver and in  Salmonella typhimurium, and mutagenicity
    in the Ames test.  Arch.Toxicol., 62, 103-109.

    ROBERTS, N.L., CAMERON, D.M. & FOXCROFT, G.R. (1983). Effects of
    trenbolone on plasma LH levels and histological changes following
    castration of mature male pigs. Summary report No. RSL/581 from
    Huntingdon Research Centre, Ltd., Huntingdon, Cambridgeshire, England. 
    Submitted to WHO by Roussel UCLAF, 93230 Romainville, France.

    ROBERTS, N.L., CROOK, D. & GOPINATH, C. (1986). Hormonal effects of
    oral administration of trenbolone acetate to growing pigs for fourteen
    weeks. Report No. RSL/663 from Huntingdon Research Centre, Huntingdon,
    Cambridgeshire, England. Submitted to WHO by Roussel UCLAF, 75020
    Paris, France.

    ROSS, D.B., STREET, A.E., & PRENTICE, D.E. (1980). Oral toxicity of
    trenbolone acetate to growing pigs (Sus scrofa) preliminary 14
    week feeding study.  Unpublished report RSL 357/80152 from Huntingdon
    Research Centre, Huntingdon, Cambridgeshire, England. Submitted to WHO
    by Roussel UCLAF, 93230 Romainville, France.

    SCHIFFMANN, D., HIEBER, L., SCHMUCK, G., PECHAN, R., METZEL, M. &  
    HENSCHLER, D. (1988). Trenbolone induces micronucleus formation and
    neoplastic transformation in Syrian hamster embryo fribroblasts but
    not in mouse C3H10T1/2 cells.  Arch.Toxicol., 63, 49-53.


    See Also:
       Toxicological Abbreviations
       Trenbolone acetate (WHO Food Additives Series 23)
       TRENBOLONE ACETATE (JECFA Evaluation)