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 µg/kg bw for trenbolone acetate (TBA) based on a
no-hormonal-effect level of 2 µg/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 µg 17-alpha-trenbolone (17alpha-TBOH) in
methylcellulose/kg bw/day for 23 weeks. Another group (10/sex)
received 40 µg 17-beta-trenbolone 17ß-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 µg/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
µg 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 µg/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 µg/kg bw
17alpha-TBOH (Dean, 1988; Hooks et al., 1988).
2.2.5 Special studies on macromolecular binding
ß-TBOH (purity >97%) was irreversibly bound to DNA isolated from
Salmonella typhimurium TA 100 after incubation of bacteria with [3H]
ß-TBOH (Lutz et al., 1988).
Covalent binding of ß-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).
ß-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 ß-TBOH
Concentration
Test Test of substance
system object tested Purity Results Reference
Ames test S.typhimurium 0-1000 Lutz
(both with µg/plate? ß-TBOH, et al.,
and without 333 µg/plate 1988
activation) TA 100 "positive"(1)
TA 98 negative
TA 102 negative
Cell Syrian hamster 1.0-7.5 µg ml >99% positive Schiffmann
transformation embryo ß-TBOH et al.,
fibroblasts 1.0-7.5 µg ml >99% positive 1988
assay alpha-TBOH
Cell Mouse 1-10 µg ml >99% negative (2) Schiffmann
transformation C3H10T1/2 ß-TBOH et al.,
cells 1988
assay
Micro- Syrian hamster 5x10-6-10-4M both positive Shiffmann
nuclei embryo ß- and 99% et al.,
induction fibroblasts alpha-TBOH 1988
Micro- Mouse 5x10-6-10-4M both negative Schiffmann
nuclei C3H10T1/2 ß- 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 µg/kg bw/day) and
17ß-TBOH (0.1, 1, 10, 16, 24 or 36 µg/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 17ß-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 µg 17ß-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 µg 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 µg ß-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 µg 17ß-TBOH/kg bw/day and 100 µg
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 µg/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 µg 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 µg/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 µg 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 µg/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 µg/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 ß-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 ß-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 ß-TBOH. On the basis of regression analysis,
ß-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 ß-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 ß-TBOH were re-evaluated. The previous
no-hormonal-effect levels of 10 µg/kg bw/day for ß-TBOH and 100 µg/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 µg/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 µg/kg bw/day).
A safety factor of 100 was applied to the marginal-effect level
of 2 µg/kg bw/day for TBA in the 14-week study in pigs, giving an ADI
of 0-0.02 µg/kg bw for TBA. A no-hormonal-effect level of 2 µg/kg
bw/day for TBA was supported by the no-hormonal-effect level of 2
µg/kg bw/day for TBA was supported by the no-hormonal-effect level of
2 µg/kg bw/day for ß-TBOH.
4. EVALUATION
Level causing no hormonal effect
Pig: marginal no effect level for TBA: 0.1 ppm in the diet, equal
to 2 µg/kg bw/day.
Monkey: 17 ß-TBOH: 2 µg/kg bw/day.
Estimate of acceptable daily intake
O - 0.02 µg/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.