TRENBOLONE ACETATE
EXPLANATION
Trenbolone acetate was considered at the twenty-sixth meeting of
the Joint FAO/WHO Expert Committee on Food Additives (Annex 1,
reference 59), but it could not be evaluated at that time because the
necessary documentation on residue levels, good animal husbandry in
relation to the use of the agent, and details of methods of analysis
were not available.
At the twenty-seventh meeting (Annex 1, reference 62) the
Committee provisionally accepted the use of trenbolone acetate as an
anabolic agent for the production of meat for human consumption in
accordance with good animal husbandry practice, and requested the
submission of the results of a study known to be in progress to
establish a no-hormonal-effect level in non-human primates.
This monograph contains the data previously considered by the
Committee, as well as data that have been submitted recently.
BIOLOGICAL DATA
Biochemical aspects
Absorption, distribution, excretion, and metabolism
Rats
Male Sprague-Dawley (bile duct cannulated) rats received single
i.v. doses of 28 mg/kg b.w. 3H-labelled trenbolone acetate (TBA).
Eighty-four percent of the administered radioactivity was excreted via
the bile in 24 hours after dosing (6% "free", 37% as the glucuronide,
and 37% as the sulfate). 3-Ketotrienic structures accounted for 66% of
biliary radioactivity; 17-alpha-hydroxytrenbolone (alpha-TBOH) was not
detected in the bile. The identified 3-ketotrienic metabolites are
presented in Figure 1 (Pottier et al., 1978).
Cattle
Two male calves, each given s.c. implantations with 140 mg TBA at
the base of the right ear, showed a high urinary elimination rate of
trenbolone (TBOH) (detected fluorometrically). Within 3 hours after
application relatively high concentrations were measured (50-80 ng/mg
creatinine); the maximum TBOH level was reached after 10 hours (about
120 ng/mg creatinine) followed by a sudden drop within two days.
Additional implantation of estradiol-17ß reduced TBOH excretion very
slightly (Bouffault, 1977).
Groups of 3 - 4 bull calves were given s.c. implantations of
20 mg 3H-estradiol-17ß or 20 mg 3H-estradiol-17ß + 140 mg TBOH.
TBOH caused a marked delay in estradiol excretion. In calves receiving
estradiol only, the maximum plasma estradiol-17ß level was 3 nmole/l,
and 95% of the applied radioactivity was excreted in the urine and
faeces within 20 days; after more than 31 days radioactivity was no
longer detectable in the urine or faeces. Calves treated with TBOH
showed a maximum plasma estradiol-17ß level of 0.33 nmole/l and
excretion of radioactivity was observed up to 107 days after
administration; at that time faecal and urinary radioactivity levels
were still 1.4 - 3 nCi/g (Riis & Suresh, 1976).
Twelve calves weighing 150 - 200 kg each received s.c. implants
in the ear containing 200 mg 3H-TBA. Half of the animals were
sacrificed at 15 days, the other half at 30 days after implantation.
Blood samples were taken at intervals between dosing and sacrifice. At
sacrifice, the liver, kidneys, and samples of muscle, fat, and bile
were taken for analysis. Concentrations of radioactivity in the plasma
were fairly constant during the experimental period, with mean levels
of 4 to 5 ng equivalents/ml. Tissue concentrations of radioactivity
were either similar at 15 and 30 days or were higher at 30 days.
Highest concentrations were found in the liver (42 and 49 ng
equivalents/g at 15 and 30 days, respectively). Lower concentrations
were found in the kidneys (15 - 20 ng equivalents/g) and muscle and
fat (2 - 3 ng equivalents/g). High concentrations of radioactivity in
the bile (1073 and 736 ng equivalents/ml at 15 and 30 days,
respectively) indicate its importance in excretion of this compound.
Comparison of total and non-volatile radioactivity showed that only a
small amount of tritiated water was produced. About 10% of the
radioactivity in the liver samples was extracted by diethyl ether or
ethyl acetate, and this proportion increased to about 20 - 30%
following incubation with ß-glucuronidase, indicating the presence of
a glucuronide(s) (Hawkins et al., 1984).
Two heifers were given single s.c. implantations with 300 mg
3H-labelled TBA. One heifer was killed 60 days after implantation;
the implant was removed from the other heifer after 60 days and the
animal was killed 16 days later. The H content in the liver, kidneys,
muscle, and fat varied from 0.5 to 25 ppb. Of these residues, 1 - 5%
was TBA, TBOH, and trenbolone glucuronide; up to 5% was found in other
organic-soluble material. Of the remaining radioactivity, about 50%
was water soluble, and the insoluble residue could be made water
soluble by treatment with the proteolytic enzymes pepsin and trypsin
(Ryan & Hoffman, 1978).
Two heifers were given single s.c. implantations with 300 mg
3H-labelled TBA. After 60 days the implants, which still contained
31% of the initial radioactivity, were removed. One heifer was killed
immediately, the other was maintained for 16 days after implant
removal and then killed. Ethyl acetate-extractable radioactivity in
blood plasma could largely be ascribed to TBOH; in most cases no TBA
was found in plasma. Plasma concentrations during days 1 - 55 after
dosing were 5 - 13 ppb; after 58 days a large increase in both total
and nonvolatile radioactivity was observed (17 - 20 ppb). The
half-lives for plasma disappearance of total and non-volatile
radioactivity were 32 and 29 days, respectively, during the
implantation period and 18 and 14 days, respectively, during the
withdrawal period. Plasma ethyl acetate-extractable radioactivity
amounted to 10 - 74% of the total radioactivity during days 1 - 55
after implantation, and this declined to 5% at 16 days after implant
removal. In the 16 days from implant removal to sacrifice,
radioactivity decreased by 58% in muscle, 75% in liver, 77% in
kidneys, and 74% in fat (Chasseaud et al., 1976).
Heifers (aged 15 months, number not given) were given daily oral
doses of 0.4 or 8 mg TBA per animal for 9 weeks. After 1 and 2 weeks
TBA was detected in the urine. Two weeks after drug withdrawal the
compound was detected in some urine samples, whereas after 3 weeks no
TBA was detected (Stephany et al., 1976).
A 14-month-old heifer, after i.v. administration of 10 mg/kg b.w.
TBA, excreted 80% of the administered radioactivity in the bile during
the first 24 hours; 3.5% was in the free form, 30% was excreted as
glucuronides, and 30% as sulfates. Metabolites with the 3-ketotrienic
structure that were identified in the bile are presented in Figure 2.
Three compounds that had lost their ketotrienic structure were also
isolated; these metabolites are presented in Figure 3. Less than 1% of
the administered radioactivity was isolated as tritiated water
(Pottier et al., 1978).
Specimens of muscle from the back and rear leg and specimens from
the liver were taken from two heifers that had been implanted two
months earlier with 300 mg 3H-TBA. In addition, bile was collected
by catheterization of one heifer on days preceding slaughter. The
radioactivity content of muscle, independent of its location, was
one-tenth the level in liver, whereas radioactivity levels in the bile
were 15 times higher than in liver tissue, alpha-TBOH and ß-TBOH
concentrations were determined by reverse isotopic dilution. On
average, the concentration of ß-TBOH was 0.05 to 0.1 ppb in various
tissues, whereas that of alpha-TBOH, which was only 0.005 ppb in the
muscle, reached 0.88 ppb in the liver. Following enzymolysis, ß-TBOH
was not detected in the bile, which contained, by contrast, nearly
200 ppb alpha-TBOH. Thus, alpha-TBOH represented 10% of total TBOH in
muscle, 90 - 95% in the liver, and more than 99% of the total in bile
(Pottier, 1979).
3H-TBA was implanted in the ears of two heifers (300 mg;
388 mCi) and the distribution of the radioactivity in liver and muscle
tissue was determined, applying rigorously standardized organic or
aqueous extraction procedures, either directly or following enzymatic
hydrolysis and proteolytic procedures. These steps yielded almost 100%
recovery of the radioactivity and indicate that only 5 to 15% of the
total residues were extractable with organic solvents. The remaining
radioactivity was either soluble in aqueous media or remained bound to
tissue structures. In another experiment, liver tissue from a calf
treated with 3500 mg TBA 68 days prior to slaughter was examined by
applying radioimmunoassay techniques to determine TBA/TBOH ratios.
Trienic-steroid type residues were obtained only from fractions
containing residues extractable with organic solvents (Hoffman et al.,
1984).
Two barren cows, after i.v. administration of 10 mg 3H-TBA per
animal, displayed very rapid hydrolysis of 3H-TBA in the blood
plasma; after 0.1 hour, only 2% of the radioactivity was recovered as
TBA, whereas 70% was recovered as TBOH. After 2 hours, radioactivity
could no longer be extracted, and in the extracted fraction polar
components predominated. From 3 - 8 hours TBOH disappeared from the
blood (half-life, 1.5 hours) (Pottier et al., 1975).
In two barren cows after s.c. implantation of 300 mg 3H-TBA per
animal at the base of the ear, slow resorption from the implant
occurred; the half-life of disappearance from the implant was 68 - 84
days. About 33% of the radioactivity was extracted in the blood plasma
over the 3-month period after implantation, 70% of which was accounted
for by TBOH. The main routes of excretion were via the bile and urine.
Tissue levels after 3 months were about 1 ppb, except in the liver
(6.5 ppb) and kidneys (4.5 ppb). Twenty-five percent of the tissue
radioactivity was extractable, 40% of which was TBOH. In the liver and
kidneys, however, only 10% was extractable, while in perirenal fat up
to 88% of the radioactivity was extractable. In perirenal fat 50% of
the radioactivity was TBA. Radioactivity levels in the implantation
zone were 8 - 21% of the implanted quantity (Pottier et al., 1973;
Pottier et al., 1975).
Slow resorption from s.c. implants of 300 mg 3H-TBA occurred in
2 lactating cows. The half-life for disappearance from the implant was
approximately 60 days. About 17% of the radioactivity present in the
blood plasma over the period of 5 months after implantation was
extractable. Less than 1% of the radioactivity was excreted in milk.
Ten percent of the milk radioactivity was extractable and 25% of this
extractable radioactivity was TBOH. Tissue levels after 5 months were
about 1 ppb, except in the liver (3.4 ppb) and kidneys (2.7 ppb).
About 25% of the tissue radioactivity was extractable, except in the
liver and kidneys (both 10%); about 40% of this extractable
radioactivity was TBOH. In contrast, 88% of total radioactivity in
perirenal fat was extractable, of which 50% was TBA. Unchanged TBA was
found in no other tissues. Radioactivity levels in the implantation
zone were 8 - 21% of the implanted quantity after 5 months (Pottier
et al., 1973; Pottier et al., 1975).
Two steers were given by single s.c. implantations 300 mg
3H-TBA in combination with 40 mg estradiol; the implants were
removed 60 days later, at which time 28% of the radioactivity remained
in them. Ethyl acetate-extractable radioactivity in blood plasma was
primarily ascribed to TBOH; in most cases no TBA was found in the
plasma. One animal was killed immediately after removal of the
implant. Plasma concentrations in this animal declined with half-lives
of 26 days for both total and non-volatile radioactivity; ethyl
acetate-extractable radioactivity in the plasma of this animal ranged
between 3 - 5% of the total radioactivity. In the other animal, which
was killed 16 days after removal of the implant, plasma concentrations
declined during days 1 - 60, with half-lives of 50 and 55 days for
total and non-volatile radioactivity, respectively. In the 16 days
from implant removal to sacrifice, radioactivity decreased by 46% in
muscle, 2% in liver and kidneys, and 29% in fat (Chasseaud et al.,
1976).
Relay bioavailability
Groups of 3 rats were fed freeze-dried or ethyl acetate-extracted
liver, kidney, or muscle obtained from two heifers killed 60 days
after s.c. implantation with 300 mg 3H-TBA. 3H-TBA levels in the
heifers averaged 30 ng equivalents/g in the liver, 24 ng equivalents/g
in the kidneys, and 3.2 ng equivalents/g in muscle. Radioactivity
excretion during the 3 days after feeding these tissues to rats is
presented in Table 1 (Hawkins et al., 1979).
Groups of 3 bile duct-cannulated rats that had been fasted for 24
hours were fed during 1 hour freeze dried liver, kidney, or muscle
from the two heifers described in the previous paragraph.
Radioactivity disposition during 48 hours after feeding of these
tissues is presented in Table 2 (Hawkins et al., 1979).
Table 1. Excretion of radioactivity by rats after being fed tissues
from heifers implanted with 3H-TBA
Excretion in percent of
administered radioactivity
Treatment Tissue Urine Faeces Total
Freeze-dried tissue Liver 3 81 84
Kidney 2 93 94
Muscle 6 85 91
Extracted tissue Liver 5 78 83
Kidney 2 103 105
Muscle 2 73 75
Table 2. Excretion of radioactivity by bile duct-cannulated rats
after feeding of tissue from heifers implanted with 3H-TBA
Excretion in percent of administered radioactivity
Tissue Bile Urine Faeces GI tract + contents Total
Liver 7 5 59 2 74
Kidney 3 1 31 60 95
Muscle 3 2 56 not detected 61
Effects on protein binding
The affinity of alpha-TBOH and ß-TBOH for corticosteroid binding
globulin, measured in vitro using the human plasma of elderly women,
was very low, less than 0.1% bound, compared with 10% for testos-
terone. The affinity of alpha-TBOH and ß-TBOH for testosterone and
estradiol binding globulin was 1% of that measured for testosterone.
When alpha-3H-TBOH was incubated in vitro with female human plasma,
it readily bound to the albumin fraction; only 4% was present
as free TBOH. The total blood clearance of ß-TBOH was twice that of
testosterone (Philibert & Moguilewsky, 1983).
Effects on estradiol-17ß excretion and nitrogen retention
Cattle
Plasma residues of estradiol-17ß in cattle were affected by the
presence of TBA in the s.c. implant. Plasma levels of estradiol-17ß
remained greater than 0.05 ppb for nine weeks in steers after
treatment with 200 mg TBA in combination with 40 mg estradiol-17ß,
whereas the residual levels decreased below 0.05 ppb within 5 weeks
after implantation of 40 mg estradiol-17ß alone (Heitzman & Hardwood,
1977).
Implantation of 40 mg TBA in the dewlap of Friesian bulls
(11 - 16 weeks of age) did not affect nitrogen retention. Implantation
of 140 mg TBA in combination with 20 mg estradiol-17ß at the same
site, however, resulted in a 47% decrease in nitrogen retention
(van der Wal, 1975).
Pigs
Pigs (males, females, and castrated males) were given s.c.
implantations with either 20 mg estradiol-17ß or 20 mg estradiol-17ß
in combination with 140 mg TBA. At 5 weeks after implantation, steroid
estrogens were hardly detectable in the faeces, and serum values for
estradiol-17ß were very low in both groups. Urine estradiol-17ß levels
were 6 - 82 µg/l in the estradiol-17ß group and 16 - 135 µg/l in the
combination group (Kroes et al., 1976a).
Toxicological studies
Special studies on carcinogenicity potential
Rats
Male Wistar rats (number not specified) were injected i.p.
with 15 µg/kg b.w. 3H-estradiol-17ß (53.6 Ci/mmole), 19 µg/kg
b.w. 3H-testosterone (54.0 Ci/mmole), 17 µg/kg b.w. 3H-TBA,
(57.0 Ci/mmole), or 30 µg/kg b.w. 3H-zeranol (50.0 Ci/mmole), all in
95% ethanol solution. The animals were sacrificed 16 hours after
injection and the Covalent Binding Indices (CBI, Lutz, 1979) of the
chemicals to DNA in the liver were quantitated. The CBIs were 11.4,
4.80, 5.62, and 1.65 for estradiol-17ß, testosterone, TBA, and
zeranol, respectively (weak carcinogens have a CBI approx. or equal
10, Lutz, 1979). The positive control, N-hydroxy-acetylaminofluorene,
had a CBI value of 262 (Barraud et al., 1983).
The CBI of TBA as a function of time was measured by
administering 0.83 mCi (22 - 40 µg/kg b.w.) 3H-TBA i.p. to 8 male
rats. The animals were killed at 4, 8, 12, 20, 24, 36, 48, and 96
hours. The highest CBI, 7.82 was obtained after 24 hours; after 96
hours the CBI was 1.11 (Barraud et al., 1983).
Treatment of rodents with initiators of liver cancer can give
rise to phenotypically altered cells which, under suitable conditions,
will develop into foci of potentially pre-neoplastic cells. These foci
may either regress or develop into malignant nodules, but because they
only take a few weeks to become apparent, induction of such foci
represents a useful short-term indication of tumour-initiating
capacity.
alpha-TBOH or ß-TBOH (2.5, 5, or 10 mg/kg b.w.), ethinyl
estradiol (0.05 mg/kg b.w.), testosterone (10 mg/kg b.w.),
nitrosomorpholine (25 mg/kg b.w.), or diethylnitrosamine (200 mg/kg
b.w.) were administered by i.p. injection approximately 18 hours after
partial hepatectomy to Fisher 344 CDF rats (5 males and 5 females per
group). Two groups presented only with vehicle and one untreated group
of 5 males and 5 females each were used as controls. The animals were
allowed to recover for a further 13 days after treatment with the test
agent. The animals then were supplied with tap water and powdered diet
containing 0.02% 2-acetylaminofluorene, except that the diet supplied
to animals in one of the vehicle control groups contained no
acetylaminofluorene. Seven days after commencing the new dietary
regime the animals were treated with carbon tetrachloride at 2 ml per
kg b.w. by intragastric gavage (animals in the vehicle control group
not given acetylaminofluorene were not treated with carbon
tetrachloride). Seven days later the animals were killed by cervical
dislocation and the livers were removed for microscopic examination.
Most of the animals showed moderate lethargy and other clinical
signs for two or three days following the operative procedure, but no
compound-related adverse signs were evident. No significant
treatment-related effects on body or liver weight were reported. Only
animals treated with nitrosomorpholine or diethylnitrosamine showed
significant increases in liver foci compared with the vehicle control
or untreated groups.
None of the steroids examined in this study (including alpha-TBOH
and ß-TBOH) showed any evidence of inducing pre-neoplastic liver foci
at the dose levels tested. The authors concluded that none of these
steroids showed any evidence of being a liver rumour initiator in this
assay (Allen & Proudlock, 1987).
Special study on immunoresponse
Cattle
Antibody production in male and female calves (about 25 animals
per group) was investigated after s.c. implantation of placebo
(lactose), 20 mg estradiol-17ß, 140 mg TBA, or 140 mg TBA + 20 mg
estradiol-17ß. A slight, non-significant immunodepressive effect was
seen in male calves treated with either estradiol-17ß or TBA alone. In
the males treated with the combination, this effect was significant.
In female calves the immunoresponse was unaffected (Gropp et al.,