TRENBOLONE ACETATE



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.

[pic]

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).

[pic]

[pic]

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.,

1975).

Special studies on mutagenicity

The results of mutagenicity assays on TBOH and TBA are summarized

in Table 3.

Special studies on no-hormonal effect levels

Pigs

Groups of 3 - 7 (11 in the control group) mature male large white

hybrid pigs, 8 - 10 months of age, were administered orally 0.1, 1,

10, 16, 24, or 36 µg ß-TBOH/kg b.w./day or 0.1, 10, 100, 160, 240, or

360 µg alpha-TBOH/kg b.w./day in gelatine capsules with the feed for

14 consecutive days after castration. They were maintained for 14 more

days, then killed and examined post mortem. Serial blood samples

were collected prior to castration (day 0) and on days 7, 14, 21, and

28; LH was determined on days 0 and 14 in the animals treated with

alpha-TBOH and on days 0, 14, and 28 in the animals treated with

ß-TBOH. After sacrifice, the pituitary, prostate, and seminal vesicles

were examined by gross pathology and histopathology. Based on changes

in plasma luteinizing hormone (LH) concentrations, distinct hormonal

effects occurred at 160 µg alpha-TBOH/kg b.w./day and at 16 µg

ß-TBOH/kg b.w./day. Thus, the no-observed-effect levels in this study

were 10 µg ß-TBOH/kg b.w./day and 100 µg alpha-TBOH/kg b.w./day. The

morphological examinations performed in this study are of limited

value, since a 14-day period elapsed between final treatment and

slaughter, allowing regression of likely effects. Thus, the

observation of treatment-related androgenic effects in the 16, 28, and

36 µg ß-TBOH/kg b.w./day group points toward the high hormonal

activity of this compound (Roberts & Cameron, 1985).

Four groups of 4 male and 4 female large white hybrid pigs were

given orally TBA incorporated into the diet for 14 consecutive weeks

at 0, 0.1, 2, or 20 ppm (equal to 0, 2.0-3.1, 40-62, or 400-620 µg/kg

b.w./day). Blood samples were obtained for steroid hormone assays

before treatment commenced and after 6 and 12 weeks of treatment.

Table 3. Results of mutagenicity assays on TBOH and TBA

Concentration

Test Test of substance

system object tested Results Reference

Ames S. typhimurium 10-10000 µg/ Negative Hossat et al., 1978

test1 TA98, TA100, plate TBA or

TA1535, TA1537 7:1 TBA:

TA1538 estradiol-17ß2

Ames S. typhimurium 1000, 2000, Negative Ingerowski et al., 1981

test1 TA98, TA100, 3000 µg/plate at 1000

TA1535, TA1537 TBOH µg/plate;

TA1538 equivocal at

cytotoxic

concentrations

Ames S.typhimurium 0.5 - 500 µg/ Negative Richold et al., 1982a

test1 TA98, TA100, plate alpha-TBOH;

TA1535, TA1537 15 - 1500 µg/

TA1538 plate ß-TBOH

Ames S. typhimurium 0.06 - 2 µg/ Negative Schiffman et al., 1985

test1 TA98, TA100 plate TBOH

Clastogenic Human lymphocytes 6, 30, or 60 Negative Richold et al., 1982b

potential in vitro µg/ml alpha-TBOH

or ß-TBOH2,3

Cell Mouse lymphoma 15 - 45 µg/ml Equivocal6 Richold et al.,

mutation L5178Y cells alpha-TBOH; 15 - 1982c, 1983

assay4 65 µ/ml ß-

TBOH5

Table 3. Results of mutagenicity assays on TBOH and TBA (cont'd).

Concentration

Test Test of substance

system object tested Results Reference

Cell Syrian hamster 5, 10, 15 µg/ Equivocal7 Schiffman et al., 1985

transformation embryo fibroblasts ml TBOH

assay

Cell Mouse C3H1OT1/2 2 - 25 µg/ml Equivocal Henderson et al., 1987a

transformation cells ß-TBOH (- act.)

assay1 (-S-9 mix)5

5 - 20 µg/ml Positive

ß-TBOH (+ act.)

(+S-9 mix)8

Forward Chinese hamster 25 - 100 µg/ml Negative Edgar et al., 1985

mutation ovary cells ß-TBOH

assay1 (HGPRT locus) (-S-9 mix)9

25 - 150 µg/ml

ß-TBOH

(+S-9 mix)5

Forward Chinese hamster 25 - 500 µg/ml Negative Henderson et al.,

mutation ovary cells ß-TBOH5 1986a

assay1 (HGPRT locus)

Forward Chinese hamster 3 - 75 µg/ml Negative Henderson et al.,

mutation V79 cells ß-TBOH 1987b

assay1 (HGPRT locus) (-S-9 mix)9

12 - 125 µg/ml

ß-TBOH

(+S-9 mix)

Table 3. Results of mutagenicity assays on TBOH and TBA (cont'd).

Concentration

Test Test of substance

system object tested Results Reference

Micronuclei Chinese hamster 1 - 10 µg/ml Equivocal Henderson et al.,

induction1 ovary cells ß-TBOH (- act.) 1986b

(-S-9 mix)10

6 - 60 µg/ml Negative

ß-TBOH (+ act.)

(+S-9 mix)10

Chromosome Chinese hamster 1 - 10 µg/ml Negative Allen et al., 1985

aberration ovary cells ß-TBOH

assay1 (-S-9 mix)3

6 - 60 µg/ml

ß-TBOH

(+S-9 mix)11

Unscheduled HeLa cells and 2.5 - 15 µg/ml Negative Schiffman et al.,

DNA Syrian hamster 1985

synthesis embryo cells

DNA Cultured human 1 - 512 µg/ml Negative13 Allen & Proudlock,

repair epithelioid alpha-TBOH or 1983

assay1 cells ß-TBOH12

In vivo Rat bone marrow 100 mg/kg b.w. Negative Richold & Richardson,

cytogenetics alpha-TBOH or 1982

assay ß-TBOH once;

25 or 50 mg

alpha-TBOH or

ß-TBOH 4 times3

Table 3. Results of mutagenicity assays on TBOH and TBA (cont'd).

Concentration

Test Test of substance

system object tested Results Reference

In vivo Erythrocytes 100 mg/kg b.w. Negative Allen et al., 1980

micronucleus ß-TBOH3

test

1 Both with and without rat liver S-9 fraction.

2 Dimethylsulfoxide was used as the solvent.

3 Mytomycin C was used as a positive control.

4 Only with rat liver S-9 fraction.

5 20-Methylcholanthrene was used as a positive control.

6 alpha-TBOH at > 22 µg/ml and ß-TBOH at > 15 µg/ml were toxic to the

cells. Both substances induced 2-fold increases in mutation frequency,

but with alpha-TBOH this occurred only at highly toxic concentrations.

7 There was an inverse dose relationship, with the largest number of

transformations occurring at the lowest dose.

8 2-Acetamidofluorene was used as a positive control.

9 Ethylmethanesulfonate was used as a positive control.

10 Colchicine was used as a positive control.

11 Cyclophosphamide was used as a positive control.

12 4-Nitroguinoline-1-oxide (-S-9 mix) and 2-aminoanthracene (+S-9 mix)

were used as positive controls.

13 Increases in nuclear grain count were observed in a limited number of

cultures in one experiment only.

A detailed post-mortem examination was carried out on all pigs

at termination, and tissues were retained for histological

examination. All animals remained generally in good health throughout

the study, and no abnormal clinical signs were noted that could be

associated with experimental treatment. No significant effects of

treatment on body weight, food consumption, or ophthalmoscopy were

noted. There was a marked, dose-related reduction in serum

testosterone levels in male pigs, mean values in the highest-dose

group at week 6 and in pigs fed 2 and 20 ppm TBA at week 12 being

significantly lower than control values (p < 0.01). Testosterone

levels in female pigs remained low at all 3 assay points.

Serum progesterone levels in females were variable. This is not

unexpected, since both progesterone and estradiol show a marked cycle

variation and the assay samples were obtained at arbitrary time points

without regard to the stage of the estrus cycle of individual pigs.

However, mean values at week 12 did indicate a dose-related reduction

in serum progesterone levels in female pigs, and on statistical

examination there was found to be a significant trend with dose

(p < 0.05). Progesterone values for male pigs were less variable and

lower on average than for females and no consistent treatment-related

differences were noted.

Levels of estradiol-17ß in female pigs were generally low,

although there was evidence of some increase in estrogenic secretion

at week 12 in pigs from all groups; no appreciable differences between

groups were observed. Estradiol levels in males were in general higher

than in females; a dose-related reduction in estradiol mean values was

noted at weeks 6 and 12. The results of the hormone assays point to a

no-hormonal-effect level below 2 µg/kg b.w./day.

Treatment-related reductions in mean weights of testes, ovaries,

and uteri were noted. Apparently, the most sensitive organs are the

testes and ovaries. At the lowest dose, no effects on any tissue

weights were observed. The main findings associated with treatment at

the 2 higher levels were atrophy of testicular interstitial cells,

suppression of cyclic ovarian activity, the consequent absence of

glandular development of the uterine endometrium, and lack of alveolar

development and secretion in the mammary glands (highest group only).

Treatment-related changes were not seen in the gonads, uteri, or

mammary glands in any of the animals examined in the lowest-dose group

(Roberts & Cameron, 1985).

Four groups of 5 male and 5 female pigs were given orally TBA as

solutions in corn oil in gelatine capsules with the food for 14

consecutive weeks at 0, 5.0, 7.5, or 10.0 µg/kg b.w./day. Venous blood

samples were taken before treatment began and at weekly intervals

throughout the test period. Plasma radioimmunoassays for testosterone,

estradiol-17ß, and progesterone were carried out. Pigs were sacrificed

after 14 weeks and examined post-mortem. Tissues processed for

histological examination included the testes, ovaries, seminal

vesicles, uterus, and mammary gland. In males, comparison with the

control group showed some differences in testosterone levels at weeks

1 and 5, which reached statistical significance in the two highest

dose groups. However, these differences were not sustained and were in

the opposite direction (i.e. increases compared with the control) to

the dose-related changes (i.e. suppression) noted in the study

described in the previous paragraphs. Testosterone levels in females

generally remained low in all groups.

No significant group differences were found in estradiol levels

in males. In females, estradiol levels generally remained low in all

groups. Mean progesterone levels in males were statistically

significantly lower than in controls between weeks 7 and 10 in the

highest-dose group and at week 10 in the group administered 7.5 µg

TBA/kg b.w./day. However, the mean pre-dose levels were also lower in

pigs in these two groups compared with the controls and analysis of

mean levels over weeks 6 to 14 showed no significant effects. Thus,

these differences were not considered by the authors of the report to

represent a real treatment effect. In female pigs, mean progesterone

values were variable due to cyclic changes, but no consistent group

differences were apparent. Examination of the time of onset of cycling

and the peak level of progesterone recorded did not show significant

effects. No significant differences in organ weights were noted in any

groups. No treatment-related changes in the morphology of the organs

examined were noted in any groups (Roberts & Cameron, 1985).

Monkeys

The antigonadotropic activity of ß-TBOH was tested in acutely

castrated male rhesus macaque monkeys aged 8 - 17 years. A seminal

vesicle biopsy was obtained at the end of the 30-day treatment period

to examine possible changes in androgenic activity induced by TBOH.

ß-TBOH was given orally at 0, 1, 20, or 400 µg/day (three control

animals and two animals per treatment group). From 17 days after

castration until the end of the experiment, the lowest-dose group was

given 1600 µg/day ß-TBOH. Administration of the compound did not

suppress the post-castration elevation in either LH or follicle

stimulating hormone (FSH) secretion, which occurs after removal of the

testes. Although TBOH and testosterone showed no antigonadotropic

activity in this model system, the monkeys receiving 400 and

1600 µg/day TBOH maintained partial or complete seminal vesicle

morphology consonant with an androgenic effect. The expected reduction

in the serum levels of testosterone and estradiol occurred after

castration, and TBOH treatment did not alter the serum concentrations

of these hormones or the typical diurnal pattern of activity within

the hypothalmic-pituitary-adrenal axis. The authors concluded that the

no-hormonal-effect level in this study was 20 µg/day, equivalent to

2 µg/kg b.w./day (Hess, 1983).

TBA was administered in Sustagen/Jello/bran diet cubes for three

cycles, or a maximum of 122 days, to three groups of six mature female

rhesus macaque monkeys weighing 6 kg each at 60, 240, or 960 µg/day.

Blood samples were obtained on a daily basis from all animals during a

pretreatment menstrual cycle, at 3-day intervals during the first two

treatment menstrual cycles, and daily during the last treatment

menstrual cycle. Serum concentrations of estradiol, progesterone, LH,

and FSH were determined by radioimunoassay. Treatment with 960 µg/day

TBA resulted in maximum average serum levels of 2.3 ng/ml of ß-TBOH,

and this dose may have inhibited gonadotropin secretion and ovarian

function in 3 of 16 reproductive cycles.

It was concluded that 960 µg TBA/day had an inhibitory effect on

the pituitary gonadal axis. The anovulatory stage was reached rather

suddenly and from the data presented no conclusions with respect to

changes in the endogenous hormone concentrations, which might signal

this effect, can be drawn. In the 240 µg/day group one animal

exhibited anovulation, which may have been related to treatment. No

effects were observed at 60 µg/day, equivalent to 10 µg/kg b.w./day

(Hess, 1984).

Special studies on relay toxicity

Rats

Female veal calves were given s.c. implantations of 0, 140, or

3500 mg/animal TBA and killed after 10 weeks. A homogenate of veal

meat and veal organs (tongue, heart, lungs, spleen, liver (partial),

and one kidney) from these calves was mixed with the diet of rats in a

two-generation reproduction study. The total length of the experiment

was 114 weeks. The F1a generation was used to study teratogenic

effects. In the group of rats receiving a diet containing 230 ppb TBA,

a slight growth depression was seen. No effects were seen on other

parameters, which included mortality, feed consumption, growth,

fertility, reproduction (copulation, conception rate, duration of

gestation, mean litter weight, litter size, fetal body weight,

mortality rate, and fetal body weight after 3 weeks), haematology,

biochemistry, organ weight, and gross- and histopathology

(Gropp et al., 1978).

Special studies on reproduction

Rats

Groups of 40 male and 80 female rats weighing 133 - 143 g each

were given diets containing 0, 0.5, 1, 4, or 16 ppm TBA from week 9

before mating until day 21 post-partum. An additional group was

handled similarly, in which females were fed 50 ppm TBA from day 1 of

pregnancy until day 21 post-partum. At day 21 post-partum all parent

animals were killed without further examination. There were no major

differences between nominal and detected dietary levels of TBA at the

beginning and end of the study. In females in the 4 and 16 ppm groups,

growth was increased throughout the study (10 - 20%); in the 50 ppm

group growth was increased after mating (10%). A dose-related decrease

in pregnancy rate was seen in the 1, 4, 16, and 50 ppm groups (maximum

-30%). At day 4 post-partum each litter was reduced in size to 4

females and 4 males; up to that time pup mortality was increased in

all dosed groups compared with controls. Litter size and litter weight

on days 0, 4, 12, and 21 post-partum were decreased in the 4 and 16

ppm groups (maximum about -10%) and in the 50 ppm group (about -25%).

Mean pup weights were decreased only in the 50 ppm group from day 4

post-partum onward (maximum -15%) (Hunter et al., 1982).

Groups of 12 male and 12 female rats were fed diets containing

0, 25, 50, or 100 ppm TBA for 63 days and then mated. In these groups,

12/12, 10/12, 4/12, and 1/12 females, respectively, were pregnant

after mating (Ross, 1980).

In a multi-generation reproduction study in CRL:COBS CD(SD)BR

Charles River rats, dietary concentrations of 0, 0.5, 3, or 18 ppm TBA

were fed to F0-generation male rats for nine weeks and female rats

for two weeks prior to mating, then through to termination. Two groups

of F1 generation rats were selected, reared, and mated. One group

was treated continuously at the same dietary concentrations as rats in

the F0 generation ("treated" group) and one was removed from

exposure to TBA at 3 weeks of age and maintained without treatment

throughout ("untreated" group).

Treatment with TBA at 18 ppm was associated with the following

effects: a) generally higher body weights affecting both F0-and

F1-generation treated males and females and females of the untreated

F1 generation; b) depression in mean body weight gain during

gestation affecting both matings of the F0 generation; c) signs of

virilization, namely coarseness of the coat and discoloration of the

skin in F0-generation animals and treated F1-generation females;

d) clitoral prominence in treated F1-generation females and to a

lesser extent in untreated F1-generation females. Similar effects

were observed in F2-generation females from treated F1 parents,

but not in offspring of the untreated F1 generation; e) the presence

of occlusive strands in the vagina and/or precocious/incomplete

vaginal opening affecting treated F1 pups and F2 pups from the

treated F1 generation; f) a delay in the occurrence of testicular

descent affecting F2 pups from the treated F1 generation; g) a

marked reduction in pregnancy rate affecting the second mating of the

F0 generation and the treated F1 generation; h) an increase in

pre-coital time for the second mating of the F0 generation and for

the treated F1 generation; i) a marginal extension of the duration

of gestation affecting the F0 generation and the treated F1

generation; j) a marked increase in the incidence of extended

parturition and total litter loss in the treated F1 generation and a

significant increase in the percentage of males per litter; k) lower

litter size and litter weight, either at birth or at 20-day sacrifice,

after both matings of the F0-generation and of the treated F1

generation; 1) increased post-implantation/pre-birth losses in the

F0 and treated F1 generations; m) at terminal autopsy, findings

additional to those previously described, namely an increase in the

incidence of depression in the forestomach epithelium affecting F0-

and F1-generation males; n) significant reductions in seminal

vesicle/prostate weights in F0- and F1-generation males and

increases in mean ovary weights among F0- and F1-generation

treated females; o) significant decreases in weights of seminal

vesicles/prostate, testes, and epididymes in F1-and F2-generation

male pups at six weeks of age. F1- and F2-generation female pups

showed reduction of adrenal weight at six weeks of age; p) a

significant reduction in anogenital distance among male fetuses and a

marginal increase in the incidence of skeletalvariants after the

second mating of rats of the F0-generation (teratology phase).

Treatment with TBA at 3 ppm was associated with the following

effects: a) retarded body weight gain at the first mating of the

F0-generation; b) coarseness of the coat and discoloration of the

skin affecting one female in the F1-generation; c) a significant

delay in the mean age of vaginal opening in females in the

F1-generation and in F2 offspring from treated F1 parents; d)

the occurrence of incomplete vaginal opening or occlusive strands in

the vagina affecting occasional animals of the F1-generation at 6

week autopsy; e) a slight, but not statistically significant, delay in

testicular descent affecting F2 male offspring from treated F1

parents; f) significantly lower litter size at birth after the first

mating of the F0-generation; g) lower litter weight after the second

mating of the F0-generation; h) marginally reduced litter size in

offspring of the treated F1-generation; i) a significant decrease in

the weight of seminal vesicles/prostate, testes, and epididymes in

F1 and F2 male pups at six weeks of age.

The only apparent effects of treatment at 0.5 ppm were as

follows: a) higher group mean body weights of males from the F0

generation; b) a slight but not statistically significant delay in the

mean age of vaginal opening in F1 pups and F2 pups from the

treated F1 generation (subsequent mating performance and resulting

litter parameters were comparable to those of controls); c) a

statistically significant decrease in seminal vesicles/prostate weight

in treated F1 male pups and in F2 male pups from treated parents

at six weeks of age; d) a significant decrease in weight of epididymes

in F2 males.

The authors concluded that, in terms of reproductive performance

of the two generations examined in this study, as assessed by the

ability of parents to produce and sustain their litters, TBA exerted a

marked effect at 18 ppm and some effect at 3 ppm. At the lowest dose

level examined (0.5 ppm), slight effects were observed, which were

more marked in F2 pups than in F1 pups of a comparable age.

However, the authors concluded that in terms of reproductive

performance TBA exerted no effect at 0.5 ppm. The reproductive

performance for all groups of F1 animals following withdrawal from

treatment showed no marked differences from those of the control group

(James et al., 1985).

Histological examination of the testes, epididymes, seminal

vesicles, and prostate of 6-week-old male F2-generation rats from

treated F1 animals in the study described above revealed no

morphological abnormalities, although lower group mean weights were

recorded in treated groups compared to controls. All rats were

considered normal for that age, when they are not quite fully sexually

mature, with spermiogenesis proceeding to tailed spermatids in the

testes but no spermatoozoa in the epididymes (Offer, 1985).

In a study in rats, dietary concentrations of 0, 0.1, 0.3, 0.5,

3, or 18 ppm TBA were fed to F0 males and females from two weeks

prior to mating until termination of pregnancy. The F1 litters were

reared through the weaning period. On day 22 post-partum the male

offspring were sacrificed, examined macroscopically, and the testes,

seminal vesicles/prostate, and epididymes from each pup were weighed

and preserved. Female offspring were sacrificed on day 24 post-partum

and examined macroscopically.

Treatment with TBA at 18 ppm was associated with the following

effects: a) slightly higher mean body weights of F0 females, but

lower body weight gain during gestation and slightly lower weight gain

of males over the last three weeks of treatment; b) clitoral

prominence at autopsy in 22/29 F0 females and in all F1 female

offspring from approximately 3 weeks of age; c) a statistically

significant extension in duration of gestation; d) total litter loss

in 4/29 F0 females; e) effects on litter parameters, including

reduced litter size, lower litter weight, marginally higher pup

mortality, and higher mean pup weight; f) lower testicular weight and

higher mean weight of seminal vesicles/prostate in F1 males at 22

days of age.

Treatment at 3 ppm was associated with: a) slightly higher mean

body weights of females and slightly lower weights of males in the

three weeks prior to termination; b) a marginal extension in duration

of gestation; c) effects on litter parameters, including reduced

litter size, and marginally higher pup mortality and mean pup weight;

d) lower testicular weight and higher mean seminal vesicle/prostate

weight in F1 males.

The only effects associated with treatment at 0.1, 0.3, and

0.5 ppm TBA were slightly higher mean body weights of females and the

marginal differences observed at 3 and 18 ppm in litter parameters

(James et al., 1986).

Special studies on teratogenicity

Rats

Four groups of 6 pregnant rats each were given by oral gavage

0, 2.5, 5, or 10 mg/kg b.w./day TBA from days 6 - 15 of gestation. The

vehicle was 2.5% ethanol in 1% methylcellulose. Mortality, growth,

number of corpora lutea, number and distribution of live and dead

young, litter weight, mean fetal weight, microscopic fetal

abnormalities, sex ratio, and fetal crown-rump distance were all

unaffected by treatment (James et al., 1982).

Groups of 20 pregnant rats received by oral garage 0, 5, 10, or

20 mg/kg b.w./day TBA from days 6 - 15 of gestation. The vehicle was

2.5% ethanol in 1% methylcellulose. In the 10 and 20 mg/kg b.w./ day

groups, 9/20 and 15/20 dams, respectively, showed hair loss. A

dose-related decrease in growth was seen in all dosed groups (maximum

20%). Pregnancy rate, number of liver and dead young, number of

implantations and corpora lutea, litter weight, mean fetal weight,

incidence of major malformations, incidence of minor visceral

anomalies (Wilson's technique), and fetal crown-rump distance were all

unaffected. The incidences of skeletal variants (number of ribs,

number of normal and variant sternebrae) were unaffected. Mean

anogential distances in male fetuses preserved in Bouin's solution

were slightly shorter than those of controls, with an apparent

slightly decreasing trend with increase in dosage; the difference at

20 mg/kg b.w./day attained borderline statistical significance

(P < 0.05). Male fetuses preserved in alcohol did not show the same

trend in mean values, although the variation in anogenital distance

was greater in pups in the high-dose group than in controls; ranges of

fixation weight and crown-rump distance length were also higher in the

high-dose group than in controls. Mean anogenital distances in females

preserved in Bouin's solution and in alcohol were comparable in all

groups (James et al., 1982).

Acute toxicity

Table 4 summarizes the results of acute toxicity studies on TBA.

Table 4. Acute toxicity of TBA

LD50

Species Sex Route Vehicle (mg/kg b.w.) Reference

Mouse M/F oral 40% ethanol 1500 Audegond et al.,

in corn oil 1981a

M i.p. ethanol + 10% 565 Escuret & Bas, 1978

sesame oil

F i.p. ethanol + 10% 643 Escuret & Bas, 1978

sesame oil

Rat M/F oral 10% ethanol 5000 Audegond et 1981b

in corn oil

M i.p. 10% ethanol 1601 Escuret & Bas, 1978

in corn oil

F i.p. 10% ethanol 1772 Escuret & Bas, 1978

in corn oil

Dog M/F oral via capsules 1000 Audegond et al.,

1981c

Dogs

Anaesthetized dogs were given i.v. 1, 2, 5, or 10 mg/kg b.w. TBA

as a 20 ml/mg solution in 92% acetylmethylamine. Dogs in the 2, 5, and

10 mg/kg b.w. groups showed a dose-related decrease in blood pressure;

at 10 mg/kg b.w., this was accompanied by slight bradycardia. A

decrease in blood pressure in the same groups was seen after injection

with adrenalin and noradrenalin; an increase was seen after

acetylcholine injection. Changes in reactions to histamine were not

noted in any groups (Seeger, 1971a).

Short-term studies

Mice

Groups of 8 male and 8 female mice weighing 19 - 25 g each were

given 0, 25, 50, or 100 ppm TBA in the diet for 8 weeks. Mortality,

appearance, behaviour, body weight, food consumption, and food

conversion were unaffected by treatment. Females in all treatment

groups exhibited significant decreases in absolute and relative liver

weights and significant increases in absolute and relative uterine

weights. Absolute and relative ovarian weights were decreased

significantly in females in the 50 and 100 ppm groups, while absolute

and relative testes weights were decreased significantly in males in

the highest-dose group. No effects on the weights of adrenals,

kidneys, prostate, seminal vesicles, or spleen were noted. Females

showed dose-related suppression of ovarian cyclic activity

characterized histopathologically by the absence of, or few, corpora

lutea in the gonads, a dose-related reduction in the amount of stroma,

and reduction in the number of endometrial glands in the uteri

(Hunter et al., 1976a).

TBA was administered to groups of 8 male and 8 female Swiss

albino CFLP mice for 10 weeks at levels of 0, 1, 2, 5, or 10 ppm of

the diet, equal to mean intakes of 0, 0.12, 0.24, 0.56, or 1.2 mg/kg

b.w./day for males and 0, 0.13, 0.25, 0.66, or 1.4 mg/kg b.w./day for

females. There were no signs of reaction to treatment, including no

treatment-related effects on food intake or body weight gain. No

treatment-related abnormalities were seen; the absolute and relative

weights of all organs examined were considered to be within normal

limits for mice of this strain and age. The only organs examined

histologically were the prostate, seminal vesicles, testes, ovaries,

and uterus from mice in the control and highest-dose group. All

histopathological parameters were within normal limits (Hunter et al.,

1976b).

Rats

Sixteen male rats that had been castrated between 21 and 24 days

of age were given total oral doses of 0, 0.75, 3, 12, or 48 mg TBA

from days 2 - 11 after castration in 10 daily portions. At autopsy,

one day after the last application, dose-related increases in the

absolute weight of the prostate (maximum +440%) and seminal vesicles

(maximum +400%) were seen in all treated groups. In the three

highest-dose groups a dose-related increase in the musculo levator ani

weight (maximum +250%) was noted (Schröder, 1971a).

Groups of 10 male and 10 female CFY rats were fed diets

containing TBA at concentrations of 0, 25, 50, or 100 ppm for 13

weeks. Group mean intakes during the treatment period were 0, 1.8,

3.8, or 7.6 mg/kg b.w./day for males and 0, 2.2, 4.2, or 8.4 mg/kg

b.w./day for females. At all the tested doses, the females exhibited

better efficiency of feed utilization than the males, causing higher

weight gain.

At 25 ppm, lower prostate weight (-36%), which was not associated

with morphological changes, was observed. At 50 ppm, lower prostate

and seminal vesicle weights (-50 and -30%, respectively), which were

not associated with morphological or uterine changes, were observed in

two rats.

At 100 ppm, the following changes were observed: lower neutrophil

and lymphocyte values in males at weeks 12 and 13 (-35%); lower

seminal vesicle weight, which was not associated with morphological

changes (-60%); lower prostate weight (-80%), which was associated

with small alveoli lined by cuboidal epithelium in 5 rats; and uterine

changes characterized by an apparent reduction of the endometrial

stroma with dilated uterine glands and also a corrugated appearance of

the endometrial and glandular epithelium in 6 rats (Hunter et al.,

1976c).

Groups of 10 male and 10 female rats weighing 60 g each were

given 0, 50, 100, 200, or 1000 µg/kg b.w./day TBA orally 6 days per

week for 3 months. The compound was presented as 0.5 ml of an aqueous

solution containing 0.9% NaCl, 0.4% polysorbate 80, 0.5% carboxy-

methylcellulose, and 0.9% benzyl alcohol. Determinations were

carried out in half of the animals at termination only.

Growth of females was slightly increased; in the two highest-dose

groups growth of males was decreased. Haematological parameters were

unaffected. In all dosed groups, SGOT and SGPT were decreased; total

cholesterol was decreased in the 100, 200, and 1000 µg/kg b.w./day

groups. In the highest-dose group a slight decrease in blood glucose

was seen; urea was slightly increased only in the females in this

group. Increases were observed in liver weight in males and females in

the 200 and 1000 µg/kg b.w./day groups, kidney weight in males and

females in the highest-dose group, and spleen weight in females in the

200 and 1000 µg/kg b.w./day groups. In females, ovary weight was

increased in the highest-dose group and uterus weight was decreased in

the 100 and 200 µg/kg b.w./day groups. In males, decreases were seen

in seminal vesicle weight in the 100, 200, and 1000 µg/kg b.w./day

groups and in prostate weight in the 100 and 1000 µg/kg b.w./day

groups. Gross pathological examination revealed atrophy of the

prostate, seminal vesicles, and testes in rats in the highest-dose

group. Histologically, changes in the ovary (cysts and released

follicles in all test groups) and in the uterus ("dentelle uterine" in

the 200 and 1000 µg/kg b.w./day groups) were observed in females. In

males in the highest-dose group a delay in spermatogenesis and aplasia

in the seminal vesicles and prostate were observed (Seeger, 1971a, b).

Female rats fed diets containing 0.01, 0.1, 2.5, 5, 10, 20, 40,

80, or 160 ppm TBA exhibited increases in uterus weight at levels

higher than 40 ppm. Vaginal smears in all groups were negative; some

proliferation of the vaginal mucosa was seen at the highest dose level

(Huis in't Veld et al., 1973).

Female rats weighing 60 - 65 g each were ovariectomized and given

s.c. doses of 0, 0.2, 1.0, or 5.0 mg/day TBA in sesame oil for 4 days.

At termination, on day 5, a dose-related increase in uterus weight was

seen in all dosed groups (maximum +550%). The estrogenic activity of

TBA was less than 0.1% of the estrogenic activity of estradiol-17ß

(Schröder, 1971b).

Male castrated rats weighing 100 g each were given daily s.c.

doses of 4, 20, or 100 µg TBA, 4, 20, or 100 µg ß-TBOH, or 20, 100,

500, or 1000 µg alpha-TBOH for 9 days. Dosing was started 1 day after

castration. One control group was used. At sacrifice, one day after

the last injection, the weights of the prostate, musculo levator ani,

and seminal vesicles were recorded. In animals dosed with both TBA and

ß-TBOH, organ weights were increased in a dose-related manner in all

test groups. In the animals administered alpha-TBOH, organ weights

were increased at the three highest dose levels (Escuret & Bas, 1978).

Male castrated rats weighing 65 - 75 g each were given daily s.c.

doses of 0, 0.02, 0.1, or 0.5 mg TBA as a solution in sesame oil for

10 days after castration. At sacrifice on day 11, dose-related

increases were seen in the weights of muscolo levator ani (maximum

+250%), prostate (maximum +1400%), and seminal vesicles (maximum

+2500%) in all groups. In this experiment TBA showed distinct anabolic

and androgenic activity that was 5 times higher than that of

testosterone and 20 times higher than that of 17-ethynyl-19-nor-

testosterone (Schröder, 1971b).

Groups of 10 male and 10 female rats weighing 123 - 131 g each

were dosed 6 times per week s.c. with 0, 200, 1000, or 5000 µg/kg

b.w./day TBA over a period of 2 months. The compound was applied as a

solution in 1:1 syncortyl and arachis oil.

Growth of females was increased in all groups; by contrast,

growth of males in the highest-dose group was decreased. Haematology

and blood biochemistry determinations, which were performed at

termination in 5 rats/sex/group, revealed slight increases in Hb and

haematocrit values and slight leucocytopenia (due to lymphocytopenia)

in all test groups. Other blood abnormalities that were noted were a

decrease in glucose in females in the 5000 µg/kg b.w./day group (not

determined in other groups), a decrease in BUN in females in the 1000

and 5000 µg/kg b.w./day groups and in males in the 5000 µg/kg b.w./day

group, and a decrease in cholesterol in males and females in the 1000

and 5000 µg/kg b.w./day groups.

In all test groups, absolute and relative kidney weights were

increased and absolute and relative adrenal and thymus weights were

decreased. Ovary weights were decreased in a dose-related manner in

females in all test groups. In females in the 200 and 1000 µg/kg

b.w./day groups uterus weights were decreased. All treated females

showed an increase in absolute liver weight. Males showed a decrease

in testes weight in all treated groups and in seminal vesicle and

prostate weight in the 1000 and 5000 µg/kg b.w./day groups. Besides

these weight changes, atrophy was noted in the thymus, ovaries, and

testes; hypertrophy was seen in the seminal vesicles and prostate

(Sovetal, 1970; Seeger, 1971a).

Rabbits

Groups of 4 - 6 rabbits weighing 2 kg each were administered s.c.

during 4 days 0, 0.05, 0.5, 2, or 5 mg/kg b.w./day TBA. Liver function

was examined (SGOT activity and BSP excretion). At 2 mg/kg b.w./day

SGOT activity was increased slightly, while at 5 mg/kg b.w./day SGOT

activity was increased significantly. BSP excretion was not affected

in any of the test groups (Seeger, 1971a).

Pigs

Male, female, and castrated male pigs were fed diets containing

1-2 ppm TBA, alone or in combination with 2 ppm estradiol-17ß or 2 ppm

ethynylestradiol, for 5 - 8 weeks. At 5 and 6.5 weeks after compound

withdrawal TBOH was not detected in the urine. Urinary excretion of

total steroid estrogen was not increased at 7 weeks after dose

withdrawal (Kroes et al., 1976a).

Groups of 4 male and 4 female domestic pigs (Sus scrofa) were fed

diets containing 0, 0.1, 2, or 20 ppm TBA (equivalent to 0, 4, 80, or

800 µg/kg b.w./day TBA, respectively) for 14 weeks. No treatment-

related effects on mortality, body weight, or ophthalmoscopy were

seen. After 6 and 12 weeks several haematological (i.e., Hb, RBC, PCV,

WBC, diff. WBC, and prothrombin index) and blood biochemical

parameters (i.e., glucose, protein, albumin, albumin/globulin ratio,

SAP, calcium, and creatinine), were unaffected. At 2 and 20 ppm,

dose-related increases were seen in platelets (maximum +70%, not

significant), urea (maximum +3%, significant) and cholesterol (maximum

+100%, significant). SGOT increased in a dose-dependent manner in all

test groups (maximum, +100%, significant). There were dose-related

decreases in blood levels of testosterone and estradiol in males in

all dosed groups (both maximum -95%, significant); progesterone was

markedly decreased (maximum -99%, significant) in females in the two

highest-dose groups. In the same groups, there were dose-related

changes in the absolute and relative weights of the liver (maximum

+30%, significant), uterus (maximum -50%, significant), kidney

(maximum +25%, significant), and testis (maximum -55%, significant).

In the highest-dose group changes were observed in the weights of the

pituitary (-15%, significant) and seminal vesicles (+280%,

significant). There was an increase in thyroid weight at all three

dose levels (maximum +20%, not significant).

Histopathological examination showed the following dose-related

abnormalities in the 2 and 20 ppm groups: in the liver, enlargement of

the hepatocytes with associated ground glass appearance of the

cytoplast; in testes, moderate to complete interstitial cell atrophy

(with normal spermatogenesis within the seminiferous tubules); in

ovaries, evidence of suppressed or abnormal cyclic activity

characterized by the absence of maturing follicles and/or mature or

early regressing corpora lutea; and, in the uteri, absence of

glandular development in the endometrium. This last observation and

the lack of alveolar development and secretion noted in the mammary

glands of the 2 ppm group were probably associated with the anestrous

state suggested by the findings in the ovaries (Ross et al., 1980).

Cattle

Groups of 8 male calves were given s.c. implants of 140 mg TBA +

20 mg estradiol-17ß at 8 or 4 weeks before slaughter. An additional

group was dosed with 200 mg/kg b.w. testosterone + 20 mg/kg b.w.

estradiol-17ß on both occasions. In all treatment groups histological

examination of prostates showed increased secretory activity and

hyperplastic and metaplastic changes (Verbeke et al., 1975).

Six weeks after single s.c. application of 20 mg estradiol-17ß to

male calves, distinct changes in seminal vesicles were found after

histological examination. Treatment of calves with 20 mg estradiol-17ß

combined with 140 mg TBOH, applied s.c., caused very slight changes in

seminal vesicles in 4/11 calves, while in 3/7 animals with unchanged

seminal vesicles, androgenic stimulation was observed (Kroes, 1972).

Male calves that were 11 weeks of age received by s.c.

implantation 20 mg estradiol-17ß alone or 20 mg estradiol-17ß in

combination with 140 mg TBA. Urinary secretion of total steroid

estrogens was high in the animals given estradiol-17ß alone during the

first 12 days after application. After 3 weeks normal values were

reached. In the estradiol-17ß/TBA combination group, gradual and

prolonged excretion of steroid estrogens for up to 42 days after

implantation occurred; after 56 days normal values were reached.

Qualitative determinations of estradiol-17ß and estradiol-17ß in urine

showed that the alpha epimer was present in almost all urine samples;

estradiol-17ß was found in the urine of those calves administered

estradiol-17ß alone. In the combination group, estradiol-17ß was found

in urine at day 21 only. Histological examination of prostates

revealed squamous metaplasia of gland epithelium after both treatments

(Kroes et al., 1976b).

A total of 1480 female calves, aged 7 weeks, were administered by

percutaneous implantation 0, 140, or 3500 mg TBA (groups 1 - 3,

respectively) or 140 mg TBA + 20 mg estradiol-17ß (group 4), 1400 mg

TBA + 200 mg estradiol-17ß (group 5), or 3500 mg TBA + 500 mg

estradiol-17ß (group 6). The calves were slaughtered 10 weeks after

implantation.

Blood parameters (glucose, GOT, GPT, AP, LDH, cholesterol,

bilirubin, Hb, and PCV), urinary density, and pH were unaffected in

all groups. Calcium and phosphorus levels in serum and bone were not

changed; however, magnesium levels in serum and deposition of

magnesium in bone were decreased in groups 3, 5 and 6. In group 3

slight, and in groups 4, 5 and 6 marked, increases in uterus weight

were seen, which were accompanied by proliferation of uterine

glandular cells, while the lumen of the uterus was partially filled

with a watery liquid. In dosed groups ovarian weights were reduced,

accompanied by a diminution in follicular size. These weight changes

were most marked in groups 2, 3 and 6. The diminution of follicular

size, which was accompanied by a decrease in the number of follicles,

was most marked in groups 5 and 6. In all groups, a dose-related

decrease in thymus weight was seen. In group 3 abnormal development of

the clitoris was noted. Histopathological examination showed a

non-dose related proliferation and secretion of glandular tissue in

the mammary glands of animals in groups 4, 5 and 6. Heart, lever,

kidneys, pituitary, pineal gland, adrenal glands, thyroid, and

skeletal muscle did not show abnormalities (Gropp et al., 1975).

Steers and heifers were seven 300 mg TBA by s.c. implantation. An

additional group of steers was given implants containing 140 mg TBA +

20 mg estradiol-17ß. In all groups, plasma urea was decreased during

the 9-week observation period after treatment. Other plasma parameters

(glucose, calcium, phosphorus, magnesium, sodium, potassium, and total

protein) were unaffected. No changes in plasma concentrations of

insulin or growth hormone were seen. A decrease in thyroxine levels in

steers was observed during the 9-week observation period after

treatment; the decrease was most marked in those administered TBA and

estradiol-17ß in combination. A marked reduction, about -50%, in

thymus weight was observed in steers (Heitzman, 1975).

Steers and oxen given, by s.c. implantation, 140 mg TBA alone or

140 mg TBA in combination with 20 mg estradiol-17ß. Control animals

received implantations with the carrier. The combination of drugs

affected urinary excretion of exogenous estradiol-17ß in oxen and

possibly in steers. Histological examination of the prostate revealed

squamous metaplasia in the combination group. In both treated groups,

more active epithelium of the prostate was seen, compared to the

control group (Kroes et al., 1976c).

Long-term studies

Mice

Groups of 64 male and 64 female Swiss albino CFLP mice, weighing

22 - 25 g each, were given diets containing 0, 0.5, 1.0, 10 or 100 ppm

TBA (equal to 0, 0.004, 0.09, 0.86, or 8.6 mg/kg b.w./day TBA for

males, respectively, and 0, 0.005, 0.10, 0.96, or 9.5 mg/kg b.w./day

TBA for females, respectively) for 95 - 104 weeks (the test was ended

when survival was 20% in males or females in the control group). After

13 weeks 12 mice/sex were killed. At that time significant increases

were observed in the absolute and relative weights of the kidneys in

males and females at 100 ppm (20 - 40% increases). Significant

decreases were seen in the weights of the spleen of top-dose females

(-20%) and significant increases were seen at 1.0, 10, and 100 ppm in

males (+25%). Dose-related relative decreases in weights of the uterus

were observed in all dosed females (maximum -25%). At interim

sacrifice, inhibition of ovulation, characterized by a lack of corpora

lutea in all females in the highest-dose group, was observed.

Follicular development proceeded to the mature follicle stage. The

uteri of all females in the 100 ppm group were consistent with the

diestrous stage of the cycle. In the spleens of 6/12 males in the

100 ppm group an increased number of polymor-phonuclear leucocytes in

the red pulp was seen (0/12 in controls); congested sinuses were noted

in 2/12 males of this group (0/12 in controls).

At terminal sacrifice no organ weights were recorded. Terminal

gross- and histopathological examination showed an increase in liver

nodular hyperplasia and dose-related tumours in the male dose groups;

these increases were statistically significant at the two highest

doses. The incidence of liver tumours was also increased in females in

the highest-dose group (8/52 versus 4/51 in controls). There was an

increase in incidence of hepatocyte vacuolation in males in the

100 ppm group. In 100 ppm females, gross pathological examination

showed an increase in the incidence of enlarged and swollen kidneys,

accompanied by a marginal increase in the incidence of nephritis. In

the same group, increases in ovarian cysts (in 10/20 animals versus

1/11 in controls) and enlarged, abscessed, or cystic preputial glands

(in 4/20 females versus 0/11 controls) were seen. The spleens of 4/20

females in the 100 ppm group appeared small (Hunter et al., 1981).

Rats

Six groups of 65 male and 65 female Sprague-Dawley CFY rats

weighing 150 - 200 g each were given diets containing 0, 0.5, 1.0,

4.0, 16, or 50 ppm TBA (equal to 0, 0.02, 0.04, 0.14, 0.56, or

1.80 mg/kg b.w./day TBA for males, respectively, end 0, 0.02, 0.04,

0.16, 0.64, or 1.92 mg/kg b.w./day for females, respectively) for 112

weeks. The parents of the test animals had been dosed at the same

levels from week 9 before mating until day 21 post-partum (except for

the 50 ppm group in which only the dams had been dosed from day 0 of

pregnancy until day 21 post-partum).

The females in the 4.0, 16, and 50 ppm groups showed prominent

pudendum. A dose-related incidence of pendulous anogenital skin was

seen in all female dose groups (maximum incidence, 85%). In males a

dose-related reduction in testes size was seen in all dose groups,

except at 0.5 ppm (maximum incidence, 45%). Growth and food

consumption were decreased throughout the study in males administered

50 ppm TBA (-15 and -10%, respectively). Water consumption was

decreased in the same group up to week 51 (-15%). Urinalysis (9

parameters measured in 5 rats/sex of the 0, 16, and 50 ppm groups on 6

occasions throughout the study) showed no treatment-related changes.

Haematology (10 parameters measured in 10 rats/sex of the 0, 16,

and 50 ppm groups on 6 occasions throughout the study) showed dose-

related slight increases in some parameters in females in the 16 and

50 ppm groups. Male haematology values were generally unaffected by

treatment. Blood biochemistry (13 parameters) showed no compound-

related changes.

At interim sacrifice at week 78, 13 - 14 rats/sex/group were

killed. Macroscopic examination revealed a prominent clitoris in 5/14

females fed 16 ppm TBA and in 13/13 females fed 50 ppm TBA. The males

of the 50 ppm group showed atrophy of the testes, seminal vesicles,

and prostate. Interim organ weighings revealed, in the males fed 16

and 50 ppm TBA, dose-related decreases in absolute testes and prostate

weights. Adrenals and pituitary weights were decreased in both sexes

in the 50 ppm group. Weights of the ovaries were increased in all dose

groups, but without dose relation.

At the end of the experiment, similar changes in the absolute

weights of the testes, prostate, and adrenals were found. In the males

of the highest-dose group, decreases were observed in the weights of

the pituitary (-30%), thyroid (-20%), kidney (-30%), spleen (-30%),

and liver (-20%). The weight of the ovaries was markedly decreased in

the highest-dose females (-60%).

Terminal gross- and histopathological evaluation revealed a

slightly increased incidence of foci or areas of ground-glass

hepatocytes in the livers of the highest-dose females. Urinary bladder

calculi and associated epithelial hyperplasia joined by cystitis and

pyelitis were seen in females in the same dose group. Both the male

and female reproductive tracts were affected. Testicular, prostatic,

and vesicular atrophic changes were seen in males in the 16 and 50 ppm

groups. Absence of corpora lutea, vaginal inflammation and

mucification, uterine endometritis, dilatation, decreased endometrial

thickness, and the development of clitoral bone were seen in the

50 ppm females. Clitoral enlargement was seen in females in the two

highest-dose groups. Neoplastic histopathology showed an increase in

the incidence of pancreatic islet cell tumours in the 50 ppm group

(Hunter et al., 1982).

Observations in human beings

Male and female human volunteers were given i.m. doses of 5 or

10 mg TBA every-other-day during 14 days. In the subjects administered

5 mg TBA, nitrogen balance was disturbed, including retention of

nitrogen. In the 10 mg group, some women showed disturbances of the

menstruation cycle. The same dose caused a slight but significant

reduction of excretion of 17-ketosteroids. No effects on

17-hydroxy-corticosteroid excretion were seen. Blood parameters

(protein, cholesterol, coagulation factors, and prothrombin and

thrombin time) were unaffected by treatment (Krüskemper et al.,

1967).

COMMENTS

TBA is a synthetic steroid with anabolic properties. At the

17-position in the molecule two epimers, alpha and ß, are possible.

The ß-epimer of TBA is the commercial product. It is administered to

cattle either alone or in combination with estradiol-17ß or zeranol as

a subcutaneous implant in the ear to improve body weight, feed

conversion, and nitrogen retention. It is usually implanted 60 - 90

days before the intended date of slaughter.

After administration to cattle, TBA is rapidly hydrolyzed to

TBOH, the major metabolite being alpha-TBOH, occurring in the excreta,

bile, and liver. In muscle most of the TBOH is present as ß-TBOH.

Experiments with implantation of 200 mg of radiolabelled TBA in calves

and heifers showed that maximum levels of residues occurred about 30

days after implantation. The highest mean concentration of residues as

TBOH equivalents was 50 µg/kg in liver, while muscle contained

3 µg/kg.

The results of the studies requested at the twenty-seventh

meeting were submitted for consideration by the present Committee. In

addition, new toxicological data were available on reproduction and

mutagenicity. The Committee also reviewed previously available data

including metabolic, teratogenicity, and carcinogenicity data.

Acute toxicity studies in several species showed TBA to be of low

toxicity, when given orally.

Experiments had been performed in rats to assess the effect of

TBA on pregnancy, on the reproductive function of multiple

generations, and on the development of the offspring to weaning.

Treatment at 0.3 and 0.5 ppm in the diet, equivalent to about

20-30 µg/kg b.w./day, was associated with mean weekly body weights for

female rats that were only slightly higher than those of controls, and

marginal differences in litter parameters, while treatment with TBA at

3.0 and 18.0 ppm in the diet was associated with a hormonal effect. It

was considered that TBA exerted no effect on reproductive performance

in the rat at 0.5 ppm in the diet, equivalent to 30 µg/kg b.w./day. No

teratogenic effect was seen in two feeding studies in rats at very

high doses of TBA. In a comprehensive range of in vivo and in vitro

mutagenicity studies, all tests were negative for TBA, ß-TBOH, and

TBA's major metabolite, alpha-TBOH, with the exception of the mutation

assay in mouse lymphoma cells, which gave equivocal results with

ß-TBOH and alpha-TBOH. The Committee also noted a report of an

equivocal result in a transformation study with ß-TBOH in Syrian

hamster embryo fibroblasts, and took into account the recognized

difficulty in interpreting results from this type of study.

The Committee reaffirmed the opinion expressed at its

twenty-seventh meeting regarding the results of long-term feeding

studies with TBA with rats and mice (Annex 1, reference 62). It

considered that the liver hyperplasia and tumours in mice fed high

doses of TBA (0.9 - 9 mg/kg b.w./day) and the slight increase in the

incidence of islet-cell tumours of the pancreas of rats fed TBA at

1.85 mg/kg b.w./day (the highest dose in the study) arose as a

consequence of the hormonal activity of TBOH.

The Committee therefore concluded that its safety assessment

could be based on establishing the no-hormonal-effect level. It

reviewed a study with castrated male rhesus macaque monkeys

administered ß-TBOH orally, and considered that this model could be

relevant to the human population. The castrated male rhesus monkey is

highly sensitive to compounds with antigonadotropic activity; the

Committee therefore adopted a conservative approach by using this

study as the basis for establishing an ADI for human beings. Despite

the small numbers in each group of monkeys studied, and the advanced

age of the animals used, the Committee set 2 µg/kg b.w./day as a

no-hormonal-effect level, based on assessment of histological changes

in the seminal vesicles. In the intact female rhesus monkey, TBA had a

clear no-hormonal-effect level of 10 µg/kg b.w./day. The Committee

also considered that the pig was a sensitive model for assessing

hormonal effects and noted that, here too, TBA had a no-hormonal-

effect level of 2 µg/kg b.w./day based on assessment of pathological

changes in the testes. Another study in the pig demonstrated that the

hormonal activity of ß-TBOH was about ten times that of alpha-TBOH. No

data on individual animals were available for any of the pig studies.

In the absence of satisfactory toxicological data the Committee

was unable to establish a separate no-effect level for the alpha-TBOH

metabolite. It also noted that this metabolite was not produced in

significant amounts in the rat, which made it inadvisable to

extrapolate from data generated from ß-epimer experiments in that

species.

EVALUATION

Level causing no hormonal effect

Pig: 0.1 ppm in the diet, equal to 2 µg/kg b.w./day.

Monkey: 2 µg/kg b.w./day.

Estimate of temporary acceptable daily intake

0 - 0.01 µg/kg b.w.

Further work or information

Required (by 1990):

( a) the final reports, with supporting data, for the tissue residue

studies in which TBA was administered to heifers and TBA in

combination with estradiol-17ß was administered to steers;

( b) histopathological data on individual animals from the three

hormonal studies in pigs that were reviewed by the Committee;

( c) results from a 90-day study in an appropriate species, with

orally administered alpha-TBOH.

REFERENCES

Allen, J.A. & Proudlock, R.J. (1983). Autoradiographic assessment of

DNA repair in mammalian cells after exposure to 17-alpha-hydroxy-

trenbolone and to 17-ß-hydroxytrenbolone. Unpublished report

No. RSL 619/83731 from Huntingdon Research Centre, Huntingdon,

England. Submitted to WHO by Roussel Uclaf, Paris, France.

Allen, J.A., Brooker, P.C., & McCaffrey, K.J. (1985). Analysis of

metaphase chromosomes obtained from CHO cells cultured in vitro and

treated with 17-ß-hydroxytrenbolone. Unpublished report No. RSL

676/85631 from Huntingdon Research Centre, Huntingdon, England.

Submitted to WHO by Roussel Uclaf, Paris, France.

Allen, J.A., Proudlock, R.J., & Pugh, L.C. (1986). Micronucleus test

on 17-ß-hydroxytrenbolone. Unpublished report No. RSL 728/861306 from

Huntingdon Research Centre, Huntingdon, England. Submitted to WHO by

Roussel Uclaf, Paris, France.

Allen, J.A. & Proudlock, R.J. (1987). Assessment of 17-alpha- and

17-ß-hydroxytrenbolone in the Solt-Farber system for induction of

liver foci. Unpublished report No. RSL 711/861252 from Huntingdon

Research Centre, Huntingdon, England. Submitted to WHO by Roussel

Uclaf, Paris, France.

Audegond, L., Collas, E., & Glomot, R. (1981a). R 1697 (trenbolone

acetate). Single administration study by oral route in the mouse.

Unpublished report No. 81213-A-JL/36-A from Roussel Uclaf Research

Centre. Submitted to WHO by Roussel Uclaf, Paris, France.

Audegond, L., Collas, E., & Glomot, R. (1981b). R 1697 (trenbolone

acetate). Single administration study by oral route in the rat.

Unpublished report No. 81212-A-JL/37-A from Roussel Uclaf Research

Centre. Submitted to WHO by Roussel Uclaf, Paris, France.

Audegond, L., Collas, g., & Glomot, R. (1981c). R 1697 (trenbolone

acetate). Single administration study by oral route in the dog.

Unpublished report No. 81216 from Roussel Uclaf Research Centre.

Submitted to WHO by Roussel Uclaf, Paris, France.

Barraud, B., Lugnier, A., & Dirheimer, G. (1983). In vivo covalent

binding to rat liver DNA of trenbolone as compared to 17-ß-estradiol,

testosterone and zeranol. In: Anabolics in animal production,

International Office of Epizootics, Paris, pp. 193-206.

Bouffault, J.C. (1977). Expert statement concerning the excretion of

trenbolone and oestradiol-17-alpha in urine of calves after

subcutaneous implantation of trenbolone acetate and oestradiol 17-ß.

Unpublished report submitted to WHO by Roussel Uclaf, Paris, France.

Chasseaud, L.F., Hawkins, D.R., Elsom, L.F., Parsonage, M.T.,

Ross, D.B., & Roberts, N.L. (1976). Plasma and tissue concentrations

of radioactivity in cattle after implantation of pellets containing

3H-trenboloneacetete, 3H-oestradiol or combinations of radioactive

drugs with the non-radioactive form of the other. Unpublished report

No. SVT/11/76351 from Huntingdon Research Centre, Huntingdon, England.

Submitted to WHO by Roussel Uclaf, Paris, France.

Edgar, D.H., Bosworth, H.J., Ransome, S.J., & Banks, S.J. (1985). An

assessment of the mutagenic potential of 17-ß-hydroxytrenbolone in

mammalian cells in vitro using the Chinese hamster ovary/HGPRT locus

assay. Unpublished report No. RSL 677/85642 from Huntingdon Research

Centre, Huntingdon, England. Submitted to WHO by Roussel Uclaf,

Paris, France.

Escuret, P. & Bas M.N. (1978). DL-50 chez le rat et chez le souris.

Unpublished report Recherches Veterinaires R.U.-essai 5.77.71 R.

Submitted to WHO by Roussel Uclaf, Paris, France.

Gropp, J., Herlyn, D., Boehncke, E., Schulz, V., Sanderleben, J.V.,

Hanichen, Th., & Geisel, O. (1975). Physiological data including

evaluation of immunoresponse in relation to anabolic effects on veal

calves. Published in "Anabolic agents in animal production",

FAO/WHO Symposium, pp. 131-141, March 1975.

Gropp, J., Buntenkotter, S., & Kaemmerer, K. (1978). Relay toxicity of

anabolic agents, general considerations and some experimental data.

Published at Relay Toxicity and Bioavailability Symposium,

5 October 1978. Report submitted to WHO by Roussel Uclaf,

Paris, France.

Hawkins, D.R., Weston, K.T., Ray, J., Ross, D.B., & Cameron, D.M.

(1979). The bioavailability of trenboloneacetate residues in the

edible tissues of heifers after oral administration to rats.

Unpublished report No. RSL/328/7959 from Huntingdon Research Centre,

Huntingdon, England. Submitted to WHO by Roussel Uclaf, Paris, France.

Hawkins, D.R., Waller, A.R., Moore, D.H., Jordan, M.C., Roberts, N.L.,

& Cameron, D. (1984). Tissue residues of radioactivity at 15 and 30

days after implantation of 3H-trenboloneacetate in calves.

Unpublished report No. HRC/RSL/636 from Huntingdon Research Centre,

Huntingdon, England. Submitted to WHO by Roussel Uclaf, Paris, France.

Heitzman, R.J. (1975). The effectiveness of anabolic agents in

increasing rate of growth in farm animals; report on experiments in

cattle. Published in "Anabolic agents in animal production", FAO/WHO

Symposium, pp. 89-98, March 1975.

Heitzman, R.J. & Hardwood, D.J. (1977). Residue levels of trenbolone

and oestradiol-17-ß in plasma and tissues of steers implanted with

anabolic steroid preparations, Br. Vet. J., 133, 564.

Henderson, L.M., Bosworth, H.J., Ransome, S.J., Banks, S.J., & Ozel,

K.J. (1986a). An assessment of the mutagenic potential of 17-alpha-

hydroxytrenbolone in mammalian cells in vitro using the Chinese

hamster ovary/HPRT locus assay. Unpublished report No. RSL 713/86713

from Huntingdon Research Centre, Huntingdon, England. Submitted to WHO

by Roussel Uclaf, Paris, France.

Henderson, L.M., Brooker, P.C., & Howell, A. (1986b). An assessment of

the mutagenic potential of 17-ß-hydroxytrenbolone in mammalian cells

in vitro using a Chinese hamster ovary micronucleus test.

Unpublished report No. RSL 729/861287 from Huntingdon Research Centre,

Huntingdon, England. Submitted to WHO by Roussel Uclaf, Paris, France.

Henderson, L.M., Ransome, S.J., Banks, S.J., & Bosworth, H.J. (1987a).

An assessment of the carcinogenic potential of 17-ß-hydroxytrenbolone

and testosterone in an in vitro cell transformation assay using

mouse C3H10T1/2 cells. Unpublished report No. RSL 691/861257 from

Huntingdon Research Centre, Huntingdon, England. Submitted to WHO by

Roussel Uclaf, Paris, France.

Henderson, L.M., Ransome, S.J., Banks, S.J., Bosworth, H.J.,

Bottoms, M., & Williams, J. (1987b). An assessment of the mutagenic

potential of 17-ß-hydroxytrenbolone in mammalian cells in vitro

using the Chinese hamster V79/HPRT locus assay. Unpublished report

No. RSL 712/861249 from Huntingdon Research Centre, Huntingdon,

England. Submitted to WHO by Roussel Uclaf, Paris, France.

Hess, D.L. (1983). Determination of a hormonal no-effect dose level

for 17-ß-trenbolone Rhesus Macaque. Unpublished report from Oregon

Regional Primate Research Center, Beaverton, OR, USA. Submitted to WHO

by Roussel Uclaf, Paris, France.

Hess, D.L. (1984). Determination of the hormonal no-effect dose level

for trenbolone acetate in the female Rhesus Macaque. Unpublished

report from Oregon Regional Primate Research Center, Beaverton, OR,

USA. Submitted to WHO by Roussel Uclaf, Paris, France.

Hoffman, B., Schopper, D., & Karg, H. (1984). Investigations on the

occurrence of non-extractable residues of trenbolone acetate in cattle

tissues in respect to their bioavailability and immunological

reactivity. Food Additives and Contaminants., 1, p. 253.

Hossack, D.J.N., Richold, M., & Jones, E. (1978). Ames metabolic

activation test to assess the potential mutagenic effect of trenbolone

acetate, 17-ß-oestradiol and 7:1 mixture of trenbolone with

17-ß-oestradiol. Unpublished report No. RSL 351/781136 from Huntingdon

Research Centre, Huntingdon, England. Submitted to WHO by Roussel

Uclaf, Paris, France.

Huis in't Veld, L.G., Have, J. ten, & Kok, G.L. (1973). Hormonale

werkingen van trenbolone-acetaat. Unpublished report No. U 146/73 ENDO

from the National Institute of Public Health, The Netherlands.

Hunter, B., Macrae, S., Prentice, D.E., & Brooks, P.N. (1976a).

Preliminary assessment of the toxicity of trenboloneacetate when

incorporated in the diet of mice. Unpublished report No. RSL 235/76316

from Huntingdon Research Centre, Huntingdon, England. Submitted to WHO

by Roussel Uclaf, Paris, France.

Hunter, B., Macrae, S., Prentice, D.E., & Majedd, S.K. (1976b).

Preliminary assessment of the toxicity of trenboloneacetate when

incorporated in the diet of mice. Unpublished report No. RSL 213/75992

from Huntingdon Research Centre, Huntingdon, England. Submitted to WHO

by Roussel Uclaf, Paris, France.

Hunter, B., Bathman, P., Heywood, R., Street, A.E., Prentice, D.E., &

Majeed, S.K. (1976c). Trenboloneacetate oral toxicity to rats by

dietary administration for 13 weeks. Unpublished report No. RSL

237/76426 from Huntingdon Research Centre, Huntingdon, England.

Submitted to WHO by Roussel Uclaf, Paris, France.

Hunter, B., Graham, C., Gibson, W.A., Read, R., Gregson, R., Heywood

R., Offer, J., Prentice, D., & Woodhouse, R. (1981). Long-term feeding

of trenboloneacetate to mice (final report). Unpublished report volume

I, II and III, No. RSL/267/80122 from Huntingdon Research Centre,

Huntingdon, England. Submitted to WHO by Roussel Uclaf, Paris, France.

Hunter, B., Batham, P., Heywood, R., Street, A.E., Gibson, W.A.,

Prentice, D., Gregson, R., & Offer, J. (1982). Trenboloneacetate

potential tumorigenic and toxic effects in prolonged administration to

rats following "in utero" exposure (final report). Unpublished report

volume I, II, III and IV, No. RSL 295-G/80974 from Huntingdon Research

Centre, Huntingdon, England. Submitted to WHO by Roussel Uclaf,

Paris, France.

Ingerowski, G.H., Scheutwinkel-Reich, M., & Stan, H.J. (1981).

Mutagenicity studies on veterinary anabolic drugs with the

Salmonella/microsome test. Mutagen. Res., 91, 93-98.

James, P.A., Cox, R., Clark, R., Palmer, A.K., & Offer, J. (1982).

Effect of trenboloneacetate on pregnancy of the rat. Unpublished

report No. RSL 458/81553 from Huntingdon Research Centre, Huntingdon,

England. Submitted to WHO by Roussel Uclaf, Paris, France.

James, P., Smith, J.A., John, D.M., Gibson, W.A., Offer, J.M., &

Anderson, A. (1985). Effect of trenbolone acetate on reproductive

function of multiple generations in the rat. Unpublished report

No. RSL 634/841004 from Huntingdon Research Centre, Huntingdon,

England. Submitted to WHO by Roussel Uclaf, Paris, France.

James, P., Smith, J.A., & Parker, C.A. (1986). Supplementary study of

the effect of trenbolone acetate on pregnancy of the rat and

development of the offspring. Unpublished report No. RSL 695/851572

from Huntingdon Research Centre, Huntingdon, England. Submitted to WHO

by Roussel Uclaf, Paris, France.

Kroes, R. (1972). De invloed van oestradiol en een combinatie van

oestradiol en trienbolone op de vesiculae semenalis van mestkalveren.

Unpublished report No. 162/72 Path. from the National Institute of

Public Health, The Netherlands.

Kroes, R., Huis in't Veld, L.G., & Stephany, R.W. (1976a). Onderzoek

naar de invloed van ethynylestradiol, 17-alpha-oestradiol en

trenboloneacetaat alleen of in combinatie op beren, borgen en geiten.

Uitscheiding in urine en faeces en residuen in serum (ILOB proeven

98.06 98.07, 98.08, 98.09, 118.04 en 118.05). Unpublished report

No. 216/76 Path. from the National Institute of Public Health,

The Netherlands.

Kroes, R., Huis in't Veld, L.G., & Stephany, R.W. (1976b). Onderzoek

naar de invloed van combinatie-preparaten van mannelijke en

vrouwelijke hormonen of hormonaal werkende stoffen of mannelijke

mestkalveren (Een biologisch, chemisch en histologish onderzoek, ILOB

proef 25.14 P 72/82). Unpublished report No. 193/76 Path. from the

National Institute of Public Health, The Netherlands.

Kroes, R., Huis in't Veld, L.G., Both-Miedema, R., & van de Berg, R.

(1976c). Onderzoek naar de invloed van 17-ß-oestradiol, trenbolone-

acetaat en testosteron alleen of in combinatie, op stieren en ossen

(Uitscheiding in urine, aanwezigheid in serum en histologisch

onderzoek prostaat, ILOB-proeven 134-02 a, 134.02 b en 134.03).

Unpublished report No. 218/76 Path. from the National Institute of

Public Health, The Netherlands.

Kruskemper, H.L., Morgner, K.D., & Noell, G. (1967). Klinische

pharmakologie von Trienolonen, eine Gruppe neuartiger anabol wirksamer

Oestran-Derivate, Arzneim. Forsch., 17, 449-454.

Lutz, W.K (1979). In vivo covalent binding of organic chemicals to

DNA as a quantitative indicator in the process of chemical

carcinogenesis. Mutation Res., 65, 289-356.

Offer, J.M. (1985). Effect of trenbolone acetate on the reproductive

function of multiple generations in the rat. Unpublished report

No. RSL 634/85959 (Addendum to RSL 634/841004) from Huntingdon

Research Centre, Huntingdon, England. Submitted to WHO by Roussel

Uclaf, Paris, France.

Philibert, D. & Moguilewsky, M. (1983). Liaison de la testosterone de

la 17-ß-OH-trenbolone (RU 2341) et de son isomere, la 17-alpha-

OH-trenbolone (RU 27423) au niveau du plasma humain. Unpublished

report No. AN 39 from Roussel Uclaf Research Centre. Submitted to WHO

by Roussel Uclaf, Paris, France.

Pottier, J. Busigny, M., & Gradadam, J.A. (1973). Tissue residues in

cows after implantation of trenbolone acetate. J. Anim. Sci.,

37, 256 (Abstract No. 110F).

Pottier, J., Busigny, M., & Gradaclam, J.A. (1975). Plasma kinetics,

excretion in milk and tissue levels in the cow following implantation

of trenbolone acetate. J. Anim. Sci., 41, 968.

Pottier, J., Cousty, C., Heitzman, R.J., & Reynolds, I.P. (1978).

Metabolites trenbolone acetate R in the bile of the cow and the rat.

Report presented during the 1978 Joint Meeting of the American Dairy

Association and the American Society of Animal Science, Michigan State

University, East Lansing, 9-13 June. Submitted to WHO by Roussel

Uclaf, Paris, France.

Pottier, J. (1979). Trenboloneacetate tissue and biliary

concentrations of the position of 17 epimers of trenbolone in heifers

after 2 months of implantation. Unpublished report No. 799 from

Roussel Uclaf. Submitted to WHO by Roussel Uclaf, Paris, France.

Richold, M. & Richardson, J.C. (1982). Metaphase analysis on

17-ß-hydroxytrenbolone and 17-alpha-hydroxytrenbolone. Unpublished

report No. RSL/512/81962 from Huntingdon Research Centre, Huntingdon,

England. Submitted to WHO by Roussel Uclaf, Paris, France.

Richold, M., Jones, E., & Hales, J.F. (1982a). Ames metabolic

activation test to assess the potential mutagenic effect of

17-alpha-hydroxytrenbolone, 17-ß-hydroxytrenbolone and

17-alpha-oestradiol. Unpublished report No. RSL 567/82767 from

Huntingdon Research Centre, Huntingdon, England. Submitted to WHO by

Roussel Uclaf, Paris, France.

Richold, M., Richardson, J.C., Proudlock, R.J., & Fleming, P.M.

(1982b). Effect of 17-ß-hydroxytrenbolone and 17-alpha-hydroxy-

trenbolone on cultured human lymphocytes. Unpublished report No. RSL

544/82136 from Huntingdon Research Centre, Huntingdon, England.

Submitted to WHO by Roussel Uclaf, Paris, France.

Richold, M. Dehnel, J.M., & Ransome, S. (1982c). An assessment of the

mutagenic potential of 4 compounds using an in vitro mammalian cell

test system. Unpublished report No. RSL 513/8286 from Huntingdon

Research Centre, Huntingdon, England. Submitted to WHO by Roussel

Uclaf, Paris, France.

Richold, M., Edgar, D.H., Ransome, S.J., & Banks, S.J. (1983). An

assessment of the mutagenic potential of four compounds using an

in vitro mammalian cell test system. Unpublished report from

Huntingdon Research Centre, Huntingdon, England. Submitted to WHO by

Roussel Uclaf, Paris, France.

Riis, P.M. & Suresh, T.P. (1976). The effect of a synthetic steroid

(trenbolone) on the rate of release and excretion of subcutaneously

administered estradiol in calves. Steroids, 27, 5-15.

Roberts, N.L. & Cameron D.M. (1985). Hormonal effects of orally

administered trenbolone. Findings from three studies in the domestic

pig. Unpublished report No. RSL 591/83499 from Huntingdon Research

Centre, Huntingdon, England. Submitted to WHO by Roussel Uclaf,

Paris, France.

Ross, D.B. (1980). Toxicology and residues of trenboloneacetate.

Published in: Conference on the Use, Residues and Toxicology of Growth

Promoters, Dublin, 9 April 1980, pp. 34-48.

Ross, D.B., Roberts, N.L., Cameron, D.M., Street, A.E., Crook, D.,

Prentice, D.E., & Cherry, C.P. (1980). Oral toxicity of trenbolone

acetate to growing pigs (Sus scrofa)- preliminary 14 weeks feeding

study. Unpublished report No. RSL 357/80152 from Huntingdon Research

Centre, Huntingdon, England. Submitted to WHO by Roussel Uclaf,

Paris, France.

Ryan, J.J. & Hoffman, B. (1978). Trenbolone acetate: experiences with

residues in cattle tissues. J.A.O.A.C., 61, 1274-1279.

Schiffman, D., Metzler, M., Neudecker, T., & Henschler, D. (1985).

Morphological transformation of Syrian hamster embryo fibroblasts by

the anabolic agent trenbolone. Arch. Toxicol., 58, 59-63.

Schroder, R. (1971a). R 1697: Prüfung der androgenen und

muskelbildenden Wirkung bei oraler Verabreichung. Unpublished report

from Hoechst A.G. Submitted to WHO by Roussel Uclaf, Paris, France.

Schroder, R. (1971b). R 1697: Untersuchung auf androgene und anabole

Wirkung, Untersuchung auf oestrogene Wirkung. Unpublished report from

Hoechst A.G. Submitted to WHO by Roussel Uclaf, Paris, France.

Seeger, K. (1971a). Anabolikum R 1697 (A 50273) as us vet.

Unpublished report from Hoechst A.G. Submitted to WHO by Roussel

Uclaf, Paris, France.

Seeger, K. (1971b). R 1967: Chronische Toxizitat per oral. Unpublished

report from Hoechst A.G. Submitted to WHO by Roussel Uclaf,

Paris, France.

Sovetal (1970). Toxicité cronique par voie sous-cutanée chez le rat

male et chez la femelle. Unpublished report No. R 1697 from Sovetal.

Submitted to WHO by Roussel Uclaf, Paris, France.

Stephany, R.W., Bosch, D., van den Sahertian, E.Th., & Kroes, R

(1976). Onderzoek naar de invloed van orale toediening van

trenboloneacetaat op vaarzen (ILOB-proef 134-01) (Eenchemisch

onderzoek). Unpublished report No. 215/76 Path. From the National

Institute of Public Health, The Netherlands.

Verbeke, R., Debackere M., Hicquet, R., Lauwers, H., Stevens, J.,

Pottier, G., Moer, D., Van Hoof, J., & Vermeersch, G. (1975). Quality

of the meat after the application of anabolic agents in young calves.

Published in: Anabolic Agents in Animal Production, FAO/WHO Symposium,

March 1975, pp. 123-130.

van de Wal, P. (1975). General aspects of the effectiveness of

anabolic agents in increasing protein production in farm animals, in

particular in bull calves. Published in: Anabolic Agents in Animal

Production, FAO/WHO Symposium, March 1975, pp. 60-78.

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