FUNDAMENTAL AND APPLIED TOXICOLOGY 11,640-65 1 ( 1988)

ln Vivo Carcinogenesis Assay of m-Methadone. HCI in Rodents’

HARRIS ROSENKRANTZ AND ROBERT W. FLEISCHMAN

Department of Biochemical Pharmacology, EG&G Mason Research Institute, Worcester, Massachusetts 01608

Received December I, 1987; accepted June 20, I988

In Vivo Carcinogenesis Assay of or.-Methadone.HCl in Rodents. ROSENKRANTZ, H., AND

FLEISCHMAN, R. W. (I 988). Fundam. Appl ToxicoL 11,640-65 1. The extensive clinical use of
methadone encouraged the performance of a carcinogenesis bioassay to support risk assessment
in man. An oral LDSO of 178 mg/kg was obtained in B6C3Fr mice. Physiologic changes induced
by mean oral doses of 15, 30, and 60 mp/kg for 90 days included dose-related central nervous
system (CNS) stimulation, fighting, tolerance development, sex-related alteration of food con-
sumption, and no drug-related pathology. In the chronic study dosages were 15 and 60 mg/kg
for mice, 16 and 28 mg/kg for male rats, and 46 and 88 mg/kg for female rats. Survival incidences
for treated and control rodents were 72-86% for mice and 80-90% for rats. Deaths related to
morphologic changes of aging occurred in all groups. CNS stimulation and fighting were more
common to male rodents. Growth rates were unchanged for mice but a dose-related inhibition
occurred for rats. Higher doses stimulated food intake in both species. Neither the type nor
incidence of neoplasia was drug related but a few nonneoplastic lesions may have been. Prelimi-
nary plasma methadone levels at necropsy were dose related in the rat. o I988 society ofToxicology.

Many of the narcotic agonists and antago-
nists in clinical use have not undergone in
vivo carcinogenic assessment. In particular
methadone,* which has been used for more
than two decades, was untested (Senay,
1983). Genetic screening assays revealed little
action in the Escherichia coli DNA repair test
and in the mouse lymphoma forward-muta-
tion assay (Brusick et al., 198 1). Questionable
effects on germ cells have been reported
(Zimmering, 1979; Badr et al., 1979).
Recently the pure antagonist naltrexone
was found to be noncarcinogenic (Rosen-
krantz, 1984). On the other hand, the con-
gener of methadone, L-cr-acetylmethadol
(LAAM), induced an increase in liver neo-
’ The major portion ofthese studies was funded under
Contract 271-78-3502 from the National Institute on
Drug Abuse (NIDA).
’ Methadone is 6-dimethylamino-4,4diphenyl-3-hep-
tanone, racemate form.
plastic nodules and possibly in the incidence
of liver carcinoma in Fischer 344 rats but not
in B6C3F1 mice (Rosenkrantz and Fleisch-
man, 1988). These adverse findings provided
an additional reason for conducting a carci-
nogenic assay on methadone.
The protocol used to evaluate methadone
was identical to that applied to LAAM except
for dosages. Gross toxic signs and morpho-
logical anomalies permitted differentiation
between the effects of methadone and
LAAM.
METHODS
The carcinogenic evaluation of methadone was per-
formed under identical circumstances to that of LAAM
using the earlier protocols of the National Cancer Insti-
tute (NCI, 1976; Rosenkrantz and Fleischman, 1988).
Three phases of investigation were mandated: (1) an
acute oral toxicity in B6C3Fi mice, (2) a 90day oral
study in the same murine strain using three doses, and

0272-0590188 $3.00 640

Copytight 0 1988 by the society of Toxiwlogy.
All rights of reproduction in any form reserved.
 CARCINQGENICITY
(3) a 24-month oral bioassay in B6C3F1 mice’ and Fi-
scher 344 rats3 using two doses of drug (NCI, 1976; Na-
tional Toxicology Program, 198 1). There exists an exten-
sive data base on both rodent strains (Cameron et al.,
1985).
Quarantined and randomized rodents were ear
punched for individual identification and segregated by
species and sex in a static environmental arrangement
in one room of a biohazard facility as described for the
LAAM studies (Rosenkrantz and Fleischman, 1988).
The room was regulated for a 12-hr light/dark circadian
cycle, an ambient temperature of 23 ? 2”C, a relative hu-
midity of 50 + 15%, and 12-15 filtered air changes per
hour.
Rodents in the acute study were housed in 18 X 24
X 1S-cm wire-mesh suspension cages and received com-
mercial food pellets4 and water from bottles ad libitum.
Those in the prolonged studies were in 32 X 24 X 15cm
(90 day) or 56 X 32 X 20-cm (24 month) polycarbonate
cages containing wood chips and fitted with filter bon-
nets. Feed meal4 was used in order to introduce drug to
the diet and an automatic system provided water. Hous-
ing, feeding, and cleaning procedures were the same as
detailed previously (Rosenkrantz and Fleischman,
1988).
The stability and purity of bulk methadone. HCl’ at
the beginning and termination of studies were deter-
mined by ultraviolet and infrared spectroscopy and thin-
layer and gas chromatography. The homogeneity and
concentrations of methadone. HCl in monthly drug/feed
admixtures prepared for rodent consumption were mon-
itored by gas chromatography using a nitrogen-phospho-
rus detector and lidocaine . HCl as an internal standard.
Drug/feed admixtures were prepared by initial homog-
enization of sufficient drug for a dosage group with 50 g
of feed and then transferred to a steel V-blender to make
6 kg of mix. The percentage concentration of metha-
done. HCl in each drug/feed preparation was determined
by gas chromatography, and in combination with quan-
tity of food consumption and body weight permitted cal-
culation of free base dosage as milligrams per kilogram
or parts per million.
Oral LD50 trial in mice. Mice weighing 18-22 g were
grouped 6/sex/cage and were gavaged with 12.5-1600
mg/kg using stock solution concentrations of 2.2-141.2
mg/ml and gavage volumes of 0.1 O-O.27 ml per mouse.
Control mice received the largest volume of vehicle. Le-
3 Procured from Charles River Breeding Laboratories,
Wilmington, MA.
4 Wayne Lab Blox meal and pellets, Allied Mills, Inc.,
Chicago, IL.
5 r&-Methadone. HCl was primarily obtained from
the Penick Corp., New York, NY, and smaller batches
identified as BL-1635 and QCD-82179 came from a
NIDA repository at the Research Triangle Institute, Re-
search Triangle Park, NC.
OF METHADONE 641
thality, behavior, body weight, and appetite were ob-
served for 7 days and gross pathology was performed on
approximately 50% of survivors.
Oral 90-day study in mice. Male mice weighing 19
+ 1 g and females weighing 16 f 1 g were arranged as lO/
sex/group housed S/cage and were given a drug/feed mix
containing methadone dosages of 15,30, or 60 mg/kg or
feed without methadone. Physiological parameters in-
cluded twice daily observations of behavior and mortal-
ity counts; initial, weekly, and final body weights; and
weekly food consumption. Body weight and food intake
permitted measurement of methadone consumption.
Gross pathology findings on all major organs were re-
corded and tissues were preserved for histopathology, if
needed.
Oral 24-month studies in mice and rats. Rodents were
procured as 4-week weanlings and after 3 weeks quaran-
tine male mice weighed 23 f 1 g and females 19 f 1 g
whereas male rats weighed 12 1 rt 2 g and females 101
-t 2 g. Groups of 50 animals/sex/species were fed a low
or high dose of methadone or served as diet controls. The
doses for mice were 15 and 60 mg/kg, which were ap-
proximately 8 and 33% ofthe oral LD50. For the rats the
doses were 20 and 60 mg/kg, which were approximately
13-20 and 40-60% of known oral LD50s (Finnegan et
al., 1948; NIDA, personal communication). Because of
the desire to use large nonlethal doses of methadone and
in the absence of subchronic data on the rat, the precau-
tion was taken to increase rat doses by 10 mg/kg every
other week until the low and high dosages were attained
(Week 3 for the low dose and Week 1 I for the high dose).
The concentration of methadone in feed was determined
as dose was increased to confirm the changes in dose.
Thereafter, each monthly drug/feed admixture was ana-
lyzed before use and the average intake of each sex was
calculated on the basis of food consumption and body
weight. For both species treatment was prolonged 2-4
weeks beyond the 24-month assayperiod in order to pre-
clude possible withdrawal signs before necropsy.
Mortality, viability, and gross manifestations of cen-
tral nervous system (CNS) and autonomic anomalies
were monitored twice daily. Body weights and food in-
take were recorded monthly. The size and appearance of
developing lumps and gross lesions were followed
weekly. Blood specimens were collected under anesthesia
from external jugular vein before necropsy and while ro-
dents were still under treatment. Plasma samples were
analyzed elsewhere by GC/MS using deuterium-labeled
analogs as internal standards and ammonia positive ion
chemical ionization.6 Gross and histopathology were
performed on approximately 46 tissues and masses from
both deceased and killed animals.
The significance of neoplastic and nonneoplastic le-
6 Courtesy of Dr. Rodger L. Foltz, Center of Human
Toxicology, University of Utah and Dr. Richard Hawks
of the National Institute on Drug Abuse (NIDA).
642 ROSENKRANTZ
sions was evaluated elsewhere using a computerized sta-
tistical program developed for carcinogenesis bioassays
(NCI, 1976).’ Statistical methods applied to neoplasm
data included fatal: life trend test and probability ofdeath
with tumor (one-sided pvalues); incidental: Peto trend
test, Cochran-Armitage test to assess dose-response
trends, and Fisher exact test. The latter was used for as-
sessing significance of rate changes of nonneoplastic le-
sions. The Student t test was used for other biological
data.
RESULTS
For the sake of comparison, the predomi-
nant responses evoked by methadone are pre-
sented in the format used for LAAM (Rosen-
krantz and Fleischman, 1988).
The purity of methadone. HCl was estab-
lished by melting point (233-236”(Z), a major
absorption band at 292 nm, a bifurcated
band near 262 nm, a single zone (20- to 40-
pg samples) on thin-layer chromatograms,
and an infrared spectrum in conformity with
chemical structure and superimposable with
that of an analytical sample. Similar results
were obtained on methadone. HCl recovered
from drug/feed admixtures (various locations
of the mix) monitored at 5 and 23°C for 3
weeks and on bulk drug at the cessation of
studies. Gas chromatographic analysis also
confirmed purity and stability of bulk drug
throughout the studies as well as methadone
concentrations in drug/feed preparations. A
linear dose (5-40 pg/ml extract)-response
plot was obtained. The mean peak area ratio
(methadone. HCl:lidocaine . HCl) was 1.145
+ 0.014 (SD) for 10 pg/ml with a coefficient
of variation of 1.2%. Linear regression data
included a slope of 0.1122, a Y intercept of
+0.0276 and a correlation coefficient of
0.9999.
Short- Term Findings
Acute oral treatment of mice evoked cir-
cling behavior, Straub tail, convulsions, and
’ Dynamac Corp., Rockville, MD.
AND FLEISCHMAN
prostration at doses above 50 mg/kg. Hyper-
activity was associated with lower doses. The
effects were dose related and were dissipated
by the end of the first or second day. Among
survivors a second phase of hypoactivity was
evident. Deaths occurred within lo-20 min.
The LD50 was 178 mg/kg for both sexes. No
gross anatomical changes were found at nec-
ropsy.
Prolonged treatment with methadone for
90 days elicited a slowly developing hyperac-
tivity in the first 4 weeks which was accompa-
nied by noise sensitivity in both sexes. The
behavioral derangements were dose related in
females and occurred only in high-dosed
males along with episodic fighting. Tolerance
developed to hyperactivity and hypersensitiv-
ity between Weeks 7 and 10. Although body
weights were not significantly altered, female
mice consumed more food at all doses
throughout the study while there was a de-
crease in food intake for high-dosed males in
the last month of treatment. The actual daily
mean consumption of methadone was 12,23,
and 53 mg/kg for males and 15, 29, and 62
mg/kg for female mice. Mortality did not oc-
cur in any group. Except for a mass in the
spleen of one low-dose male, gross pathology
findings were not remarkable. Fighting
among high-dose males was confirmed by
bite lesions in 20-25% of mice. No drug-re-
lated histopathology was evident.
24-Month Carcinogenesis Findings
I. Behavioral changes. The actual mean
f SD doses consumed by mice were 15.4
+ 0.3 and 60.3 f 0.7 mg/kg for both sexes.
Within 2-4 weeks dose-related hyperactivity
and fighting appeared in treated groups,
mainly among males. Tolerance to CNS
stimulation developed rapidly but sporadic
fighting occurred in 20% of low-dose males
throughout treatment. Controls behaved nor-
mally.
Mean f SD doses consumed by rats were
16.1 f 0.4 and 28.1 4 0.9 mg/kg for males
 CARCINOGENICITY OF METHADONE 643
TABLE 1
GROWTH RATE CHANGES IN B6C3F, MICE AND FISCHER344 RATS FED METHADONE FOR 24 MONTHS”

Percentage change ofgrowth rate for treated versus control groupsb

B6C3F, mice Fischer 344 rats

Month Male Female Male Female Male Female Male Female

in study 15 mg/kg 15 w/k 60w/kg 60mg/kg 16w/kg 28mg/k 46 w/kg 88 mg/kg

1 0 0 +5 -4 -1 +7 -2 +-I
3 0 +3 +4 0 -2 +4 -6 +6
6 -2 f10 +2 +3 -3 +3 -4 +1
9 -4 +8 -1 +6 -5 +2 -8 0
12 0 +8 +5 0 -4 +2 -9 -3
15 -3 +8 +2 0 -5 +4 -9 -4
18 -3 +8 +2 -2 -9 -2 -11 -11
21 -4 +6 -3 -2 -7 -2 -12 -10
24 -3 +2 -3 -4 -10 -2 -14 -14

a Growth rates (monthly body weight:initial body weight) of treated groups were divided by those of corresponding
control groups and converted to percentage changes. To savespace values for 3-month periods are given.
b In general, values exceeding +8% were significant (p < O.Ol-<0.05) by the Student t test.

and 45.8 f 0.5 and 88.1 + 1.5 mg/kg for fe- radically exhibited by high-dose females dur-
males. High-dose males displayed hypersen- ing the second year. Control rats behaved
sitivity or hyperactivity to which tolerance normally.
developed rapidly. Similar behavior was spo- 2. Growth rates and food consumption.

TABLE 2
FOOD CONSUMPTION CHANGES IN B6C3F, MICE AND FISCHER 344 RATS FED METHADONE FOR 24 MONTHS’

Percentage change of food consumption for treated versus control groupsb

B6C3F, mice Fischer 344 rats

Month Male Female Male Female Male Female Male Female

in study 15 mg/kg 15 w/kg 60 mg/kg 60 mg/kg 16 mg/kg 28w/kg 46 w/kg 88w/kg
1 0 0 +3 0 -23 -14 +6 +21
3 -2 -2 -13 -10 -5 +14 -1 +25
6 +13 +22 +22 +20 -6 +22 -5 +23
9 +16 +10 +11 +21 -3 +35 -2 +17
12 +6 +20 +3 +30 -5 +40 -6 +27
15 +6 +5 -3 +5 -5 +41 -4 +38
18 +13 +I -8 +6 -20 +31 +11 +34
21 +7 +10 -5 +19 -12 +31 -15 +28
24 +2 +17 +9 +17 -19 +18 -10 +48

a Food intakes of treated groups divided by those of corresponding control groups and converted to percentage
changes. Values of 3-month periods instead of monthly measurements are given to save space.
b In general, values exceeding f 10% were significant (p < O.Ol-40.05) by the Student t test.
644 ROSENKRANTZ AND FLEISCHMAN

TABLE 3
TEMP~RALPA~ERNSOFMORTALITYAMONGB~C~F~MICEANDFISCHER~~~RATSFED
METHADONEFOR~~MONTHS'
Spontaneous (unscheduled) deaths plus moribund deaths

B6C3F, mice (dose in mg/kg) Fischer 344 rats (dose in mg/kg)

Diet Diet
control control
Months 15 15 60 60 16 28 46 88
in study M F M F M F M F M F M F

1-3 0 0 1 0 0 0 0 0 0 0 0 I
4-6 0 1 0 0 0 0 0 0 0 0 0 0
l-9 0 0 0 0 1 1 0 0 0 0 0 0
10-12 0 0 1 0 0 0 0 0 0 0 0 0
13-15 0 0 3 1 1 1 1 1 0 1 1 1
16-18 4 0 3 1 1 3 2 2 2 2 0 3
19-21 4 4 2 1 5 2 1 1 1 4 0 1
22-24 2 4 4 4 2 2 5 1 6 2 4 1
256 I 0 0 0 0 0 1 2 0 0 0 0
Total 11 9 14 7 10 9 10 7 9 9 5 7

’ There were 50 animals per group. Exact days of death are known as are which animals died naturally and which
were killed due to morbidity. The data have been telescoped to save space.
‘This month reflects deaths in the final observation period until necropsy could be performed.

That actual dosages consumed affected masses (two to four mice) was similar in
growth rates of each species is revealed by treated and control groups. Neither neo-
data in Table 1. Growth rates of both sexes plasms or nonneoplastic lesions could be as-
of mice were generally unchanged although signed as the major cause of death of mice.
there was a trend toward increased rates Deaths among rats occurred predomi-
among low-dose females. In contrast male nantly in the second year (Table 3). Survival
and female rats progressively had reduced rates were 80-90% for males and 82-86% for
rates which were dose related. females in all groups. About 30% of dying
More revealing was food intake (Table 2). males in each group were killed because of
A similar increase occurred for female mice morbidity while palpable masses were found
at both doses whereas food increments only in female groups. The exact causes of
among males were more common at the low death were not established.
dose. The response was more definitive 4. Neoplastic findings. The number of
among rats. Males at both doses consumed mice with specific neoplasms in control and
less food than controls whereas female rats treated groups is given in Table 4. Common
consumed more. Spillage of food was not a tumors of low frequency occurred in the pitu-
factor. itary (males), thyroid, ovary, uterus, hemato-
3. Mortality patterns. Except for an occa- poietic organs, subcutaneous tissue (females),
sional demise in each group in the first year, Harderian gland, and tumors of multiple or-
most deaths appeared related to the aging gans. At higher frequencies, common neo-
process (Table 3). Survival rates were 72-80% plasms were liver hepatocellular carcinoma
for male mice and 82-86% for female mice. and adenoma, lung alveolar/bronchiolar ade-
The number of deceased mice with palpable noma, lymphoma of multiple organs, and
 CARCINOGENICITY OF METHADONE 645

TABLE 4
TYPES AND INCIDENCES OF NEOPLASIA IN B6C3F, MICE FED METHAWNE FOR 24 MONTHS

50 Males per group 50 Females per group

Diet 15 60 Diet 15 60
Organ system, type neoplasm control w/kg w/k control mg/kg w/k

Endocrine
Pituitary, (anterior), adenoma 2 0 1 5 15O 4
Thyroid, follicular cell adenoma/
carcinoma 2 I 2 1 0 3
Reproductive
Ovary, fibroma/cystadenoma 0 0 0 2 0 1
Uterus, endometrial stromal polyp 0 0 0 0 2 0
Uterus, angiosarcoma 0 0 0 1 1 0
Digestive
Liver, hepatocellular carcinoma 12 11 17 5 6 5
Liver, hepatocellular adenoma 9 2 8 3 6 8
Hematopoietic
Multiple organs, Iymphoma 1 2 5 10 14 9
Spleen. lymphoma 0 1 0 1 1 0
Thymus, Iymphoma 1 1 0 I 1 0
Lymph nodes, lymphoma/hemangioma 3 0 0 0 0 1
Peyer’s patch, lymphoma 1 0 1 0 0 1
Respiratory
Lungs, alveolar/bronchiolar adenoma 8 5 3 1 5 6
Lungs, alveoIar/bronchioIar carcinoma 5 0 1 1 0 0
Other organ systems
Subcutaneous, sarcoma/tibrosarcoma/
fibroma 5 6 4 0 0 1
Multiple organs, sarcoma/mesothelioma 1 1 I 0 1 0
Harderian gland, adenoma 0 2 2 2 2 3

n Significant change at p < 0.05 (fatal: life trend test; incidental: peto trend test, and Fisher’s exact test) for treated
versus control rates.

subcutaneous tumors of male mice. There pheochromocytoma. A uterine stromal sar-

was a significant increase in pituitary adeno-
mas in low-dose females.
Not shown in Table 4 were single occur-
rences of neoplasms in mice. A testicular ade-
noma, a preputial gland adenoma, a liver
cholangiocarcinoma, a basal cell adenoma
and carcinoma of the stomach, or an adeno-
carcinoma of the kidney were found among
control males. Control females exhibited in-
dividual cases of cervical sarcoma, uterine
hemangioma, liver angiosarcoma, mediasti-
nal lymphoma, and brain lymphoma. One
low-dose male had an adrenal cortical ade-
noma and one high-dose male exhibited
coma and a liver angiosarcoma were ob-
served in low-dose females. Individual high-
dose females had a pancreatic islet cell
adenoma, a spleen hemangioma, or an hem-
angioma of the urinary bladder. The occur-
rence of isolated cases of tumors precludes as-
sessment of significance.
The number of rats with various tumors in
control and treated groups is outlined in Ta-
ble 5. Common neoplasms of high incidence
included pituitary adenoma, adrenal pheo-
chromocytoma, testicular adenoma, mam-
mary gland adenocarcinoma/fibroadenoma,
uterine polyp, liver neoplastic nodules, and a
646 ROSENKRANTZ AND FLEISCHMAN

TABLE 5

50 Males per group 50 Females per group

Diet 16 46 Diet 28 88
Organ system, type neoplasm control m/kg as/kg control m&3 w/kg

Endocrine
Pituitary, adenoma 16 10 11 22 18 18
Thyroid, follicular cell adenoma/carcinoma 0 0 0 2
Thyroid, C-cell adenoma/carcinoma 4 4 0 3
Adrenal, cortical adenoma 2 0 0 0
Adrenal, pheochromocytoma 10 4 6 3
Reproductive
Testis, interstitial cell adenoma 46 47 47 0 0 0
Mammary gland, adenocarcinoma/fibroma 2 20 9
Uterus, endometrial stromal polyp 0 0 0 16 18 14
Uterus, endometrial stromal sarcoma 0 0 0 0
Clitoral gland, adenoma/carcinoma 0 0 0 3 3 0
Digestive
Liver, hepatocellular carcinoma 0 1 0 0 0
Liver, neoplastic nodules 10 9 8 3 1
Pancreas, islet cell adenoma 4 2 0 0 0
Nervous
Brain, astrocytoma/oligodendrolioma 3 2 0 0 0
Hematopoietic
Multiple organs, leukemia 4 3 3 5 4 3
Multiple organs, lymphoma 2 0 2 1 0
Respiratory
Lungs, alveolar/bronchiolar adenoma 2 0
Other organ systems
Subcutaneous, sarcoma/lipoma/fibroma 6 6 3
Skin, papilloma

mix of various subcutaneous tumors. Of bronchiolar carcinoma (male and female), an

lesser frequency were neoplasms of the thy-
roid, adrenal cortex (males), clitorial gland,
pancreas (males), brain (males), lungs, and
skin. Leukemia and lymphoma were also
common. There were no significant changes
related to dose or drug treatment. The nega-
tive trend of frequency for mammary tumors
in female rats was significant. However, it
cannot be concluded that methadone was an
anti-cancer agent since the design of the pro-
tocol was only pertinent to increments in
neoplasia.
Not given in Table 5 were isolated inci-
dences of tumors in either control or treated
rats. Individual control rats had a subcutane-
ous fibrosarcoma (female), a lung alveolar/
ileual leiomyosarcoma (male), or a mesothe-
lioma of multiple organs.
Single low-dose males had a mesothelioma
of the tunica albuginea or an adenocarci-
noma of the jejunum. A parathyroid ade-
noma, a trichoepithelioma of skin, and a
squamous cell papilloma of the skin were
found among high-dose males. In the low-
dose female group, a sebaceous gland ade-
noma, a basal cell carcinoma of the skin, a
vaginal sarcoma, and an adenocarcinoma of
the kidney were identified. Single high-dose
females exhibited a pituitary carcinoma, an
adrenal cortica1 carcinoma, a stomach sar-
coma, a leiomyosarcoma of the jejunum, a
mediastinal mesothelioma, or a transitional
 CARCINOGENICITY
cell papilloma of the urinary bladder. A pre-
putial gland adenoma was discerned in one
male each in control and treated groups.
5. Nonneoplastic findings. A myriad of
nonneoplastic lesions were found in control
and treated mice. The number of mice with
the more predominant anomalies is listed for
each group in Table 6. Common lesions of
similar frequency in control and treated mice
were found in heart (males), kidneys, lungs,
hematopoietic tissues, and some reproduc-
tive organs. None of the incidences of mor-
phologic change were significant. Also insig-
nificant were sex-related lesions: for male
mice, cardiomyopathy, nephropathy, blad-
der degeneration, adrenal angiectasis/hyper-
plasia, angiectasis of lymph nodes, and min-
eralization of brain; for female mice, degener-
ative lesions of the liver and omentum and
myelofibrosis of bone marrow.
Although not dose related, a significant in-
crease in thyroid follicular cell hyperplasia
was discerned in both mice sexes, Aberra-
tions tending to be dose related, but of bor-
derline significance, included pituitary hyper-
plasia (females), testicular degeneration, and
uterine hydrometra.
The number of rats with a variety of non-
neoplastic lesions is presented in Table 7.
Predominant but common morphologic al-
terations included cardiomyopathy, mineral-
ization of the pulmonary artery, arterial
vasculitis (males), nephropathy, degenerative
and/or hyperplastic changes in lungs, and
anomalies in endocrine glands, reproductive
tissues, and digestive organs. Of significance
were the dose-related increases in frequencies
of pituitary hyperplasia in males and angiec-
tasis in the adrenal of males. Also significant
but not dose related were greater incidences
of C-cell hyperplasia of the thyroid and uter-
ine hyperplasia in female rats. Eosinophilia
cytoplasmic cell changes in liver were evi-
dent.
Isolated morphological changes of a di-
verse nature occurred in various organs of
one or two rodents of each species in either
OF METHADONE 647
the control or treated animals or both, which
did not permit interpretation.
6. Plasma drug levels. The plasmas of only
four animals/sex/group/species were ana-
lyzed and therefore the results are considered
to be preliminary. The levels of plasma meth-
adone for both sexes of mice were 17 + 5 (SD)
rig/ml at 15 mg/kg and 18 +- 3 rig/ml at 60
mg/kg. Corresponding values for male rats
were 26 +- 5 rig/ml at 16 mg/kg and 45 +- 8
rig/ml at 46 mg/kg. Female rats values were
47 f 4 rig/ml at 28 mg/kg and 62 * 8 rig/ml
at 88 mg/kg.
DISCUSSION
The racemic form of methadone is used
clinically in order to sustain efficacy and re-
duce toxicity (Chen, 1948; Kreek et al., 1979;
Leander and McCleary, 1982). Human oral
dosages have been in the range of 1-3 mg/
kg and the rodent doses in the present studies
provide a suitable excess to enhance possible
expression of carcinogenesis (Thomas et al.,
1979; Senay, 1983). That rodents are suitable
animal models is suggested by similar drug
binding to opiate receptors and methadone
metabolism as found in man (Pohland et al.,
197 1; Horng et al., 1976).
The current findings have revealed the sim-
ilar behavioral effects of methadone and
LAAM and the lesser toxicity of the former
(Rosenkrantz and Fleischman, 1988). Toler-
ance developed to signs of CNS stimulation
but was not always complete. Others have
distinguished opioid from nonopioid behav-
ioral responses (Leander and McCleary,
1982; Middaugh et al., 1983). Methadone
evoked an oral LD50 significantly larger than
that for LAAM and did not induce deaths at
larger doses given for 90 days. There was also
an absence of drug-related deaths in the
chronic study on methadone, unlike LAAM.
Survival rates of all groups were in confor-
mity with general experience (Cameron et al.,
1985).
Except for male rats, methadone, like
648 ROSENKRANTZ AND FLEISCHMAN

TABLE 6

TYPES AND INCIDENCES OF NONNEOPLASTIC LESIONS IN B6C3FI MICE FED METHADONE FOR 24 MONTHS

50 Males per group 50 Females per group

Diet 15 60 Diet 15 60
Types of Morphologic lesions control mtikg w/kg control w/kg w/k

Cardiovascular
Cardiomyopathy 5 4 I I 1
Arteries, vasculitis 2 I 2 1 4
Arteries, hyperplasia (pulmonary) 0 1 4 0 2
Kidneys
Nephropathy, inflammation, degeneration 10 14 13 6 5 3
Nephropathy, hyperplasia/regeneration 9 13 I1 I 4 1
Bladder, intlammation/necrosis 6 4 2 1 0 0
Lungs
Congestion/histiocytosis 11 7 12 8 8 6
Inflammation/hemorrhage/fibrosis 8 9 II 4 9 6
Hyperplasia 5 3 3 2 6 1
Endocrine glands
Pituitary, hyperplasia 4 6 6 5 I 11
Adrenal, hyperplasia 2 1 4 0 0 0
Thyroid, hyperplasia (follicular cell) 4 1 17” I 10’ 5”
Hematopoietic
Thymus, lymphoid depletion 4 2 3 4 0 2
Thymus, cyst 2 7 6 0 3 2
Spleen, lymphoid depletion 2 0 2 2 1 2
Spleen, extramedullary hematopoiesis 6 6 5 4 5 2
Spleen, hyperplasia 19 3 3 12 12 8
Lymph nodes, lymphoid depletion 1 7 I 0 1 1
Lymph nodes, hemonhage/angiectasis 20 22 24 4 5 4
Lymph nodes, hyperplasia 4 3 4 3 3 4
Reproductive
Testis. hyperplasia 12 0 1 0 0 0
Testis. degeneration 14 22 21 0 0 0
Preputial gland, intlammation 5 0 3 0 0 0
Prostate. inflammation 2 3 I 0 0 0
Uterus, hyperplasia 0 0 0 39 40 38
Uterus, hydrometra 0 0 0 0 3 5
Uterus, inffammation 0 0 0 4 3 8
Ovary, intlammation/cyst 0 0 0 7 15 12
Digestive
Forestomach, metaplasia/hyperplasia 4 2 3 2 2
Forestomach, acanthosis/kemtosis 1 1 2 2 3
Liver, fatty metamorphosis/necrosis/
inflammation 12 4 12 20 27 17
Pancreas, atrophy I 2 1 1 6 1
Pancreas, hyperplasia 7 4 2 0 I 2
Integument and miscellaneous
Omentum, inflammation/necrosis 8 6 1 6 19 10
Subcutaneous, fibrosis 4 2 1 1 0 3
Skin, inflammation/necrosis/keratosis 2 7 0 0 0 1
Peritoneum, inIlammation/
hemoperitoneum 1 0 0 4 2 1
Bone marrow, hyperplasia 1 3 0 3 I 0
Bone marrow, myelohbrosis 0 0 0 33 41 39
Multiple organs, inflammation 41 34 31 39 36 40
Nervous
Brain, hemorrhage 1 I 1 1 1 3
Brain, mineralization 26 21 26 13 15 19

’ Significant change at p < 0.05 (Fisher’s exact test) for treated versus control rate.
 CARCINOGENICITY OF METHADONE 649

TABLE I
TYPES AND INCIDENCESOF NONNEOPLASTIC LESIONS IN FISCHER344 RATS FED METHADONE FOR 24 MONTHS

50 Males per group 50 Females per group

Diet 16 46 Diet 28 88
Types of morphologic lesions control Wkg w/k control m/k m&k

Cardiovascular
Heart, cardiomyopathy 40 46 44 45 40 39
Pulmonary artery, mineralization 21 31 27 22 17 17
Arteries, inflammation (aorta/others) 11 4 19 3 2 2
Kidneys
Nephropathy, inflammation, degeneration 49 48 49 39 44 45
Nephropathy, mineralization 8 3 8 16 14 16
Nephropathy, hyperplasia 26 20 25 16 15 19
Lungs
Congestionfhistiocytosis 14 18 14 15 17 22
Inflammation/hemorrhage/fibrosis 9 5 4 39 16 39
Hyperplasia I 4 6 1 3 4
Endocrine glands
Pituitary. hyperplasia 3 5 9a 5 1 7
Pituitary, angiectasis/hemo. cyst 8 4 3 26 19 14
Adrenal, angiectasis 17 26" 32" 27 34 28
Adrenal, fatty metamorphosis 4 1 2 12 9 5
Adrenal, hyperplasia 5 4 8 7 8 1
Thyroid, hyperplasia (C-cell) 3 4 4 8 21" 17”
Hematopoietic
Thymus, lymphoid depletion 36 39 42 37 40 37
Spleen, lymphoid depletion 5 3 2 5 8 2
Lymph nodes, lymphoid depletion 3 0 2 0 3 0
Lymph nodes, pigmentation/hemorrhage 2 4 5 4 2 2
Lymph nodes. hyperplasia 2 2 2 1 0 0
Reproductive
Testis, hyperplasia 2 I 3 0 0 0
Testis, degeneration 14 11 6 0 0 0
Testis, tubule degeneration 36 39 41 0 0 0
Prostate, inflammation 19 20 15 0 0 0
Prostate, hyperplasia 12 7 10 0 0 0
Uterus, hyperplasia 0 0 0 IO 22" 17
Uterus, inflammation 0 0 0 3 7 3
Uterus, hydrometra 0 0 0 9 4 6
Ovary, inflammation/cyst 0 0 0 9 6 7
Mammary, galactocoele 0 0 0 13 9 3
Mammary, hyperplasia 3 0 0 13 7 14
Digestive
Forestomach, hyperplasia 17 15 18 14 4 5
Forestomach, inflammation/necrosis 3 3 0 9 1 2
Bile duct, hyperplasia 45 42 43 25 38 32
Liver, fatty metamorphosis 29 29 47 6 5 9
Liver, inflammation/necrosis 5 6 1 18 5 3
Liver, eosinophilic cyto. change 8 14 22" 0 8" 9"
Liver, basophilic cyto. change 26 24 33 39 43 34
Pancreas, atrophy/inflammation 17 13 28 15 17 15
Nervous
Brain, hemorrhage 11 8 11 8 6 7

a Significant change at p i 0.05 (Fisher’s exact test) for treated versus control rate.
650 ROSENKRANTZ
LAAM, tended to inhibit growth and stimu-
late food consumption in rodents (Smith et
al., 1977; Middaugh et al.. 1983). It is not es-
tablished whether energy production from
excess food intake was utilized for develop-
ment and growth of tumors and/or was di-
rected into detoxication processes. In con-
trast to the findings with LAAM, methadone-
treated rodents did not exhibit an increase in
liver neoplastic nodules.
The spectrum and incidence of neoplasia
did not definitively differentiate treated from
control rodents. Even rare neoplasms in a
particular group could not be assigned to
drug treatment or environmental factors
(Goodman et al., 1979; Ward et al., 1979). In
regard to nonneoplastic lesions there was less
similarity than congruence in pattern or fre-
quency to those found in the LAAM study.
One might expect some correlation because
of the relationship of chemical structures.
Methadone and LAAM induced thyroid hy-
perplasia, follicular cell for mice and C-cell
for female rats, in the absence of thyroid tu-
mors. Some suggestion of pituitary aberra-
tion in male rats was also noted for both nar-
cotic agonists. Others have detected impair-
ment of the neuroendocrine system in rats
and in man (Kreek, 1978; Lafisca et al., 198 1;
Kuhn and Bartolome, 1985).
The metabolism of methadone has been
elucidated and the major metabolites had lit-
tle analgesic action (Beckett et al., 1971; Ger-
ardy et al., 1986). In the present chronic
study, plasma methadone levels were similar
to those in rats and monkeys but somewhat
less than those in man at steady state (Holms-
trand et al., 1978; Snyder et ai., 1978; Liu et
al., 1983). The presence of methadone in
plasma and its confirmed stability in feed and
the absence of drug-related neoplasia docu-
ment the lack of carcinogenicity of metha-
done.
ACKNOWLEDGMENTS
The authors express their sincere appreciation to
Jeffrey Grant for monitoring certain biological parame-
AND FLEISCHMAN
ters, to Mark Lammi and Susan Roberts for drug formu-
lations, and to Dr. M. Hagopian and Richard Norlin for
drug/feed analyses. The support of John J. Metterville
and the carcinogenesis necropsy technicians as well as
the histology staff are also appreciated. The authors also
express sincere gratitude to Janet Kasputis for typing lo-
gistics of reports and manuscripts. A special thanks is ex-
tended to Dr. Heinz Sorer of the National Institute on
Drug Abuse for encouragement and support.
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