DIAZEPAM

This substance was considered by previous working groups in October 1976 (lARC,
1977) and March 1987 (IARC, 1987). Since that time, new data have become available,
and these have been incorporated into the monograph and taken into consideration in the
evaluation.

1. Exposure Data

1.1 Chemical and physical data

1. 1 . 1 Nomel1clature
Chem. Abstr. Serv. Reg. No.: 439-14-5
Deleted CAS Reg. No.: 11100-37-1; 53320-84-6
Chem. Abstr. Name: 7-Chloro- 1 ,3-dihydro- 1 -methyl-5-phenyl-2H- 1 ,4-benzodiazepin-
2-one
IUPAC Systematic Name: 7-Chloro- 1 ,3-dihydro- 1 -methyl-S-phenyl-2H-l ,4-benzo-
diazepin-2-one
SYl1ol1ym: Methyldiazepinone

1.1.2 Structural al1d molecular fonnulae and relative molecular mass

CH,

CI
~)o
-N

Ci6H1,ClN20 Relative molecular mass: 284.75

1.1.3 Chemical al1d physical properties of the pure substal1ce

(a) DescriptiOI1: Off-white to yellow, odourless, crystalline powder (Gennaro, 1995)
(b) Meltil1g-poil1t: i 25- 126°C (Budavari, 1995)
(c) Spectroscopy data: Infrared, ultraviolet, nuclear magnetic resonance and mass
spectral data have been reported (MacDonald et al., 1972).

-37-
 38 lARe MONOGRAPHS VOLUME 66

(d) So!uhility: Slightly soluble in water (1 g/333 mL); soluble in acetone, benzene,
chloroform (1 g/2 mL), diethyl ether (1 g/39 mL), dimethylformamide and etha-
nol (1 g/16 mL) (Gennaro, 1995)
(e) Stahility: Stable in air (Gennaro, 1995)
(f Dissociatiol1 COl1stal1t: pK" = 3.4 (American Hospital Formulary Service, 1995)

1.1.4 Techl1ical products and impurities

Diazepam is available as 2-, 5- and 10-mg tablets, IS-mg extended release capsules,
2- and 5-mg/5 mL oral solutions, 5-mg/mL concentrated oral solution, 5-mg/mL paren-
teral injection, 5-mg/mL emulsion injection, 2- and 4-mg/mL rectal tube solutions and
10-mg suppositories. Preparations may also contain acetylated monoglycerides, anhy-
drous glucose, benzoic acid, benzyl alcohol, corn starch, ethanol, flavouring, fractionated
egg phospholipids, fractionated soya bean oil, glycerol, lactose, magnesium stearate,
methyl hydroxypropylcellulose, polyethylene glycol, propylene glycol, saccharin,
sodium benzoate, sodium hydroxide, talc, D&C Yellow 10 (Quinoline Yellow), FD&C
Blue 1 (Brilliant Blue FCF) or FD&C Yellow 6 (Sunset Yellow FCF). Sodium benzoate,
benzoic acid and sodium hydroxide are added to the commercially available injection
products to adjust pH (Thomas, 1991; Farmindustria, 1993; British Medical Asso-
ciation/Royal Pharmaceutical Society of Great Britain, 1994; American Hospital For-
mulary Service, 1995; Medical Economics, 1996).
Trade names and designations of the chemical and its pharmaceutical preparations
include: Aliseum; Alupram; Amiprol; An-Ding; Anksiyolin; Ansiolin; Ansiolisina;
Antenex; Apaurin; Apozepam; Armonil; Assival; Atensine; Atilen; Avex; Bensedin;
Betapam; Bialzepam; Calmocitene; Calmpose; Canazepam; Cercine; Ceregulart; Con-
dition; Deprestop; Diacepan; Diaceplex; Dialag; Dialar; Diapam; Diatran; Diaz; Diazem;
Diazemuls; Diazepam-Lipuro; Diazidem; Dienpax; Dipam; Dizac; Dizam; Domalium;
Doval; Drenian; Ducene; Duksen; Duxen; E-Pam; Eridan; Erital; Eurosan; Euphorin;
Evacalm; Faustan; Gewacalm; Hexalid; Horizon; Kiatrium; LA 11 1; Lamra; Lembrol;
Levium; Liberetas; Lizan; Lorinon; Mandrozep; Metil Gobanal; Méval; Morosan; Néo-
Calme; Neosorex; Nervium; Neurolytril; Noan; Notense; Novazam; Novodipam;
Paceum; Pacipam; Pacitran; Pax; Paxate; Paxel; Pro-Pam; Psychopax; Q-Pam; Quétinil;
Quievita; Relaminal; Relanium; Relivan; Remedium; Renborin; Rival; Ro 5-2807; Saro-
met; Scriptopam; Sedapam; Sedipam; Seduxen; Serenak; Serenamin; Serenzin; Servi-
zepam; Setonil; Sibazon; Sibazone; Sico Relax; Solis; Somasedan; Sonacon; Stesolid;
Stesolin; Stress-Pam; Tensium; Tensopam; Tiromne; Tranimul; Tranquase; Tranquirit;
Tranquo-Puren; Tranquo-Tablinen; Umbrium; Unisedil; Valaxona; ValCaps; Valclair;
Valeo; Valibrin; Valiquid; Valitran; Valium; Valrelease; Vatran; Vival; Vivol; Wy 3467;
Zepam; Zetran.

1.1.5 Analysis

Several international pharmacopoeias specify potentiometric titration with perchloric

acid as the assay for purity of diazepam, and thin-layer chromatography for determining
impurities and decomposition products. Assay methods for diazepam in capsules, tablets
 DIAZEPAM 39

and injection solutions include liquid chromatography or ultraviolet/visible absorption

spectrometry using standards. Assays for heavy metal impurities are also specified
(Society of Japanese Pharmacopoeia, 1992; British Pharmacopoeial Commission, 1993;
United States Pharmacopeial Convention, 1994). Other spectrophotometric (Mañes et al.,
1987; El-Brashy et al., 1993) and mass spectrometric (McCarley & Brodbelt, 1993)
methods of analysis for diazepam in pharmaceutical preparations have been reported.
Diazepam and its metabolites (including oxazepam (see pp. 1 16-117) and temazepam
(see pp. 162-163)) can be analysed in biological fluids and tissues by radioimmunoassay
(Takatori et al., 1991), gas chromatography (GC) (Löscher, 1982), GC-mass spectro-
metry (GC/MS) (Maurer & Pfleger, 1987), GC with electron capture detection (Peat &
Kopjak, 1979; Beischlag & Inaba, 1992) and high-performance liquid chromatography
(Peat & Kopjak, 1979; Lensmeyer et al., 1982; Komiskey et al., 1985; Mura et al., 1987;
Fernández et al., 1991; Chiba et al., 1995).

1.2 Production and use

1.2.1 Productiol1

A method for preparing diazepam was first reported in 1961 (Sternbach & Reeder,
1961; Sternbach et al., 1961); commercial production of diazepam in the United States of
America was first reported in 1963 (United States Tariff Commission, 1964).
Diazepam is prepared by reacting 2-(methylamino)-5-chlorobenzophenone in ethereal
solution with bromoacetyl bromidç to form 2-(2-bromo-N-methylacetamido)-5-chloro-
benzophenone. The latter is then reacted with ammonia in methanol solution to form the
2-amino-N-methylacetamido compound, which is cyclized with dehydration to produce
diazepam. The crude diazepam may be purified by recrystallzation from diethyl ether
(Gennaro, 1995).

1.2.2 Use

Diazepam is a benzodiazepine with anxiolytic, sedative, muscle-relaxant and anti-

convulsant properties. The active metabolite is N-desmethyldiazepam, which has a long
duration of action (Reynolds, 1993). The therapeutic effects of the benzodiazepines are
believed to be due to their binding to the protein receptor complex for the inhibitory
neurotransmitter, y-aminobutyric acid (GABA). This complex has binding sites for both
phenobarbital and the benzodiazepines (Barnard et aL., 1984). Binding of benzodia-
zepines to the ex subunit of the complex affects chloride conductance within long-fibre
neurons and interneurons in the central nervous system and enhances the efficiency of
GABAergic transmission (Richards et aL., 1986). Central benzodiazepine receptors have
been found in human fetal brain tissues by 18 weeks of conceptual age (Brooksbank
et aL., 1982). Besides this receptor in the central nervous system, there also appears to be
a benzodiazepine receptor in peripheral organs (Krueger & Papadopoulos, 1992). This is
a mitochondrial protein which may be involved in the regulation of steroid biosynthesis
(see Section 4.2.2(c)).
 40 IARC MONOGRAPHS VOLUME 66

Diazepam is used in the management of severe, disabling anxiety disorders, as a

hypnotic in the short-term management of insomnia, in treating convulsions, particularly
status epilepticus and febrile convulsions, and in controlling alcohol withdrawal
symptoms. It is also used as a premedication and sedati ve before surgical and other
procedures, and for the relief of muscle spasm as in cerebral paisy (Reynolds, 1993).
Diazepam is a common adjunct in cancer therapy and may be provided as a pre-
admission drug before cancer diagnosis (Derogatis et al., 1979).
The oral dose for anxiety states usually ranges from 2 mg three times daily up to
30 mg daily in divided doses. Similar doses may be sufficient for control of mild to
moderate symptoms of alcohol withdrawaI. A single dose of 5-30 mg before retiring is
given for insomnia associated with anxiety. ln muscle spasm, 2-15 mg may be given
daily in divided doses and increased, in severe spastic disorders, such as cerebral paisy,
to up to 60 mg daily. A similar dosage range has been recommended for the adjunctive
use of diazepam in some types of epilepsy. Diazepam at 5-20 mg may be given as a
single oral dose or in divided oral doses as a premedication before dental, minor surgical
or other procedures. A slow-release oral formulation of diazepam is available in some
countries; a dose of 15 mg daily is considered to be equivalent to 5 mg three times daily
of the conventional oral formulation. A suggested initial oral dose of diazepam for
children is 100-200 iig/kg bw, but up to 800 iig/kg daily has been given. Dosage recom-
mendations are not generally given for premature infants or infants 30 days of age or
younger, since safety and efficacy have not been established for these groups (Reynolds,
1993; Medical Economics, 1996).
Diazepam may be given rectally as suppositories in doses similar to the oral doses.
A rectal solution of 2-4 mg/mL diazepam may be particularly useful for the control of
convulsions; the dose for adults and children over three years of age is 10 mg, and the
dose for children aged one to three years is 5 mg. If there is no response after five
minutes, the dose may be repeated (Reynolds, 1993).
Diazepam may be given by deep intramuscular injection, although absorption is
erratic and gives rise to lower blood concentrations than those obtained after oral admi-
nistration. lt may also be given by intravenous injection, carried out slowly into a large
vein of the antecubital fossa at a recommended rate of no more than 1 mL of a 0.5%
solution (5 mg) per minute. ln cases of severe anxiety or acute muscle spasm, diazepam
(10 mg) may be given intramuscularly or intravenously and repeated after 4 h. Higher
doses may be required for the treatment of delirium tremens. Patients with tetanus may
be given 100-300 iig/kg bw intravenously, repeated every 1-4 h; alternatively, a conti-
nuous infusion of 3- 10 mg/kg bw every 24 h may be used or similar doses may be given
by nasoduodenal tube. Considerably higher doses have been used for extremely severe
cases of tetanus. For premedication or sedation before dental, surgical or other proce-
dures, 100-200 iig/kg bw (usually 10-20 mg for adults) may be given by injection.
A suggested parenteral sedative or muscle-relaxant dose for children is up to 200 iiglkg
bw (Reynolds, 1993).
Diazepam may be given parenterally, preferably by the intravenous route, for the
control of status epilepticus or severe recurrent or febrile convulsions. ln the United
 DIAZEP AM 41

Kingdom, the usual dose is 150-250 Jlg/kg bw (or 10-:20 mg) for adults and 200-
300 Jlg/kg bw or 1 mg per year of life for children. These doses may be repeated after
30-60 min if required. Once the seizures are controlled, their recurrence may be
prevented by intravenous administration of phenytoin sodium (see monograph, pp. 178-
179) or by a slow infusion of diazepam. For adults, the maximal total dose of diazepam
is 3 mg/kg bw over 24 h (Reynolds, 1993). ln the United States, the initial dose for adults
is 5- 10 mg, repeated if required at 10-15-min intervals up to a maximum of 30 mg.
Doses for children are: infants over 30 days and under five years of age, 200-500 Jlg
every 2-5 min up to a maximum of 5 mg; children five years and older, 1 mg every
2-5 min up to a maximum of 10 mg. The above dosage regimens may be repeated after a
period of 2-4 h if necessary (Medical Economies, 1996). Elderly and debilitated patients
should be given no more th an one half of the usual adult dose. Reduction of dosage may
also be required in patients with liver or kidney dysfunction (Reynolds, 1993).
Clinical uses of diazepam and other benzodiazepines have been reviewed (HoUister
etal., 1993). Diazepam has been used extensively in children (Goodman Gilman et aL.,
1990) .
Worldwide, diazepam is the most widely prescribed of the benzodiazepines. Compa-
rative data on sales of diazepam in several countries are shown in Table 1. Overall, sales
declined by approximately 16% from 1990 to 1995.
Table 2 compares the number of prescriptions written in the United States for several
benzodiazepines, including diazepam, and for the anticonvulsant, phenytoin, in 1990 and
1995.
Table 3 compares the total sales for these same benzodiazepines and phenytoin in
1990 and 1995 in major markets worldwide.

1.3 Occurrence

1.3.1 Naturaloccurrel1ce

Wildmann et al. (1987, 1988) reported the occurrence of trace amounts of diazepam
in the brain and adrenals of rats and in wheat and potato samples.
Unseld et al. (1989) reported finding low concentrations of diazepam in brain tissue
samples from several animal species and plants using GC/MS. Diazepam concentrations
ranged from 0.005 to 0.019 ng/g wet weight in brain tissue from salmon, frog, monitor
lizard, rat, cat and dog. Traces of diazepam were detected in deer, bovine, adult human
and stilIborn human brain tissue samples. Diazepam concentrations ranged from 0.002 to
0.010 ng/g in the plant samples (potato tuber, yelIow soya beans, unpealed rice,
mushrooms).
Unseld et al. (1990) reported on the 'natural' occurrence of diazepam in human brain
samples. AlI brain samples were examined by GC/MS and the concentrations observed
ranged from 0.15 to 0.34 ng/g wet weight tissue. The human brain tissue samples had
. been stored before diazepam was first synthesized in 1963.
42 rARC MONOGRAPHS VOLUME 66

Table 1. Sales of diazepam in various countriesa (number of standard

unitsb, in thousands)

Country 1990 1991 1992 1993 1994 1995

Africa
South Africa 8227 7843 7903 6977 7657 7456
North America
Canada 119159 104 306 1 00 400 80016 84831 82 060
Mexico 90 858 83 626 81044 70177 67265 63874
United States 77 5 409 711049 667 798 678 466 697 750 764 904
South America
Argentina 97 383 106 010 107 794 100 03 7 95 962 88 942
Brazil 387 216 388 859 327 549 282 044 259 621 209 23 1
Colombia 9644 10109 12070 4575 1840 2571
Venezuela 11398 IL 959 11 153 12821 10 552 12190
Asia
J apan 533 690 520 677 511530 484280 482 458 474676
Republic of Korea 22647 22 903 23253 22 990 22 356 21310
Australia 94309 85418 82 738 81 212 80203 80653
Europe
Belgium 21044 21379 21748 21 496 21309 20 544
France 102 252 96216 88013 82 141 77 222 68 05 1
Germany 152 300 141334 118695 113702 113 156 115477
Greece 28691 22 454 20476 18719 17557 14291
Italy 265 153 252 443 242 370 22 1 544 194035 190 047
Netherlands 45821 48 460 48 497 46615 45 161 44 898
Portugal 75961 77 806 74370 75 962 78941 80 988
Spain 238 524 228 722 226454 215 093 210853 211265
Sweden 51830 46781 47 085 46 296 46 255 45 552
Switzerland 11364 11007 10616 7947 7588 7232
Turkey 13 156 14370 15507 14809 16054 18 156
United Kingdom 238 198 235 807 227 345 230 963 224 86 1 232365

"Data provided by IMS

¡, Standard dosage units, uncorrected for diazepam content

1.3.2 Occupatiol1al exposure

No quantitative data on occupational exposure levels were available to the Working
Group.
The National Occupational Exposure Survey conducted between 1981 and 1983 in
the United States by the National Institute of Occupational Safety and Health indicated
that about 20 650 employees were potentially occupationally exposed to diazepam. The
estimate is based on a survey of United States companies and did not involve
measurements of actual exposure (United States National Library of Medicine, 1996).
 DrAZEPAM 43

Table 2. Use of sorne benzo-

diazepines and phenytoin in
the United Statesa (numbers of
prescriptions, in thousands)

Drug 1990 1995

Diazepam 13056 12475

Estazolam 0 598
Oxazepam 1647 1436
Prazepam 1205 1

Temazepam 5567 5916

Phenytoin" 8 848 9 81 i

" Data provided by lMS. No sales of

doxefazepam or ripazepam in the
United States
"Dilantin'" only

Table 3. Comparative sales of several benzodiaze-

pines and phenytoin in major markets worldwidea
(number of standard units\ in milions)

1990 1995 Countries with the highest use

Diazepam 3394 2857 USA, Japan, Brazil, Spain, UK

Phenytoin 2423 2218 USA, Japan, UK, Canada
Oxazepam 1278 996 Germany, France, USA,
Netherlands
Temazepam 706 756 UK, USA, Australia
Prazepam 361 276 France, USA, ltaly
Estazolam 158 187 Japan, USA, Portugal

"Data provided by lMS

l, Standard dosage units, uncorrected for content of active
ingredient

1.4 Regulations and guidelines

Diazepam is listed in the following pharmacopoeias: Belgian, British, Brazilian,

Chinese, Czech, Egyptian, European, French, Greek, Hungarian, Indian, International,
Italian, Japanese, Mexican, Netherlands, Nordic, Portuguese, United States and former
Yugoslavian (Reynolds, 1993).
44 IARC MONOGRAPHS VOLUME 66

2. Studies of Cancer in Humans

W orldwide, diazepam is the most widely used of the benzodiazepines. For this
reason, most specific epidemiological information about potential carcinogenic effects of
the benzodiazepines relates to this drug. ln addition, reported use of unspecified
sedatives or hypnotics probably implies principally use of diazepam. This section
therefore reviews not only epidemiological studies which investigated diazepam
specifically but also those which reported risk associated with unspecified psychotropics,
tranquillizers or benzodiazepines.
Several potential biases in the epidemiological studies of benzodiazepines or
diazepam in relation to cancer deserve mention. Control selection may be problematic in
case-control studies, in that it may be difficult to select a priori a diagnostic category
unrelated to the exposure, while general population controls may be less likely to self-
report short- or long-term use. Indication for use of diazepam or other benzodiazepines
has, in general, not been considered as a potential confounding factor. If anxiety or
depression due to an underlying hormonal imbalance is involved in the etiological path-
way of a particular cancer, the use of psychotropic drugs is merely a marker for the
ter of
underlying condition, rather than indicating that the drug is the initiator or promo

the cancer. Another, possibly most important, consideration is that diazepam is a

common adjunct in cancer therapy and may be provided as a pre-admission drug before
cancer diagnosis (Derogatis et al., 1979). It is therefore essential to ascertain precisely
the date of first use of the drug in relation to the date of diagnosis of the cancer, so that
reasonable rules for censoring exposure history may be established.

2.1 Descriptive studies

A cross-sectional study examined use of psychotropic drugs for one month or more
by 250 women who had been diagnosed with breast cancer at least one year previously
and who were attending breast cancer clinics for follow-up visits at two general hospitals
in the United Kingdom (Stoll, 1976). Hypnotics, minor tranquillzers, sedatives and
antidepressants were used by 14% of the women during the 12 months before diagnosis
and by 32% in the 12 months prior to the questionnaire. Among women with metastases
at presentation or recurrence within 12 months, 22% had used such drugs before
diagnosis, compared to 13% among women with local disease at presentation or
recurrences later than 12 months (p .c 0.03). (The Working Group noted that both the
absence of comparably collected control data and a biological rationale for distinguishing
the high-usage group were limitations of this descriptive study.)

2.2 Cohort studies

Since 1969, members of the Kaiser Permanente Medical Care Program (KPMCP) in
northern California, United States, have been categorized according to their drug
exposure, as identified from prescription records, and followed during their membership
 DIAZEPAM 45

in the KPMCP. The occurrence of cancer was identified from admission records or by
cross-checking against the San Francisco Bay Area Tumor Registry. Expected numbers
of cancers were based upon age- and sex-specific rates for the entire cohort. ln the latest
report from this study with follow-up through 1984 of 12 928 diazepam users, the
standardized morbidity ratio for all cancers was 1.0 (( 95% confidence interval (CL), 1.0-
1.1); 807 observed versus 784 expected); for breast cancer, 1.1 ((0.9-1.3); 155 observed
versus 144 expected); for Hodgkin's disease, 0.0 ((0.0-0.79); 0 observed vers
us 4.7
expected) and for colon cancer, 0.7 ((0.5-0.9); 57 observed versus 79.9 expected) (Selby
et al., 1989; Friedman & Selby, 1990). (The W orking Group noted that dose response
was not addressed in these data, and that age and sex were the only confounding factors
considered. )

ln a reconstructed retrospective cohort study, breast cancer in female members of a

Group Health Cooperative (GHC) in Seattle, W A was investigated (Danielson et al.,
1982). During the period 1977-80, 302 women, aged 35-74 years, who had been
members of the GHC for at least six months and who had a newly diagnosed breast
cancer were identified. Age-specific incidence rates of breast cancer for users and non-
users of diazepam were calculated. W omen were classified as exposed to diazepam if at
least one prescription for the drug had been filed in the six months before breast cancer
diagnosis; drug taken only in the two weeks before mastectomy was not considered. Of
302 women with breast cancer, 27 had taken diazepam before their breast cancer
diagnosis; on the basis of 184 438 women-years of observation in total, the age-adjusted
risk ratio for breast cancer was 0.9 (95% Ci, 0.6-1.3). (The Working Group noted that
this study cou Id not address th~ ettect of long-term use of diazepam and that no
confounding factors had been considered.)

2.3 Case-control studies

Table 4 summarizes case-control data on use of benzodiazepines or diazepam that

were available to the Working Group. By far the most numerous studies of diazepam in
relation to human cancer are case-control studies of breast cancer.

2.3.1 Breast cal1cer

Wallace et aL. (1982) compared diazepam use in 151 new 1 y diagnosed breast cancer
cases at hospitals of the University of Iowa, United States, between 1974 and 1978 with
use in a similar number of women with non-cancer conditions selected from the general
medical and surgical wards. Matching variables included age and hospital payment
category. Ali subjects were white. The crude relative risk for breast cancer associated
with diazepam use was 1.0 (95% CI, 0.5-1.8). Adjustment for other potential con-
founders such as age at menarche, parity, type of menopause and family history of breast
cancer did not affect the association. Details on refusai rates for controls were not
provided. (The W orking Group noted the small number of cases and insufficient data on
refusaI rates for controls.)
 .l
0\
Table 4. Case-control studies of diazepam or benzodiazepine use

Study Location, No. of cases/ Source of Exposure Odds 95% CI Notes

period contro1s controls ratio
Breast cancer
Wallace Iowa, USA, 151/151 Non-cancer Diazepam, any use 1.0 (0.5-1.8) Response rate not reported
et al. (1982) 1974-78 patients
K1einerman USA, 1075/1146 Participants Diazepam, any use -.
et al. (1984)
0.7 0.6-0.9 Invasive carcinoma ? 1 cm );
1973-77 in screening
programme
0.9
1.1
0.6-1.3
0.8-1.6
Invasive carcinoma :: 1 cm
ln-situ carcinoma
n;:
Kaufman USA, Canada, 1236/728 Cancer ~
et al. (1982) Israel,
Diazepam, 4 times/week, 0.9 0.5-1.6 Similar results with 'fema1e' 0
patients during ~ 6 months, Z
1976-80 cancer or other cancer controls 0
? 18 months before 0
;:
interview );
Kaufman USA, 3078/1259 Cancer and Diazepam, 4 times/week, ""
1.0 0.6-1.7 Similar results with non-cancer ::
et al. (1990) 1981-87 non-cancer cr
during ~ 6 months, controls
patients ~
? 18 months before
interview
0r-
Toronto, 607/1214 c:
Cens us Diazepam, 4 times/week, 0.8 0.5- 1.3 ~
Canada, records during ~ 6 months, tT
1982-86 0\
? 18 months before 0\
interview
Rosenberg USA, 6056/1603 Cancer Benzodiazepine, sustained 1.0 0.8-1.3 Overlap with Kaufman et al.
et al. (1995) 1977-91 patients use
"
(1982, 1990). Similar results with
non-cancer controls. Odds ratio for
;? 5 years of benzodiazepine use:
0.8 (0.5-1.4)
Diazepam, sustained use " 1.0 0.7-1.4
Table 4 (contd)

Study Location, No. of cases/ Source of Exposure Odds 95% CI Notes

period controls contraIs ratio

Ovarian cancer
Tzonou Athens, 189/200 Visitors to 'Tranquilizers or 1.0 0.6-1.6
et al. (1993) Greece, hospitals hypnotics', any use
1989-91
Harlow & Boston, MA, 450/454 General Benzodiazepine, any use 1.8 1.0-3.1 Higher risk for use before age 50 or
Cramer USA, population ¿ 10 years before interview
(1995) 1978-81,

Rosenberg
1984-87
USA,
-0
~
767/1603 Cancer Benzodiazepine, sustained 0.9 0.6-1.4 Similar results with non-cancer N
et al. (1995) 1977-91 patients use " contro1s. Odds ratio for ¿ 5 years of rr
'i
benzodiazepine use: 0.3 (0.1-0.9) ;.
Diazepam, sustained use" 1.0 0.6- 1.6
~
Malignant melanoma
Adam & Eng1and and 150/496 General Diazepam, ¿ 1 month 1.2 0.6-2.2 General practitioners' records
Vessey Wales, practitioners
(1981 ) 1971-76 ' patients
10 1/302 General Diazepam, ¿ 1 month 1.7 0.9-3.2 Self-reported use
practitioners
, patients
Rosenberg USA, 1457/3777 Cancer Benzodiazepine, sustained 1.0 0.8- 1.4 Simi1ar results with non-cancer
"
et al. (1995) 1977-91 patients use controls. Odds ratio for ¿ 5 years of
benzodiazepine use: 0.9 (0.5-1.7)
Diazepam, sustained use" 1.0 0.7-1.5

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48 IARC MONOGRAPHS VOLUME 66
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=- ro
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N N N Q)
ro i: ~cU i: ~
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cU cr ro ro i: ~ ro r: "cU c:
¡r i: o: :: i: cU cU
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i: ..
() "~
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:: r:
o 0 ro ro ro ro r: ..
u: () U 0. U c. c: ro
U 0. ro ro
U ci
..
cr
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() cr i- r-
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"- .-8 ~ r- r-
o .. .. .. ..r- r-
r-
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o r: aa
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a~ r-
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Z 8 -~\0 N
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0\
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... "C

i.'¿
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o r. 00
E ~ 1
0\ 0\ .c -
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--
"0
~ 0
() 1:: ~ ... . Ir
ro Cl
"" r-
0\
~. r-1 ~. r-1 e . 1
.. ~ t: ~ ~. r-1
.. o cU
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.. r. r-
~~ r. r-
~~ - CI 0\
~'I ~ _
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:fI: ~
'I
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~ Ol Ç)
'o ~ i; ~
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Ç) Ol If ~'I
..-
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;; Q. ti ~
- ..
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~ ..i: '--
~ cU 0\
= ..
i: '--
~ cU 0\ o
i. 0\
:i. ..
i: '--
Q) 0\ = i. 0\
:i ..i:cU '-0\-
~oI: ~cU -.,:
I: cU -.
"0 = cr
0 ..i;
~
E-
::
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00
"3 ~ 0\
~ ~ '- - =
~ i: ~
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Õ ~ ,:
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Q) -.
o= ~ ,:
i: ~ :: i: ~
Table 4 (contd)

Study Location, No. of cases/ Source of Exposure Odds 95% CI Notes

period controls con troIs ratio

Thyroid cancer
Rosenberg USA, 111/3777 Cancer Benzodiazepine, sustained 0.9 0.4-2.4 Similar results with non-cancer
et al. (1995) 1977-91 patients use
a
controls. Odds ratio for 2: 5 years of
benzodiazepine use: 1.3 (0.3-6.0)
Diazepam, sustained use" 0.8 0.2-2.7
Liver cancer
Rosenberg USA, 37/3777 ..0
et aL. (1995) 1977-91
Cancer
patients
Benzodiazepine, sustained
"
1.2 0.3-5.2 Similar results with non-cancer ?N
use controis tT

Endometrial cancer
Diazepam, sustained use" 2.0 0.5-8.4
?'"
$:
Rosenberg USA, 812/1603 Cancer Benzodiazepine, sustained 1.2 0.8-1.9 Similar results with non-cancer
et al. (1995) 1977-91 patients use
"
controls. Odds ratio for 2: 5 years of
benzodiazepine use: 1.4 (0.6-2.9)
Diazepam, sustained use " 1.4 0.8-2.3
Testicular cancer
Rosenberg USA, 314/21 74 Cancer Benzodiazepine, sustained 1.2 0.5-3.1 Similar results with non-cancer
et al. (1995) 1977-91 patients use" controls
Diazepam, sustained usea 1.4 0.4-.7
"2: 4 times/week, during ;; 1 month, ;: 24 months before interview

.t
\0
 50 IARC MONOGRAPHS VOLUME 66

Kleinerman et al. (1984) examined the association between breast cancer and
diazepam use in white women participating in the Breast Cancer Detection
Demonstration Project in the United States between 1973 and 1977. The study included
1075 prevalent cases who had a histologically confirmed breast cancer detected during
the five-year period and 1146 controls selected from women with normal mammographic
results that did not require biopsy. Controls were matched by screening centre, age and
date at entry and length of continuation in the screening programme. Exposures were
assessed by home interview, and participation rates were 86% for cases and 74% for
controls. Only diazepam use begun at least six months before the date of the breast
cancer diagnosis was considered. The relative risk associated with diazepam use for
invasive tumours :? 1 cm was 0.7 (95% CL, 0.6-0.9), that for invasive tumours ~ 1 cm
was 0.9 (0.6- 1.3) and that for in-situ tumours was 1. 1 (0.8-1.6). (The Working Group
noted that some drug-exposed women with poor survival may not have been included in
this study.)
Data on breast cancer in relation to diazepam use are available from a hospital-based
case-control surveillance system (Kaufman et al., 1982, 1990; Rosenberg et al., 1995).
(These three studies, although reported separately, may overIap in either study
methodology or subjects included.) ln the first report from this series, diazepam use was
ascertained by personal interviews in 1236 women less than 70 years old diagnosed with
primary breast cancer in the six months before admission and 728 controls admitted to
metropolitan hospitals in the United States, Canada and Israel during 1976-80 (Kaufman
et al., 1982). Control women had other cancers including other 'female' cancers such as
endometrial or ovarian cancer. Of patients approached, 5% refused to be interviewed.
The principal analyses excluded use of diazepam in the 18 months before hospital
admission, to avoid the possibility of recording use begun after a diagnosis of breast
cancer or because of clinical symptoms preceding the diagnosis, and also focused on
'regular' use of diazepam (defined as use of the drug for at least four days per week) and
'sustained regular' use (for a total duration of at least six months). Potential confounding
factors considered were age, geographical region, education, religion, parity, age at first
pregnancy, menopausal status, age at menopause, family history of breast cancer and
alcohol use. The relative risk for breast cancer associated with regular use of diazepam
for six months or more was 0.9 (95% CI, 0.5- 1.6), with ail other cancers as the control
group. The relative risk for breast cancer associated with regular use of diazepam for less
than six months was 0.8 (0.4-1.4), with ail other cancers as the control group. The
relative risks were no greater for women with metastatic disease. Similar risk estimates
were found when women with either other cancers or 'other female' cancers were used
as the control group.
These investigators extended their study with data from 3078 breast cancer cases, 18-
69 years old, with cancer diagnosed within six months before admission, interviewed
between 1981 and 1987 in hospitals in the metropolitan United States and from three
separate control groups interviewed over the same time period (Kaufman et al., 1990).
As in the previous study, women with other cancers (754) or with other 'female' cancers
(505) were included as controls. A non-cancer control group (672) was also included,
which was composed primarily of women with ectopie pregnancy (281), appendicitis
 DIAZEPAM 51

(230) or retinal detachment (100). W omen admitted for trauma were not included
because of the possibility that diazepam increases the risk for accidents. Of patients
approached, 4% refused to be interviewed. The authors adjusted for age, geographical
region, education, religion, parity, menopausal status, age at menarche, first birth and
menopause, family history of breast cancer, alcohol, oral contraceptive and other benzo-
diazepine use. ln this study, the percentage of white women varied from 64% in the non-
cancer controls to 90% in the female cancer controls, and race was included as an
adjustment variable. Regular use of diazepam, defined as in the previous study, was
associated with a relative risk for breast cancer of 1.0 (95% Ci, 0.6-1.7), compared with
controls with any other cancer and 0.8 (0.4-1.8) compared with the non-cancer controls.
Risks were similar for 'sporadic' use of diazepam, defined as use beginning at least 18
months before interview and lasting for less than six months or use involving fewer th
an
four days per week. 'Recent' use of diazepam (beginning within 18 months of interview)
was also associated with a significantly elevated risk for breast cancer of 1.9 (1.1-3.1),
compared with controls with any other cancer and 5.6 (2.3-13.6) compared with the non-
cancer controls. (With respect to 'recent use', see the comment below.)
ln the same publication, results of a separate study of 607 cases of breast cancer
identified between 1982 and 1986 through the Ontario Cancer Institute, Canada, were
reported (Kaufman et al., 1990). Controls in this study were 1214 women selected from
municipal voting and census records matched for neighbourhood and decade of age.
Refusai rates were 21% among potential cases and 35% among potential controls. The
relative risks for breast cancer associated with categories of diazepam use, as defined
above, were 0.8 (95% Ci, 0.5- 1.3) for regular use, 1. 1 (0.8-1.5) for sporadic use and 3.1
( 1.5-6.4) for recent use. (With respect to 'recent use', see the comment below.)
ln the most recent and comprehensive report using this hospital surveillance system to
investigate possible effects of benzodiazepine exposure, not only breast cancer but also
other cancers were analysed (Rosenberg et aL., 1995). The 6056 breast cancer cases
included some previously reported cases from 1977 to 1987 as weIl as new cases
admitted between 1988 and 1991. The primary control group was 1603 women with
other cancers excluding those of the endometrium or ovary. Participation rates were
about 96% and adjustment was made for age at menarche, first pregnancy and
menopause, parity, religion, education, race, family history of breast cancer and duration
of use of oral contraceptives or oestrogen replacement therapy. ln contrast to the
previous reports from this series, 'recent use was defined in relation to a two-year
interval before hospital admission, rather than an 18-month interval. 'Sustained' use of
diazepam for at least four days per week for one month initiated two or more years
before admission was associated with a relative risk for breast cancer of 1.0 (95% Ci,
0.7- 1.4). For sustained use of aIl benzodiazepines combined, risk did not vary
significantly with the number of years since last use or with a duration of use of five
years or more.
(The Working Group noted that the elevated risks for breast cancer associated with
benzodiazepine use within the 'recent' period before hospital admission observed in
some of these studies could be attributed to drug use begun after the diagnosis of breast
cancer. More precise information on the timing of the drug exposure in relation to the
 52 IARC MONOGRAPHS VOLUME 66

date of diagnosis of breast cancer rather than the interview date might have clarified this
potential bias.)

2.3.2 Ovarial1 cal1cer

Tzonou et al. (1984) studied 150 women with malignant epithelial ovarian tumours
newly diagnosed during 1980 and 1981 in 10 hospitals in Athens, Greece, and compared
them with 250 women in the Athens Hospital for Orthopedic Disorders. No contrais
approached were said to have refused, but participation rates for cases were not stated.
'Frequent' use of 'psychotropic' drugs was reported by eight cases and two contrais,
giving a crude odds ratio of 7.0 (95% CI, 1.8-27) for ovarian cancer associated with use
of these drugs. (The W orking Group noted that the indications gi ven by the authors for
'frequent use of 'psychotropic' drugs in this study suggest that benzodiazepines may
have been used infrequently.)
ln a subsequent report, the same investigators compared drug use in 189 women less
than 75 years old with malignant epithelial ovarian cancer diagnosed during 1989-91 in
two hospitals in greater Athens with that of 200 visitors to the same hospitals (Tzonou
et al., 1993 ). Participation rates were 90-94%. The relative risk associated with ever use
of 'tranquillizers or hypnotics' over an 'extended' period was 1.0 (95% CI, 0.6-1.6) after
adjustment for age, education, weight, age at menarche and menopause, parity, smoking
and other study variables.

(The Working Group noted the small number of exposed subjects in both studies, a
possible lack of appropriateness of the control selection and the lack of specificity of
information on the agent and its duration of use.)
Benzodiazepine use was investigated in a study which combined two case-control
studies conducted previously from ten hospitals in Boston, MA, United States (Harlow &
Cramer, 1995). The study included 450 cases of malignant epithelial ovarian cancer
diagnosed between 1978-81 and 1984-87 in women 18-80 years old and 454 contraIs
identified from the general population during the same period and matched for age, race
and precinct of residence. Participation rates for cases and controls were araund 70%.
Any use of a benzodiazepine tranquilizer was associated with a relative risk for ovarian
cancer of 1.8 (95% CI, 1.0-3.1) after adjustment for parity, oral contraceptive use,
religion, body mass, prior hysterectomy and therapeutic abortion. Risk appeared to be
confined to women whose first use of the drug either was before the age of 50 years,
where the relative risk was 2.7 (1.3-5.6), or occurred 10 or more years before the age at
diagnosis (3.2; 1.4-7.6). (The Working Group noted that more specifie data on risk for
ovarian cancer by frequency or duration of drug use wou
Id have been helpful in
establishing the validity of this association.)
Data on diazepam use in relation to ovarian cancer are also available from the study
by Rosenberg et al. (1995), described previously. Among 767 women with ovarian
cancer, sustained use of diazepam was reported by 25 (4.3%), giving a relative risk of 1.0
(95% CI, 0.6- 1.6).
 DIAZEPAM 53

2.3.3 Other cal1cers

A case-control study of malignant melanoma in women, aged 15-49 years, in relation

to diazepam use was conducted from a survey of general practitioners in southern
England between 1971 and 1976 (Adam & Vessey, 1981). Controls were selected from
the practice lists of the same doctors and matched by age and marital status. The relative
risk for malignant melanoma associated with use of diazepam for more th
an one month,
as assessed from the general practitioners' records, based on 150 cases and 496 controls
was 1.2 (95% CI, 0.6-2.2). The risk was 1.7 (0.9-3.2) for diazepam use as assessed from
postal questionnaires completed by 101 cases and 302 controls. (The W orking Group
noted that the poor response rates to the postal questionnaire may have accounted for the
marginally greater risk for melanoma associated with diazepam use if a greater number
of responding cases recalled short-term use.)
A hospital-based case-control study of multiple myeloma was conducted in
Baltimore, MD, United States (Linet et al., 1987). A total of 121 cases of multiple
myeloma in white patients were ascertained from seven hospitals during the period
1 January 1975 to 31 December 1982. Controls were individually matched to cases on
hospital, age (:1 5 years), sex and year of diagnosis. The control group included patients
randomly selected from 11 categories of disease which included digestive and nervous
system diseases, accidents and poisoning but excluded diagnoses of cancer, diseases of
blood-forming organs, mental disorders, obstetric conditions and congenital anomalies.
Information about many factors was obtained from the study subjects with a
questionnaire by telephone; if the indi vidual concerned had died or could not be
interviewed for any other reason, information was sought froID the closest possible
relative. The response rate was 83% among cases, leaving 100 cases for analysis. Data on
drug use occurring before diagnosis were analysed. The crude odds ratio associated with
diazepam use, based on nine discordant pairs, was 2.0 (95% CI, 0.4-12). (The Working
Group noted that the diagnostic categories for controls could have been associated with
exposure to diazepam. ln addition, the greater number of proxy interviews for cases than
controls could not be adjusted for with diazepam as the exposure.)
The study by Rosenberg et al. (1995), described previously, addressed risk for other
cancers associated with diazepam use. For sustained use of diazepam, defined as use for
at least four days per week for at least one month which had been initiated at least two
years before admission, relative risks for selected sites were: 0.7 (95% CI, 0.5-1.0) for
cancer of the large bowel, 0.8 (0.5-1.2) for lung cancer, 0.9 (0.4-2.0) for Hodgkin's
disease, 0.8 (0.2-2.7) for cancer of the thyroid, 2.0 (0.5-8.4) for liver cancer, 1.4
(0.8-2.3) for endometrial cancer and 1.4 (0.4-4.7) for testicular cancer. (See Section
2.3.1 for the Working Group's comments on this study.)
Anthony et al. (1982) surveyed general practitioners in the United Kingdom and
requested information on medications used by any new patients with cancer and by age-
and sex-matched controls who were the 'next' patient with a 'new' complaint not related
to cancer. Six (possibly seven) of 211 male patients with cancer (mostly lung) had taken
barbiturates and benzodiazepines concomitantly, compared to none of 21 1 male contrais
Ip = 0.03 i. (The Working Group noted that participation by the general practitioners was
 54 IARC MONOGRAPHS VOLUME 66

voluntary and quite variable and that the study may have included drug exposures
postdating the cancer diagnosis.)

3. Studies of Cancer in Experimental AnimaIs

3.1 Oral administration

3.1.1 Mouse
Diazepam was incIuded as a reference compound in a study on the carcinogenicity of
prazepam. Groups of 100 male and 100 female albino CFI mice (control group) and sa
male and 50 female mice (diazepam group), eight weeks of age, were given a or
75 mg/kg bw diazepam (melting point, 131 - 135°C) mixed in the diet for up to 80 weeks,
when surviving animais were killed. The dose was chosen to match the high-dose level
of prazepam. The diazepam concentration in the food was adjusted weekly for changes in
body weight and food consumption. The diazepam/diet mixtures were prepared freshly
each week. ln treated mice, body-weight gains were similar to those of con
troIs
throughout the study. From graphie presentations, there appeared to be no significant
effect on mortality in male mice, but the diazepam-treated females had lower survival
(65-75% survival for control and diazepam-treated males, 70% for control females and
40% for diazepam-treated females) (statistics and exact numbers not given). Major
organs (not specified) and visually apparent lesions were examined histologically. By life
table analysis, the incidences of benign hepatocellular tumours were (tumour-bearing
mice/effective number of mice): control males, 1/93; diazepam-treated males, 2/43;
control females, 1/91; and diazepam-treated females, 0/38. Those for malignant hepato-
cellular tumours were: control males, 7/93; diazepam-treated males, 9/43 (p -: 0.05, chi-
square test); control females, 1/91; and diazepam-treated females, 2/38 (de la Iglesia
etal., 1981). (The Working Group noted that diazepam was tested only as a reference
chemical and that the study was terminated at 80 weeks.)

3.1.2 Rat

Diazepam was included as a reference compound in a study on the carcinogenicity of

prazepam. Groups of 115 male and liS female albino SPF Wistar rats (control group)
and 65 male and 65 female rats (diazepam group), eight weeks of age, were gi ven either
o or 75 mg/kg bw diazepam (melting-point, 131- 135°C) daily mixed in the diet for up to
104 weeks, when surviving animais were killed. The diazepam concentration in the food
was adjusted weekly for changes in body weight and food consumption. The diazepam/-
diet mixtures were prepared freshly each week. ln treated rats, body weight gains were
similar to those of controls throughout the study. From graphic presentations, there
appeared to be no significant effect on mortality (50-60% survival for males and about
60% for femal~s) (statistics and exact numbers not given). Major organs (not specifiedl
and visually apparent lesions were examined histologically. No significant increase in the
incidence of tumours at any site was seen for either male or female rats. The incidences
of benign hepatocellular tumours were: control males, 1/1 15; diazepam-treated males,
 o IAZEP AM 55

0/65; control females, 1/1 15; and diazepam-treated females, 0/65. Those for malignant
hepatocellular tumours were: control males, 0/1 15; diazepam-treated males, 3/65
(p = 0.054, Fisher's exact test); control females, 0/1 15; and diazepam-treated females,
0/65 (de la Iglesia et al., 1981).
To study initiating activity, groups of 10 male Fischer 344 rats weighing 150 g (age
not specified) were given 7 and 70 mg/kg bw diazepam (purity, :; 99%) suspended in
10% arabic gum solution daily by gastric instillation for 14 weeks. Fort
Y rats served as
untreated controls. Neoplastic nodules and iron-excluding foci were not found in the
livers of diazepam-treated animais nor in those of the controls (Mazue et aL., 1982;
Remandet et al., 1984).

3.1.3 Hamster

Groups of 55-56 male and 55-56 female Syrian golden hamsters were given
120 mg/kg bw diazepam mixed in the di
et for 57 weeks (females) or 79 weeks (males).
Approximately 110 hamsters per sex served as controls. Some intercurrent deaths, mostly
after week 30, occurred from severe enteritis. No significant increase in the incidence of
tumours was found (Black et al., 1987). (The Working Group noted the single dose, that
diazepam was used as a reference compound for a study on quazepam and that specific
data on survival were not given.)

3.1.4 Cerbil

Groups of 15 male and 12 female gerbils (strain and age not specified) were given
10 mg diazepam (purity not specified) per animal by gastric instillation weekly. Eleven
males and ten females receiving saline served as vehicle controls. Male and female
controls survived 80 and 69 weeks, respectively, while gerbils receiving diazepam
survived 79-81 weeks. Complete histopathology was performed as animaIs died or
became moribund, and two ovarian granulosa-cell tumours in control females were the
only tumours reported (Green & Ketkar, 1978). (The Working Group noted the small
numbers of animais and the weekly administration of diazepam.)

3.2 Administration with known carcinogens

3.2.1 Mouse

Groups of 40 male B6C3Fl mice, five weeks of age, were given a single intra-
peritoneal injection of either 0 or 90 mg/kg bw N-nitrosodiethylamine (NDEA) in
tricaprylin. At seven weeks of age, the mice received 500 or 1500 mg/kg diet (ppm)
diazepam (purity unspecified) in the diet. Eight mi
ce per group were killed at 9, 21 and
33 weeks of exposure and the remainder were killed after 53 weeks of exposure.
Complete necropsy was performed on each animal, and liver, lung, spleen, thyroid,
kidney and visually apparent lesions in other organs were examined histologically.
Between 33 and 53 weeks of exposure, there was an increase in the incidence of hepato-
cellular tumours in animaIs treated with NDEA and diazepam compared with those given
NDEA alone (see Table 5) (Diwan et al., 1986).
 56 IARC MONOGRAPHS VOLUME 66

Table 5. Incidence of lIver tumours in B6C3Fl mice

Treatment Hepatocellular Hepatocellular
adenomas carcinomas

NDEA 101 1 6 0/16

NDEA + 500 ppm diazepam 13/14 5/14"
NDEA + 1500 ppm diazepam 15/15" 9/15h
500 ppm diazepam 0/16 0/16
1500 ppm diazepam 3/15 0/15

From Diwan et aL. (i 986)

a (p ~ 0,05, Fisher's exact test compared with NDEA con
troIs)
b (p ~ 0.01, Fisher' s exact test compared with NDEA contraIs)

3.2.2 Rat

Groups of 10 or 20 male Fischer 344 rats weighing 170 g (age unspecified) were fed
basal diets or diets containing 200 mg/kg (ppm) 2-acetylaminofluorene (2-AAF) for eight
weeks. The daily dose was estimated to be approximately 15 mg/kg bw. ln one group of
10 rats, this was followed by 12 weeks' treatment with 70 mg/kg bw diazepam in 10%
arabic gum solution (purity ? 99%) daily by gastric instilation. There was no significant
increase in the incidence of neoplastic nodules of the liver: untreated controls (20 rats), 0
neoplastic nodule/liver; 2-AAF al
one (20 rats), 0.2 neoplastic nodule/liver; and 2-AAF
plus diazepam (10 rats), 0.5 neoplastic nodule/liver (Mazue et aL., 1982; Remandet et aL.,
1984). (The W orking Group noted the small number of animaIs and the single dose level
of diazepam.)
Groups of 10 male weanling Donryu rats, 21 days of age, were fed a diet containing
600 mg/kg (ppm) 3'-methyl-4-(dimethylamino)azobenzene for three weeks, then left for
a week on basal diet followed by either basal diet or a diet with 500 mg/kg diazepam
(purity unspecified) (daily intake, approximately 50 mg/kg bw) for a further 12 weeks.
The rat livers were scored for adenosine triphosphatase (A TPase)-deficient islands
greater th an 50 llm in diameter. There was no significant difference between rats on basal
di et and those given diazepam in the total number of enzyme-altered islands/cm2
(control, 9.76:t 1.32; diazepam-treated, 8.64 :t 0.80) or in the number of enzyme-altered
islands ? 400 ¡.m (control, 1.03:: 0.24; diazepam-treated, 1.33:: 0.34) (Hino &
Kitagawa, 1982).

3.2.3 Cerbil

Groups of 24 male and 16 female gerbils (strain and age not specified) were given
i 0 mg diazepam (purity not specified) per animal by gastric instilation weekly plus
30 min later weekly subcutaneous injections of 23 mg/kg bw NDEA for life. Groups of
20 male and 19 female gerbils receiving weekly subcutaneous administrations of NDEA
only served as positive controls. Groups of i 1 male and i 0 female gerbils receiving
weekly subcutaneous administrations of saline served as vehicle controls. Gerbils
receiving NDEA plus diazepam tended to survive approximately 20 weeks longer than
 D IAZEP AM 57

gerbils receiving NDEA alone. The incidence of nasal cavity adenocarcinomas was high
in both groups: 92-95% in males and 63-69% in females. ln gerbils receiving NDEA
alone, 85% males and 84% females had cholangiocarcinomas of the liver vers
us none in
the NDEA plus diazepam group (75% females and 83% males had cholangiomas of the
liver). Male gerbils receiving NDEA plus diazepam had three hepatocellular adenomas
and one male and one female had a hepatocellular carcinoma versus none in the NDEA
group (Green & Ketkar, 1978). (The W orking Group noted the small number of animaIs
and that diazepam was administered weekly. J

3.3 Carcinogenicity of metabolites

See the monographs on oxazepam (pp. 1 19- 1 23) and temazepam (pp. 164- 1 65).

4. Other Data Relevant to an Evaluation of

Carcinogenicity and its Mechanisms
4.1 Absorption, distribution, metabolism and excretion

4.1.1 Humal1s

The disposition of diazepam has been reviewed (Mandelli et al., 1978; Schmidt,
1995). Diazepam is rapidly and almost completely absorbed following oral doses of 5, 10
or 20 mg; peak plasma concentrations are usually obtained within 30-90 min and a
secondary peak during the elimination phase has been observed in some studies (Baird &
Hailey, 1972; Hillestad et aL., 1974; GambIe et al., 1975; Kanto, 1975; Korttila &
Linnoila, 1975; Schmidt, 1995). Peak plasma concentrations vary widely (30- fold range)
in different subjects given the same dose of diazepam (GambIe et aL., 1973). Oral admi-
nistration of two 5-mg tablets to 48 healthy male volunteers aged 18-44 years resulted in
peak plasma concentrations after 0.9 h (range, 0.5-2.5 h) of 406 ng/mL (range, 253-586
ng/mL) (Greenblatt et al., 1989). Results were similar among pregnant women receiving
single 10-mg doses during the first trimester (Jørgensen et aL., 1988). Intravenous
administration of 10 or 20 mg diazepam to volunteers gave peak plasma concentrations
of 700-800 ng/mL and 1100-1607 ng/mL, respectively, within 3-15 min. Intramuscular
administration is not clinically useful in adults, but, in newborn babies and children
under 12 years, doses of 0.24-1 mg/kg bw give peak plasma concentrations of 206-1400
ng/mL in 10-60 min (reviewed in Schmidt, 1995). Diazepam has a low pKa and is
lipophilic and consequently distributes quickly into lipoid tissues, and rapidly crosses the
blood-brain barrier. A distribution phase with a usual half-life of about 1 h following a
single dose precedes the elimination phase (Kaplan et aL., 1973; Klotz et al., 1976a,b;
Mandelli et aL., 1978). Plasma protein binding of diazepam is about 97% (van der Kleijn
et al., 1971; Klotz et al., 1 976b). Irrespective of the route of administration, the terminal
elimination half-life is usually within the range of 24-48 h, and me
an values of about
32 h have been obtained in several single-dose studies (reviewed by Schmidt, 1995).
Somewhat longer half-lives (mean of 44 h) were measured by Greenblatt et aL. (1989) in
58 IARC MONOGRAPHS VOLUME 66

healthy male volunteers, and longer half-lIves are also obtained following repeated admi-
nistration (Kaplan et aL., 1973; Mandelli et al., 1978). Elimination half-lives were longer
in premature newborn babies (75 :t 35 h) than in full-term newborn babies (31 :t 2 h)
(Morselli, 1977). Plasma clearance values (CLE) are typically 15-35 mL/min for diaze-
pam and 7- IL mL/min for N-desmethyldiazepam. The values tend to be lower in cases of
liver disease and, at least for N-desmethyldiazepam, after the age of 60 years (Schmidt,
1995) .
Diazepam has two major metabolic pathways in humans (see Figure 1), involving
either the loss of the Ni-methyl group, yielding N-desmethyldiazepam, which is then
oxidized at C) to oxazepam, or the direct oxidation at C), yielding temazepam. The
elimination of oxazepam and temazepam is reviewed in sections 4.1.1 of the respective
monographs in this volume. N-Desmethyldiazepam is the major circulating metabolite of
diazepam, as some 50-60% of diazepam is demethylated (Bertilsson et aL., 1990). The
plasma concentrations of N-desmethyldiazepam approach those of diazepam following a
single dose, and typically exceed those of diazepam after multiple doses, since the
elimination half-life of this metabolite is much longer (50- 120 h) than that of diazepam.
Thus, in the data tabulated by Schmidt (1995), the half-lives of N-desmethyldiazepam are
longer th an those of diazepam in every situation where both were measured (in
volunteers, psychiatric patients, epileptic patients, the elderly and patients with liver
disease). N-Desmethyldiazepam has a longer half-life (40-120 h) than diazepam (20-
54 h) in adults (Mandelli et al., 1978; Bertilsson et al., 1990). There have been few
studies of the excretion of diazepam and its metabolites, but it appears that conjugation is
important before elimination. Schwartz et al. (1965) found that approximately 71 % of
orally administered diazepam and its metabolites is excreted in the urine and 10% in the
faeces. It is not clear whether conjugated oxazepam or conjugated N-desmethyldiazepam
is the more important urinary metabolite (Schwartz et al., 1965; Morselli et al., 1973;
Kanto et al., 1974; Arnold, 1975). ln a recent, but preliminary, study (Chiba et aL., 1995),
four male volunteers aged 24-40 years were given a single dose of 4 mg diazepam orally
and urine was collected over 96 h. Following treatment of the urine with ß-glucuro-
nidase/sulfatase, diazepam was not detectable and the cumulative excretion of N-des-
methyldiazepam, temazepam and oxazepam was 3.9 :t 0.4,6.6:t 1.4 and 2.8:t 0.6% of the
dose, respectively. Enterohepatic circulation might explain the long elimination half-life
of diazepam and the secondary peak observed in some studies during the elimination
phase, but most studies indicate that diazepam is not excreted in the bile in significant
amounts (reviewed in Schmidt, 1995).
Diazepam is able to cross the placenta rapidly (deSilva et aL., 1964; Idänpään-
Heikkila et aL., 1971 a; Jørgensen et aL., 1988) and accumulates in the fetus with slow
elimination of its active metabolite. The fetal plasma levels of diazepam are equal to or
1.2 times higher than the maternaI plasma concentrations (Cavanagh & Condo, 1964;
Erkkola et aL., 1974); the level of its active metabolite, N-desmethyldiazepam, is excep-
tionally high in fetal liver (Erkkola et al., 1974). After the use of diazepam in labour,
diazepam and N-desmethyldiazepam persist in the newborn for eight days postpartum
(Cree et al., 1973). Diazepam and its active metabolite also pass from the mother' s blood
into breast milk (Erkkola & Kanto, 1972; Cole & Hailey, 1975).
 DIAZEPAM 59

Figure 1. Postulated metabolic pathways of diazepam

CI

/ OH

4'-Hydroxydiazepam:
rat

CI
. CI . CI

N-Desmethy Idiazepam
Diazepam OH
(nordiazepam): man
4'-1- ydroxydesineihyl-
diazepam: rat

CHi H
j ~--0 1 1 0
N~OH
Ci -N)-OH Ci -N
~

3-l-ydroxydiazepam (temazepam: Oxazepam: man

N-meiliyloxa/.epam): man GllIcl!onide: dog. 1l01ise.
GIlIclIonide: iioiise. raI, dog, raI. giiinea-pig, rabbit
giiinea-pig. rabbii

CHi H
j 1 . ()
N~OH
~--0
)-OH
Ci -N CI - N

OH OH
..l,-I-ydroxyieiiazepaii: rat 4'-l-ydroxyoxazepaii: ral
Aùaptcd l'rom Schmiùt ( 1995)
Major step:- are indicated hy thick arrows, Note that the chloriiie at the 7 positioii l'l'mains intact.
60 IARC MONOGRAPHS VOLUME 66

Diazepam clearance shows marked interindividual differences. Factors which might

contribute to this phenomenon include age, sex, smoking, lIver disease, enzyme induc-
tion or inhibition and genetic factors affecting the regulation of metabolism (Klotz et al.,
1975, 1977; Greenblatt et al., 1980; Ochs et al., 1981a,b; Abernethy et al., 1983; Ochs
et al., 1983; Alda et al., 1987; Bertilsson et aL., 1989; Zhang et al., 1990; Sohn et al.,
1992). With regard to the importance of the regulation of metabolism, a relationship was
observed in studies of Swedish volunteers (Bertilsson et al., 1989) and Korean volunteers
(Sohn et al., 1992) between more rapid elimination of diazepam and high S-mephenytoin
hydroxylase activity (but see Section 4.1.2). There was no relationship with debrisoquin
hydroxylation (CYP2D6) polymorphism among the Swedish subjects (Bertilsson et aL.,
1989). No significant difference in the clearance of diazepam was found between
Chinese subjects who were extensive or po or S-mephenytoin hydroxylators (Zhang et al.,
1990). Bertilsson and Kalow (1993) suggested that the racial differences can be
explained in terms of the chance selection of different proportions of heterozygotes and
homozygotes in these studies.

4.1.2 Experimel1tal systems

ln contrast to the extensive human pharmacokinetic investigations, equivalent in-vivo

studies in experimental animaIs are rare. Garattini et al. (1973) gave rats, mice and
guinea-pigs 5 mg/kg bw diazepam by intravenous injection and measured blood levels of
diazepam, N-desmethyldiazepam and oxazepam at times from 1 min up to 40 h. Maximal
diazepam concentrations of 2.33 :t 0.10, 1.36 :t 0.1 1 and 1.70:t 0.10 l1g/mL were found
at 1 min in rats, mice and guinea-pigs, respectively. The blood levels were negligible by
5 h in rats and mice and by 10 h in guinea-pigs. ln rats, N-desmethyldiazepam was
detected only at 0.10 :t 0.0 I l1g/mL after 5 min, while oxazepam was not detected at aiL.
However, in mice, N-desmethyldiazepam concentrations were significant from 1 min to
10 h, with a maximum at 30 min of 1.15 :t 0.04 l1g/mL and significant oxazepam
concentrations were found between 30 min and 10 h, with a maximum at 3 h of 0.22
:t 0.02 l1g/mL. ln guinea-pigs, concentrations of N-desmethyldiazepam were significant
from 1 min to 20 h, with a maximum at 1 h of 0.37 :t 0.07 l1g/mL, but no oxazepam was
detected.
Lukey et al. (1991) administered a single dose of 100 l1g/kg bw diazepam to six male
rhesus monkeys by intramuscular injection. The maximal serum concentration of
diazepam was 49.6 :t i 3.9 ng/mL at 28.7 :t 3.6 min, whiIe that of N-desmethyldiazepam
was 38.4 :t 8.8 ng/mL at 169.8 :t 60.5 min. The volume of distribution and systemic
clearance values were i.5 L/kg and 19.4 mL/min/kg, respectively, assuming 100% bio-
availability. Serum protein binding was about 95%.
Diazepam and N-desmethyldiazepam accumulate in adipose tissue. Garattini et al.
(1973) found that adipose tissue/blood ratios in mice given 5 mg/kg bw diazepam iiitra-
venously varied from about 4 (at 5 min) to :; 6 (at 5 h) while in mice given the metabolite
(5 mg/kg bw), the corresponding ratios were 3 (at 5 min) and:; 45 (at 5 h). Accumulation
also occurred in the brain. Maximal diazepam concentrations in the brain after intra-
venous administration of 5 mg/kg bw were 7.04 :t 0.37 l1g/g and 4.28 :t 0.14 /lg/g at
 DIAZEPAM 61

1 min in mice and rats, respectively, and 6.28 :t 0.30 ¡.g/g at 5 min in guinea-pigs. The
brain : blood ratios varied from 2.6 (at 1 h) to 5.2 (at 1 min) in mice, from 1.8 (at 1 min)
to 5.8 (at 3 h) in rats and from 2.8 (at 1 h) to 9.8 (at 5 min) in guinea-pigs.
The distribution of diazepam to certain tissues was examined in rats given 83 ¡.g/kg
bw by intraperitoneal injection (Takatori et al., 1991). Serum concentrations were 3.75 :t
0.62 ng/mL at 1 h and 0.28 :t 0.05 ng/mL at 4 h. Diazepam was also found in the saliva,
bone marrow and brain. From 1 h to 8 h after administration, the concentration in bone
marrow was higher th an that in serum by factors of about 1.2-8,0. Over the period 2-8 h
after dosing, concentrations in the serum, saliva and brain were similar. Transplacental
transfer of diazepam occurs in mice, hamsters and monkeys (Idänpään-Heikkilä et al.,
1971 b).
Early studies of the metabolism of diazepam revealed substantial interspecies diffe-
rences (see Figure 1), whether studied in vivo or by in-vitro methods using liver micro-
somes (Garattini et aL., 1973). N-Demethylation (yielding N-desmethyldiazepam) and
Ci-oxidation (yielding temazepam) occur to various extents in all species studied, but
hydroxylation at the 4'-position of the 5-phenyl substituent is a major pathway in the rat.
The resulting phenolic derivatives of diazepam, N-desmethyldiazepam, oxazepam and
temazepam are ail found in rat urine. This pathway seems to be negligible in most other
species, apart from rabbits (Jommi et al., 1964). Oxazepam can be formed either from
temazepam (by N-demethylation) or from N-desmethyldiazepam (by Ci-oxidation). The
Ci-hydroxy compounds (oxazepam and temazepam) are eliminated in urine as glu-
curonides by mice (Marcucci et aL., 1968), rats (Schwartz et aL., 1967), guinea-pigs
(Marcucci et aL., 1971), rabbits (Jommi et aL., 1964) and dogs (Ruelius et al" 1 965). The
glucuronide and/or sulfate conjugates of several metabolites have also been identified in
the intestinal contents of a rat dosed intraperitoneally with 100 mg/kg bw diazepam
(Schwartz et al., 1967).
ln single-pass experiments with perfused male CD-I mouse liver and an input
concentration of 0.5 ¡.M, diazepam was rapidly cleared, with a steady-state extraction
ratio of 0.952 (St-Pierre & Pang, 1993). The mean hepatic clearance was 1.74 :t
0.21 mL/min/g, which was very close to the perfusate flow rate (1.82 mL/min/g), The
metabolites recovered were (authors' terminology): nordiazepam (N-desmethyl-
diazepam) (47.7 :t 6.5%); nordiazepam conjugate (3.7 :t 2.1 %); oxazepam (28.6 :t 7.1 %);
oxazepam glucuronide (1.7 :t 0.7%); 4'-hydroxynordiazepam (0.6 :t 0.2%); 4' -hydroxy-
nordiazepam glucuronide (0.9 :t 0.3%); temazepam (0.7 :: 0.2%); temazepam glliCU-.
ronide (0.3 :t O. 1 %).

The metabolism of diazepam in cultiired hepatocytes and siibcellular preparations has

also been studied extensively. An early study showed that diazepam iindergoes both
N-demethylation and Ci-hydroxylation in dog and rat IIver in vitro, reactions that are
NADPH-dependent and inducible by phenobarbital (Schwartz & Postma, 1968).
Subsequently, Ackermann and Richter (1977) demonstrated that the oxidations are
mediated by cytochrome P450, using human fetal liver preparations. The CYP2C 1 1
isozyme catalyses the N-demethylation reaction, although other isozymes are also
involved, while CYP3A2 is the major catalyst for C i-hydroxylation (Reilly et a!. 1990;
 62 (ARC MONOGRAPHS VOLUME 66

Neville et aL., 1993; Yasumori et al., 1993). S-Mephenytoin does not inhibit either N-de-
methylation or Ci-hydroxylation of diazepam by human liver microsomes (Hooper e.t al.,
1992), The human S-mephenytoin hydroxylases appear to be CYP2C9 or CYP2C 1 8
enzymes (Nebert et al., i 989; Wilkinson et al., i 989; Romkes et al., 1991), so the
apparent relationship between rapid diazepam clearance and high S-mephenytoin
hydroxylase activity observed in humans (see Section 4,1.1) remains to be explained.

4.2 ToxIc effects

4.2.1 Humal1s

(a) Aciite to:ricity

ln a recent report on 215 lethal intoxications due to self-poisoning with diazepam
alone or in combination with other substances, diazepam was associated with a higher
rate of death per million prescriptions than the average for benzodiazepine anxiolytics
such as lorazepam and oxazepam. However, this may not retlect a higher toxicity of
diazepam, since diazepam was used more frequently together with alcohol than the other
tranquillizers investigated (Serfaty & Masterton, 1993). ln non-Iethal intoxications, the
symptoms consist mainly of an enhancement of the therapeutic effects, with severe
drowsiness, oversedation and ataxia, while in some cases, particularly in elderly persons,
a paradoxical excitation may be induced. Rare but severe acute adverse effects after
therapeutic intravenous administration include respiratory or cardiac arrest or both
(reviewed by Dollery et al., 1991),

(h) Chronic [Link]

ln addition to the effects associated with psychological and physical dependence and
rebound withdrawal phenomena, extremely rare but serious adverse reactions are
increases in the levels of serum aminotransferase and alkaline phosphatase, jaundice
(both hepatocellular and cholestatic), leukopenia, hypersensitivity reactions such as skin
rashes, single cases of exfoliative dermatitis and, in predisposed persons, circulatory and
lespiratory depression (reviewed by Dollery et al., 1991). Taking into account the
widespread use of diazepam over a long period, the virtual lack of adverse effects
reportcd in the literature suggests the absence of organ toxicity associated with chronic
administration.

4,2.2 Erperimeiital systl'ms

(a) !\clfl' fO-tci.r

A verage oral LD,1i values of 1901 and 1517 mg/kg bw in mice and rats. rcspectivcly,
have bccn reported. The average LD,1i values aftel intraperitoncal administration wcrc
774 mg/kg bw in mice and 661 mg/kg bw in rats (Owen et al., i 970).

(b) Suhchroiiic aiid chronic toxicit\'

Groups of 20 Charles River CD rats (10 male, 10 female) wcre admiiiistcrcd O. hO(),
1250 or 2500 mg/kg diet (ppm) diazepam orally in the diet for 20-22 wecks. No dcaths
 DIAZEP AM 63

occurred in either control or treated rats, and neither food consumption nor body weight
differed significantly between the four groups. No haematological or ophthalmological
effect of diazepam was found, but kidney and pancreas weights in males and liver
weights in males and females were greater at ail doses. ln addition to the mild renal
alterations observed in males of ail treated and control groups, some males of the high-
dose group had histopathological traces of brown, finely granular material within the
epithelial cells of the renal proximal convoluted tubules in the absence of gross toxicity.
Mild alteration of the thyroid architecture was observed in some animaIs in each treated
group, the acini appearing condensed and containing less colloid than usual (Owen et aL.,
1970).
Groups of four dogs were given 80, 127 or 200 mg/kg bw orally by capsule 30 min
before feeding daily for four weeks (Owen et al., 1970). One dog of the high-dose group
died and marked losses in weight were observed in the mid- and high-dose groups
associated with decreased food consumption due to severe sedation and somnolence; in
addition, increased emesis was induced in the three treated groups. The authors
suggested weakness and starvation as the cause of death. Haematological examination
revealed elevated haemoglobin, haematocrit, total red cell count and blood viscosity in
the mid- and high-dose groups; increased weights of the kidneys and adrenal gland were
also observed in these two groups. Most dogs treated with diazepam displayed histo-
pathological and biochemical signs of hepatobiliary dysfunction and some gonadal
changes, namely testicular atrophy.
(The Working Group noted that, since the study of Owen et al. (1970) focused on
evaluating oxazepam toxicity, with diazepam administered only for comparison
purposes, the description of the effects of diazepam is incomplcte on many points and the
number of animais treated with diazepam was very small.J

(c) Effects 011 ceU proliferatiol1 al1d diferel1tiatiol1 and 011 steroidogel1esis: the
role of peripheral bel1zodiazepil1e receptors
The anxiolytic and hypnotic effects of diazepam are mediated via GABAergic
receptors in the central nervous system. ln addition, diazepam can affect peripheral
organs, in particular the immune and endocrine system, directly through a second class of
binding sites. Although the peripheral benzodiazepine receptors are ubiquitous in the
organism, they are localized in very specifie regions of the different organs and their
density is strictly controlled by endocrine and neural mechanisms. ln generalized anxiety
disorders in humans and in chronically stressed or food-deprived experimental animaIs,
the density of the peripheral benzodiazepine receptors is decreased in most organs, while
diazepam administration has been reported to induce upregulation (Gavish et aL., 1992;
Ferrarese et aL., 1993). These peripheral benzodiazepine receptors are mitochondrial
proteins consisting of two subunits. The 'diazepam-binding inhibitor' peptide is a
putative endogenous ligand for peripheral benzodiazepine receptors. This polypeptide
has been purified from the brain and from a variety of other organs such as the liver,
kidney and adrenal glands, and probably functions as a precursor of smaller biologically
active neuropeptides that interact preferentìally with either central or peripheral benzo-
diazepine receptors (Ferrarese et al., 1993).
 64 IARC MONOGRAPHS VOLUME 66

Experimental evidence suggests that the effects of diazepam on celI proliferation and
differentiation observed il1 vitro are mediated via binding to benzodiazepine receptors.
Such effects include blockage of mitogenesis in Swiss 3T3 cells, induction of differen-
tiation in murine Friend erythroleukaemia celIs, acceleration of melanogenesis in mouse
melanoma cells, and inhibition of proliferation of human glioma cells, rat pituitary
tumour cells and cultured mouse spleen lymphocytes il1 vitro (Wang et al., 1984;
Pawlikowski et al., 1988a,b; Kunert-Radek et al., 1994). ln contrast, a single subcuta-
neous injection of diazepam induced increased thymic mitotic activity in rats in vivo
(Stepien et al., 1988). Inhibition of plasma membrane calcium influx through voltage-
dependent channels has been repeatedly discussed as a mechanism possibly involved in
the inhibitory effects on cell proliferation mediated via peripheral benzodiazepine
receptors (Pawlikowski et aL., 1988b; Ferrarese et aL., 1993).
ln adrenocortical and testicular Leydig celIs and cultured cell lines, the 'diazepam-
binding inhibitor' peptide, as well as other ligands including diazepam, stimulate
hormone-induced steroid biosynthesis, probably by binding to peripheral benzodiazepine
receptors and thus mediating the translocation of cholesterol from the outer to the inner
mitochondrial membranes and regulating cholesterol side-chain cleavage to pregne-
nolone (Krueger & Papadopoulos, 1990; Ferrarese et aL., 1993). The extent of stimu-
lation correlates with the binding affinity of the different ligands to these peripheral
receptors (Mukhin et aL., 1989; Ferrarese et aL., 1993). 'Diazepam-binding inhibitor' has
been also suggested to stimulate the release of corticotropin-releasing factor from
neurons and to stimulate the synthesis of neurosteroids in glial cells (Ferrarese et al.,
1993). Earlier studies il1 vivo demonstrated that diazepam increases corticosterone and
testosterone secretion in rats, as well as plasma levels of testosterone and 1 1 -hydrocorti-
coids in humans (Marc & Morselli, 1969; Argüelles & Rosner, 1975). ln contrast, fluni-
trazepam has been reported to antagonize the stimulatory effect of purified 'diazepam-
binding inhibitor' on steroidogenesis il1 vitro (Papadopoulos et aL., 1991).
The presence of peripheral benzodiazepine receptors in both animal and human
tumours has been explored extensively. Increased density was demonstrated in various
tumours, such as rat gliomas, human gliomas or astrocytomas, human colon adeno-
carcinomas and human ovarian and prostatic carcinomas; in contrast, peripheral benzo-
diazepine receptors were absent in renal carcinomas (Katz et aL., 1988; Ferrarese et aL.,
1989; Gorman et aL., 1989; Katz et aL., 1989, 1990). The localization of these binding
sites in the mitochondria raises the possibility that they might be involved in inter-
mediary metabolism and in respiratory control; hence quantitative and/or qualitative
changes in their function might bring about important alterations in cellular bio-
chemistry. However, the available data are fragmentary and do not allow assessment of
the role of peripheral benzodiazepine receptors in tumour formation.

(d) Effects 011 the immune system

The effects of diazepam on the immune functions have been studied both il1 vitro and
il1 vivo with conflicting results: both stimulatory and inhibitory effects have been
demonstrated. Diazepam injected one day after immunization stimulated the humoral
immune response of mi ce to sheep red blood celIs, probably as a result of T celI-
 DIAZEP AM 65

dependent antigen binding to peripheral benzodiazepine receptors on macrophages

(Ferrarese et aL., 1993). The presence of peripheral benzodiazepine receptors on the mito-
chondrial and plasma membranes of peripherallymphocytes was demonstrated immuno-
cytochemically. Compounds that act exclusively on central GABAergic receptors do not
exert immune functions (Ferrarese et aL., 1992). Further studies il1 vitro demonstrated
that binding of 'diazepam-binding inhibitor' and diazepam to the peripheral benzo-
diazepine receptors is involved in monocyte chemotaxis and enhances the production of
interleukin- 1 and tumour necrosis factor-a (Ruff et aL., 1985; Taupin et aL., 1991). ln
contrast, diazepam at micromolar concentrations, which can be achieved therapeutically
in blood, inhibited phagocytosis and kiling of Cal1dida albical1s cells by human poly-
morphonuclear cells and monocytes il1 vitro (Covelli et al., 1989). At similar concen-
trations, diazepam also suppressed the activity of natural killer cells isolated from human
peripheral blood against erythroleukaemia target cells, suggesting the possibility of
impairment of antiviral and antitumour defence in humans taking diazepam (Stepien
et aL., 1994). However, it is also possible that the demonstrated elevated levels of
'diazepam-binding inhibitor' in stress and anxiety are responsible for the reduced density
of peripheral benzodiazepine receptors on lymphocytes and mediate, together with the
increased levels of adrenal steroids, the immunosuppressive effects of stress (Ferrarese
et al., 1993). Hence, although the involvement of peripheral receptors in immuno-
modulation can be considered as proven, the role of diazepam versus 'diazepam-binding
inhibitor' remains to be elucidated (Zavala & Lenfant, 1987).

4.3 Reproductive and prenatal effects

4.3.1 Humal1s

The maternai metabolism of diazepam during pregnancy is discussed in Section 4.1.1.

Three kinds of developmental consequences of diazepam treatment during pregnancy
can be differentiated:
(i) Congenital abnormalities, i.e., the possible classical teratogenic effect of diaze-
pam used mainly in the first trimester of gestation.
(ii) Short-term functional alterations that are manifested postnatally and are related
mainly to diazepam treatment in the perinatal period.
(iii) Long-term postnatal developmental (including behavioural) effects which in
general are connected with diazepam intake in the second and third trimesters of
gestation, i.e., after the development of specific brain receptors for benzo-
diazepines.

(a) Possible teratogel1ic effect

The possible teratogenic effects are considered according to the circumstances of
exposure of the mother.

Ul1defil1ed exposure circumstances

The data of the Finnish Register of Congenital Malformations (1967-71) showed a
significant association between oral cIefts and maternai intake of antianxiety drugs
66 IARC MONOGRAPHS VOLUME 66

(Saxén, 1975). Extended analysis of the Finnish case-control material (Saxén & Saxén,
1975) indicated significantly greater use of benzodiazepines (diazepam, oxazepam,
nitrazepam or chlordiazepoxide) in the first trimester by the mothers of affected children
(14 versus 5) among 232 children with isolated cleft palate and 226 matched contrais
(p -= 0.05). Benzodiazepine use was greater, but not significantly so, in 232 children with
isolated cleft lip with or without cleft palate than in 230 matched controls (1 1 versus 4).
(The Working Group noted that benzodiazepines were not differentiated and that con-
founding factors such as maternai illness and use of other drugs were not controlled.)
ln the Metropolitan Atlanta Congenital Defects Program monitoring the incidence of
birth defects since 1967, Safra and Oakley (1975) found, by interviews of 49 women
who had infants with cleft lip with or without cleft palate, a history of diazepam
ingestion in the first trimester in seven, and in nine of 229 mothers of children with other
congenital abnormalities (relative risk, 4.1; 95% Ci, 1.5- 1 1.5). The other abnormalities
included Down' s syndrome, tracheo-oesophageal fistula and/or atresia, small-bowel,
rectal and anal atresia, omphalocele, diaphragmatic hernia and limb reductions. The
corresponding relative risk estimate for cleft palate alone was 0.9 based on one exposed
infant. Later, Safra and Oakley (1976) considered the results of their previous study to be
inconclusive because there was no association between secular trends in the prevalence
at birth of children with cleft lip with or without cleft palate and in drug sales.
ln a hospital-based study in Norway, Aarskog (1975) evaluated retrospectively 12
(1 exposed in the first trimester) cases with cleft palate and 99 (6 exposed in the first
trimester) cases with cleft lip with or without cleft palate born in 1967-71 and 362 (9
exposed in the first trimester) controls born in 1972-75. The number of subjects exposed
to diazepam in the first trimester was significantly higher in the combined group with
oral clefts than that of controls. (The Working Group noted that the validity of the study
is uncertain in view of the different study periods for cases and controls. Moreover, deft
lip with or without cleft palate is etiologically distinct from isolated cIeft palate.J
Czeizel (1976) conducted an ad-hoc population-based study using data from the
Hungarian Congenital Malformation Registry for the period 1970-75. Of 413 cases with
cleft lip with or without cleft palate, 121 cases with cleft palate alone and a control series
comprising 843 cases with neural-tube defects, 20 (4.8%), 2 (1.7%) and 37 (4.4%),
respectively, had mothers who reported having received diazepam treatment in the first
trimester of pregnancy. The differences were not statistIcally significant. .
Subsequently, Czeizel (1988) analysed the data-set of the Hungarian Case-Control
Surveilance System of Congenital Abnormalities for 1980-84. Approximately 15% of
pregnant Hungarian women used diazepam in the 1980s (Czeizel & Rácz, 1990).
Maternai diazepam use in the first, second and third months of pregnancy did not differ
significantly between 355 cases with isolated cleft lip with or without deft palate, 167
cases with isolated cleft palate and similar numbers of matched healthy controls. A pros-
pective follow-up study was based on women who visited genetic counselling clinics
between 1973 and 1980 following exposure to potentially hazardous environmental
factors during early pregnancy. Of 546 women, 33 had ingested benzodiazepines, mainly
diazepam; their 26 liveborn babies had no congenital abnormality.
 DIAZEPAM 67

Rosenberg et al. (1983) compared the use of diazepam during the first four lunar
months of pregnancy in 445 infants with cleft lip with or without cleft palate, in 166
infants with cleft palate alone and in 2498 controls with congenital abnormalities other
than oral clefts from the birth defect surveillance system in Boston, MA, and
Philadelphia, PA, United States, and Toronto, Canada, in 1976-82. The relative risk,
adjusted for several confounding factors, was 0.8 (95% CI, 0.4-1.7) for cleft lip with or
without cleft palate and 0.8 (0.2-2.5) for cleft palate alone.
ln a prospective study of 33 249 pregnant women, in the Birth Defects Study of the
National lnstitute of Child Health and Human Development and Kaiser-Permanente in
the United States, Shiono and Mills (1984) found no increase, based on 854 cases, in the
relative risk for oral clefts associated with exposure to diazepam during the first trimester
(relative risk, 1.2; 95% Cl, 0.17-9.0).
Exposure by attempted suicide
Two retrospective studies were carried out in Budapest and the surrounding area in
1960-79 (Czeizel et al., 1984, 1988) and 1980-84 (Czeizel & Lendvay, 1987; Lendvay
& Czeizel, 1992) and one prospective study between 1985 and 1986 (Czeizel &
Lendway, 1987) in self-poisoned pregnant women. (The Working Group calculated that,
in these two studies, 46 pregnancies ended in births to mothers who had used diazepam
for self-poisoning. Relatively low doses (25-45 mg) were used by 3 women, while
higher doses of 50-95 mg were used by 1 1, 100- 1 45 mg by 20 women, 150-195 mg by
3 women and more than 200 mg by 7 women. Of the children, only one was affected by
congenital abnormality (Fallot tetraIogy). Since the expected rate of congenital abnorma-
lities was about 6.5% (Czeizel et aL., 1993), it appears that single, extremely high doses
of diazepam did not cause an increase in the rate of detectable defects in the offspring. i
Gunnarskog and Källén (1993) observed that, of 70 infants born in Sweden to
mothers exposed to psychoactive drugs as a result of suicide attempts during the organ-
forming period, 20 of whom were exposed to benzodiazepines, no
ne had a congenital ab-
normality. (Specific drugs were not mentioned.)

Psychotherapeutic exposure
Several studies relate to the offspring of women who had psychiatric disorders for
which they received high doses of benzodiazepines throughout pregnancy,
Laegreid et al. (1987) reported seven cases exposed to benzodiazepines. Ali offspring
had intra- and extrauterine growth retardation, facial dysmorphism and central nervous
system dysfunctions. Of five cases wIth maternai exposure to diazepam, one had sub-
mucous cleft of the hard palate and secondary hydronephrosis, another was affected with
submucous cleft hard palate and a third one with microcephaly and left renal aplasia.
These cases resembled, but were not identical to, the fetal alcohol syndrome but abuse of
alcohol was denied by all the mothers. Ali the mothers had received 30-75-mg daily
doses of benzodiazepines throughout pregnancy, in sorne cases confirmed by the exaini-
nation of stored blood samples for diazepam. Laegreid et aL. (1989) reportcd two ncw
cases with similar findings.
68 (ARC MONOGRAPHS VOLUME 66

ln addition, Laegreid et al. (l 990) carried out a population-based study of surviving

live births born in 1985-86 in Gothenburg, Sweden. Twenty-five children were [Link]
with one or more of (a) embryopathy-fetopathy not otherwise specified, (b) oral cletts,
(c) defects of the central nervous system and (d) urinary tract malformations. lt was
possible to analyse maternai plasma in 18 of these cases (three were reported in previous
papers) and eight samples were found to be benzodiazepine-positive, including seven for
diazepam. A control series of 109 children was selected using paired sampling. Of 60
controls for whom blood analysis cou Id be carried out, two were positive, both for
diazepam. The difference in the proportion exposed to diazepam was highly significant.
Laegreid et al. (1992a) studied psychotropic drug use in the mothers of ail 73
perinatally dead infants in the city of Gothenburg, Sweden, in 1985-86 and in control
mothers of 73 surviving infants. Serum samples obtained in early pregnancy were
screened for benzodiazepines. Eighteen case-mothers had used psychotropic drugs
(benzodiazepines in nine) during pregnancy as documented from case-notes, compared
with seven control mothers (benzodiazepines in three). The association between benzo-
diazepine drug use and perinatal death was significant (p = 0.03), but confounding due to
psychiatric disorders and other drug use could not be excluded.
Bergman et al. (1990) evaluated the follow-up of the children of 4640 mothers in
1971-86 for their exposure to diazepam, oxazepam or nitrazepam during pregnancy.
Only six of the pregnant women appeared to be regular benzodiazepine users (two
diazepam, two oxazepam, one nitrazepam and one diazepam plus nitrazepam) and none
of their six children had abnormalities.
Later, Bergman et al. (1992) examined benzodiazepine use during pregnancy in
104 339 women whose deliyeries were recorded by the United States public health
insurance system, Medicaid, during 1980-83. Of 80 pregnant women who had received
l 0 or more benzodiazepine prescriptions (63 diazepam, 36 chlordiazepoxide, 8 lora-
zepam, 13 tlurazepam), three experienced fetal death and two infants were found to have
lethal congenital abnormalities. Records of 64 surviving children could be linked to these
80 pregnancies (1 1 survivors cou Id not be located), and six children had congenital
abnormalities of various types. These defects differed from the pattern described by
Laegreid et al. (1987, 1989, 1990); in particular, there were no oral cletts.

(b) Short -te rm functiol1al alte rations

There are case reports of floppy infant syndrome (namely hypotonia, hyporetlexia,
apnoeic spells, reluctance to feed, a risk for inhalation of feeds, impaired metabolic
responses to cold, hypothermia, low Apgar rating at birth) associated with diazepam
treatment of pregnant women at doses of 30 mg or more within the 15 hours before
delivery (Owen et al., 1972; Cree et al., 1973).
Acute withdrawal effects (namely depressed respiration, hypothermia and feeding
difficulties) have been documented in neonates exposed to diazepam ¡Il utero for long
periods (Cree et al., 1973). The time of onset of the symptoms, their severity and
duration were related to the dosage and fetal kinetics (including elimination) of the drug
(Mazzi, 1977; Mac New & Finnigan, 1980).
 DIAZEPAM 69

(c) Humal1 IOl1g-term postnatal studies

The behavioural development of 101 children (and their 1 17 siblings) of pregnant
women who attempted suicide has been studied (Lendvay & Czeizel, 1992). Forty-two of
the mothers had lIsed diazepam during pregnancy. Some of the children had behavioural
alterations which coiiid be explained mainly by their familial and social problems.
Laegreid et aL. (1992b,c) examined the neiirodevelopment of 17 chi Idren born to 16
mothers who used benzodiazepines throiighout pregnancy: 15 used diazepam (5-30 mg
daily) or oxazepam (15-60 mg daily) alone or in combination and one mother used lora-
zepam. The results were compared with those for 29 children born to mothers without
any known use of psychotropic drugs. A neurological investigation was performed on the
second day of life. Significant differences in the frequency of pre- and perinatal compli-
cations and in neurobehaviour were found between the two groups. The benzodiazepine-
exposed children recovered from their lower mean birth weight at an early stage, whereas
their slightly decreased head circumference at birth remained lower. Gross motor deve-
lopment was retarded at six and 10 months, but was nearly normal at 18 months.
Impaired fine motor functions were found on all follow-up occasions (at six, 10 and 18
months of age).
The cases studied by Laegreid et al. (1990, 1992b,c) were followed up prospectively
in late infancy and found to have a general delay in mental development up to 18 months
of age associated with prenatal exposure to benzodiazepines (Viggedal et aL., 1993). (The
Working Group noted that ail the mothers in the benzodiazepine group had psychiatric
disorders, and these have an important negative effect on children's development (Cox,
1988). ln addition, neuropsychological symptoms are frequent among children of abusers
of psychoactive substances (Deren, 1986; Van Baar et al., 1989).)

4.3.2 Experimel1tal systems

Few experimental data are available concerning the effect of diazepam on repro-
duction. The incidence of abnormal sperm heads was significantly higher in mice after a
daily oral dose of 0.5 mg diazepam (Kar & Das, 1983; Šrám & Kocišová, 1984).
Guerriero and Fox (1977) found a significant decrease in mating performance, with
depressed birth weights, among Swiss-Webster mice given a diet containing 500 mg/kg
diazepam.
ln A/J mice given a single intramuscular dose of 100 mg/kg diazepam on day 14 of
pregnancy, the frequency in the offspring of both cleft lip with or without cleft palate and
cleft palate only was 3.4% (Walker & Patterson, 1974). The authors observed that this
was lower than the frequency of spontaneous occurrence of these defects reported in
other series.
Miler and Becker (1975) treated Swiss-Webster mice with 50, 100, 140 or 500 mg/kg
bw diazepam by gastric instillation once daily for three days on gestation days 8- 10 or
days 11-13 or for one day only between days 8 and 15 or with 280 or 400 mg/kg bw for
one day only between days 1 1 and i 4. The highest dose was associated with a maternai
mortality rate of 50%. When 140 mg/kg bw diazepam was administered on day 13, there
was 2 i % fetal resorption. The incidence of cleft palate was significantly increased in the
 70 lARC MONOGRAPHS VOLUME 66

oftspring of mice treated with 140 mg/kg bw diazepam on days 1 1, 12 and 13, and with
single-day administrations of 400 mg/kg bw on days 1 1 - 1 4 and 500 mg/kg bw on days 9
and 11-15.
Tocco et al. (1987) reported an increase in the frequency of cleft palate in Swiss-
Webster and AJ mice following two-day dosing with 400 mg/kg bw diazepam by gastric
instillation on days 13.5 and 14.5. Maternai mortality was high (50% or more) but no
increase in resorption was observed.
ln rats, no abnormality was caused by oral administration of 20 or 80 mg/kg bw
diazepam per day on days 6-15 of gestation (Beall, 1972). Saito et al. (1984) did not find
a significant increase in any abnormalities after oral administration of 100 mg/kg bw
diazepam on gestation days 8-14 in Sprague-Dawley rats.
ln hamsters, exencephaly, cleft palate and limb defects were detected after a single
oral dose of 30, 50, 70 or 100 mg on days 8 and 10 or single intravenous injections of
10 mg diazepam on day 1 1. There was no dose-related effect (Shah et al., 1979). A
single intraperitoneal injection of 120-980 mg/kg bw diazepam on day 8 of gestation
induced a dose-related increase in the frequency of fetal malformations in hamsters,
mainly exencephaly or cranioschisis, at doses of 280 mg/kg and above (Gill et al., 1981).
No structural abnormality was observed in the offspring of two rhesus monkeys
treated orally with diazepam (0.5-3.2 mg/kg bw) twice daily during the second and third
trimesters, nor in those of three monkeys treated during the third trimester only (Jerome
et al., 1981). (The Working Group noted that there was no treatment during the first
tri mes ter. )
Specific receptors for benzodiazepines develop at the beginning of the fetal period in
the central and peripheral nervous systems of rats (Braestrup & Nielsen, 1978).
Behavioural studies have demonstrated pronounced effects in rodents following exposure
to diazepam during late gestation. ln rat models, prenatal exposure to diazepam and other
benzodiazepines resulted in behavioural deficits in pups (Kellogg et al., 1980), such as
learning and memory disabilities (Gai & Grimm, 1982), the absence of acoustic startle
reflexes and the impairment of conditioned avoidance response (Kellogg, 1992) which is
dependent on the time of treatment (Frieder et al., 1984). However, diazepam prevented
the adverse effects of maternai restraint stress in postnatal development and learning in
rats (Barlow et al., 1979).
A chick embryotoxicity screening test did not show any teratogenic effect of
diazepam (Peterka et al., 1992).

4.4 Genetic and related effects (see also Table 6 for references and Appendices 1
and 2)

4.4.1 Humal1s

The first published report of a possible association between exposure to diazepam and
chromosomal aberrations in the lymphocytes of patients using the drug as a tranquillizer
(four patients) or for its muscle-relaxing properties (19 patients) was that of Stenchever
et al. (1970). These patients were compared with eight controls. Although there was no
 Table 6 (contd) --
t'
Test system Result" Doseb Reference
(LED/HID)
Without With
exogenous exogenous
metabolic metabolic
system system
G9H, Gene mutation, Chinese hamster lung V79 cens, hprt locus in vitro - - 250 Röhrborn et al. (1984)
GIA, Gene mutation, rat primary hepatocytes, hprt locus in vitro - - (abstract) -;p
50 Swierenga et al. (1983) :;
MIA, Micronucleus test, Chinese hamster (CI-1) cells in vitro c
(abstract) n
+ NT 20 Antoccia et al. (1991) ~
MIA, Micronucleus test, Chinese hamster lung V79 cel1s in vitro +c
NT NG Bonatti et al. (1992) Z
a
MIA, Micronucleus test, Chinese hamster pulmonary (Luc 2) cel1s in vitro
MIA, Micronucleus test, Chinese hamster 1ung V79 cells in vitro
+
+c
NT 10 Lynch & Parry (1993) a0
NT 100 Seelbach et al. (1993) :;
CIC, Chromosomal aberrations, Chinese hamster 1ung CHL cells in vitro - ? 1ÒOO Matsuoka et al. (1979) ;p
CIC, Chromos omal aberrations, Chinese hamster lung CHL cel1s in vitro - ..
CIC, Chromosomal aberrations, Chinese hamster (CHE-3N) cens in vitro
NT 125 Ishidate et al. (1988) ::
(/
? NT 100 Lafi & Parry (1988)
AIA, Polyploidy, Chinese hamster (Don) cells in vitro ~
+ NT 100 Satya-Prakash et al. aL
(1984 ) C
AIA, Aneup10idy, Chine se hamster (Don) cel1s in vitro NT
AIA, Aneuploidy, Chinese hamster (CHE-3N) cens in vitro (hypodip1oidy)
+ 100 Hsu et al. (1983) ~
(+) NT 100 Lafi & Parry (1988) tT
AIA, Aneuploidy, primary Chinese hamster embryonic cel1s in vitro +d 0"
NT 10 Natarajan et al. (1993) 0"
AIA, Aneuploidy, Chinese hamster pu1monary (Luc 2p4) cel1s in vitro + NT 10 Warr et al. (1993)
(hypodiploidy and polyploidy)
*, c- Mitoses, Chinese hamster (CL-1) cel1s in vitro
+ NT 60 Antoccia et al. (1991)
TCL, Cell transformation, BHK 21 -C 13 cens in vitro - + 130 Röhrborn et al. (1984)
(abstract)
ter chromatid exchange, human fibroblast cell line in vitro
SHF, Sis - NT 28.5 Sasaki et al. (i 980)
MIH, Micronucleus test, human lymphocytes in vitro - NT 75 Migliore & Nieri
MIH, Micronucleus test, human fibroblasts in vitro c
(1991)
+ NT 25 Bonatti et al, (i 992)
MIH, Micronucleus test, human lymphocytes in vitro +' NT 30 Ferguson et al. (1993)
 Table 6 (contd)

Test system
Resulta Dose b Reference
(LED/HID)
Without With
exogenous exogenous
metabolic metabolic
system system
CHF, Chromosomal aberrations, human primary fetal fibroblasts in vitro - NT 50 Staiger (1969)
CHF, Chromosomal aberrations, human fibroblast cell line in vitro - NT 25 Staiger (1969)
CHF, Chromosomal aberrations, human fibroblast cell line in vitro - NT 28.5 Sasaki et al. (1980)
CHL, Chromosomal aberrations, human lymphocytes in vitro - NT 50 Staiger (1970)
CHL, Chromosomal aberrations, human lymphocytes in vitro - - NG Röhrborn et al. (1984)
AIH, Aneuploidy, human primary fibroblasts in vitro - NT 50
(abstract)
Staiger (1969)
..0
AIH, Aneuploidy, human fibroblast-cell line in vitro ;p
- NT 25 Staiger (1969) N
AIH, Aneuploidy, human lymphocytes in vitro (hypodiploidy) +d tT
NT 25 Sbrana et al. (1993) '"
*, c-Mitoses, human lymphocytes in vitro + ;p
NT 50 Sbrana et al. (1993)
DV A, DNA strand breaks, rat Iiver in vivo - ~
285 po x 1 Carlo et al. (1989)
DV A, DNA strand breaks, rat liver in vivo - 57poxl5 Carlo et al. (1989)
MVM, Micronucleus test,mouse bone marrow in vivo
(+) 22 po x 1 Kar & Das (1979)
MVM, Micronucleus test, mouse bone marrow in vivo
+ 20 po x 2 Das & Kar (1986)
MVM, Micronucleus test, mou se bone marrow in vivo - 150ipx 1 Adler et al. (1991)
MVM, Micronucleus test, mouse bone marrow in vivo - 30 ip x 1 Leopardi et al. (1993)
MVM, Micronucleus test, mouse bone marrow in vivo
(+) 10 x If Marrazzini et al. (1994)
CBA, Chromosomal aberrations, Chinese hamster bone marrow in vivo - 300pox 10 Schmid & Staiger
(1969)
CBA, Chromosomal aberrations, rat bone marrow in vivo - 500 po x 10 Neda et al. (1977)
CBA, Chromosomal aberrations, mouse bone marrow in vivo
(+) 75 ip x 7 Petersen et al, (1978)
CBA, Chromos omal aberrations, mouse bone marrow in vivo - (abstract)
0.85 ip x 1 Degraeve et al. (1985)
(abstract) --
lN
 Table 6 (contd) --
.t
Test system
Result" Doseh Reference
(LED/HID)
Without With
exogenous exogenous
metabolic metabolic
system system
CBA, Chromosomal aberrations, mouse bone marrow in vivo
0.85 ip x 22 Degraeve et al. (1985)
CBA, Chromosomal aberrations, mouse bone marrow in vivo (abstract)
-
;¡
(+) 1000 po x 28 Kocišová & Šrám
CBA, Chromosomal aberrations, rat bone marrow in vivo (1985) (abstract)
niO
$:
500 po x 10 Ishimura et al. (1975) 0
CBA, Chromosome aberrations, mou
se bone marrow in vivo
CBA, Chromosomal aberrations, mouse bone marrow in vivo 100 ip x 1
(abstract)
Xu & Adler (1990) 00Z
CGG, Chromosomal aberrations, mouse spermatogonia treated in vivo, 10 x if Marrazzini et al. (1994) ;:
;¡
spermatagonia observed 0.85 ip x 22 Degraeve et al. (1985) "'
::
CCC, Chromosomal aberrations, mouse spermatocytes treated in vivo, (abstract) V1

spermatocytes observed 0.85 ip x 22 Degraeve et al. (1985) ~

DLM, Dominant lethal test, mouse in vivo (+) (abstract)
0r
DLM, Dominant lethal test, mouse in vivo 22poxl5 Kar & Das (1979) c:
$:
0.85 ip x 40 Degraeve et al. (1985) tT
(abstract) 0\
DLM, Dominant lethal test, mou
se in vivo 0\
1000 po x 28 Šrám & Kocišová
*, c-Mitoses, mouse bone marrow in vivo (1985)
A VA, Polyploidy, mouse bone marrow in vivo 150ipx 1 Miller & Adler (1989)
A V A, Aneuploidy, mouse secondary spermatocytes in vivo 100 ip x 1 Xu & Adler (1990)
A VA, Aneuploidy, mouse bone marrow in vivo
(+) 150ipx1 Miler & Adler (1992)

A V A, Aneuploidy, mouse secondary spermatocytes in vivo 30 ip x 1 Leopardi et al. (1993)

A V A, Aneuploidy, mouse oocytes in vivo 30 ip x 1 Leopardi et al. (1993)
150ipx 1 Mailhes & Marchetti
(1994 )
 Table 6 (contd)

Test system Resulta Dosel, Reference

(LED/HID)
Without With
exogenous exogenous
metabolic metabolic
system system

A VA, Aneuploidy, male mou se germ cells in vivo + 150ipx 1 Gassner & Adler
(1995)
A VA, Aneuploidy, mouse bone marrow in vivo (+) 10 x if Marrazzini et al. (1994)
SLH, Sister chromatid exchange, human lymphocytes in vivo 0.2 po x 1 Husum et al. (1985)
SLH, Sister chromatid exchange, human lymphocytes in vivo + NG Huong et al. (1988)
omal aberrations, human lymphocytes in vivo
CLH, Chromos

CLH, Chromosomal aberrations, human lymphocytes in vivo

0.5 po x l'
0.3 po xil,
Cohen et al. (1969)
Stenchever et al. (1970)
-
CI
~
CLH, Chromosomal aberrations, human lymphocytes in vivo N
0.30 iv x 1 White et al. (1974) tT
CLH, Chromosomal aberrations, human lymphocytes in vivo + 'V

CLH, Chromosomal aberrations, human lymphocytes in vivo

NG Huong et al. (1988) ~
2.4 po xii van Bao et al. (1992) 3:
MVH, Micronucleus test, human lymphocytes in vivo 2.4 po xii van Bao et al. (1992)
A VH, Aneuploidy, human cells in vivo (hypodiploidy) + 2.4 po x r van Bao et al. (1992)
*, Inhibition of tubulin assembly in vitro 712 Brunner et al. (1991)
*, Inhibition of tubulin assembly in vitro + 285 Wallin & Hartley-Asp
(1993)
ICR, Inhibition of intercellular communication, Chinese hamster lung + 1.25 Trosko et al. (1982)
(V79) cells in vitro
ICR, Inhibition of intercellular communication, rat liver epithelial cells 10 Wälder &
Lützelschwab (1984)
ICR, Inhibition of intercellular communication, mou se hepatocytes in vitro + 25 Diwan et al. (1989)
ICR, Inhibition of intercellular communication, Chinese hamster lung ? 20 Toraason et al. (1992)
(V79) cells in vitro
ICH, Inhibition of intercellular communication, human hepatoma cellular 10 Rolin-LImbosch et al.
carcinoma cell line (SK-HEP-1)
( 1987)
--
lI
Table 6 (contd) ..
0\

Test system Result" Doseh Reference

(LED/HID)
Without With
exogenous exogenous
metabolic metabolic
system system

SPM, Sperm morphology, mouse in vivo + 20 po x 15 Kar & Dass (1983)

SPM, Sperm morphology, mouse in vivo + 200 po x 28 Kocišová & Šrám
(1985) (abstract) ;p
BF A, Urine of mouse, Ames test, Salmonella typhimurium TA 100 + + 200 po x 1 Batzinger et al. (1978) n;;
BF A, Urine of mouse, Ames test, Salmonella typhimurium T A98 + + 200 po x 1 Batzinger et al. (1978) 3;
o
BFA, Urine ofmouse, Ames test, Salmonella typhimurium TAIOO 200 Matula & Downie z
o
(1983) (abstract) o;;
BF A, Urine of mouse, Ames test, Salmonella typhimurium T A98 200 Matula & Downie
(1983) (abstract) v;p
*, Metabolites from canine gastric mucosa in vitro, Ames test, Salmonella NT + NG Riee et al. (1981) :c
C/
typhimurium T A98 ..
*, Metabolites from human gastric mucosa in vitro, Ames test, Salmonella NT + NG Rice et al. (1981) or
typhimurium T A98 C
3;
*Not shown on profile t'
0\
" +, positive; (+), weak positive; -, negative; NT, not tested; ?, inconclusive 0\
h LED, lowest effective dose; HID, highest ineffective dose; in-vitro tests, ¡.g/mL; in-vivo tests, mg/kg bw/day; NG, dose not given
C Kinetochore-positive
d Negative for polyploidy
e Size ratio of micronuclei to main nucleus indicates aneuploidy induction
f Both intraperitoneal and oral routes were used when no useful indication concerning the most effective route was available from the literature.
g Average daily dose from six patients treated for 36-72 months
ii Average daily dose from 23 patients teated for 0.5-36 months. One patient had a significant increase in cells with breaks following treatment

(30 mg/day x 18 months)

; Average estimated dose from 25 self-poisoned individuals. Increase in hypodiploidy but not polyploidy 6-12 h after poisoning; no increase 3 and 30
days after poisoning
 orAZEPAM 77

overall difference between the groups, three patients had elevated levels of chromosomal
aberrations and one of the se in particular showed chromosomal breakage in 15.3% of the
cells examined, but, on re-examination six months after discontinuing the drug, only
control levels of damage were found.
With regard to sister chromatid exchange, Torigoe (1979) studied 20 epileptic
children (lO boys, 10 girls; age, 4-23 years) who had taken two to six anticonvulsant
drugs for one to 18 years and 20 controls (10 boys, 10 girls; age, 6- 15 years) who did not
receive any drugs for at least six months; they found no significant difference between
control subjects and epileptic patients. ln the study of Husum et al. (1985), the peripheral
lymphocytes of 34 persons (18 men and 16 women undergoing minor surgery) were
examined before and 2-5 h after oral administration of a single 0.2 mg/kg bw dose of
diazepam; possible effects of smoking were taken into account and no indication of the
induction of sister chromatid ex change was found. ln contrast with these observations, a
cytogenetic investigation (Huong et al., 1988) of 18 self-poisoned pregnant and 16 self-
poisoned non-pregnant women and 31 controls (16 pregnant and 15 non-pregnant) found
statistically significant differences in frequencies of sister chromatid exchange per cell
between the third and seventh day after poisoning (pregnant: control, 8.55 :! 1.08;
poisoned, i 0.30 :! 1.63, p ~ 0.01; non-pregnant: control, 9.13 :! 1.32; poisoned, 11.26 :t
2.31, p ~ 0.05). ln the same population, a very highly significant difference in the
prevalence of chromosomal aberrations between self-poisoned women and controls
(pregnant: control, 4.03%; poisoned, 9.56%, p ~ 0.001; non-pregnant: control, 5.99%;
poisoned, 14.38%, p ~ 0.001) was also found; moreover, the frequency of chromatid
aberrations was significantly lower in pregnant relative to non-pregnant women
(p ~ 0.05). ln contrast, the studies of White et al. (1974) on 20 patients given a single 20-
mg intravenous injection of diazepam and of van Bao et al. (1992) on 25 patients 6- 12 h,
3 days or 30 days after self-poisoning with diazepam failed to confirm the induction of
chromosomal aberrations il1 vivo in human lymphocytes. The latter, however, reported
that hypodiploidy (but neither hyperdiploidy nor polyploidy) was observed in the indi-
viduals studied 6-12 h after self-poisoning; the effects were not observed at later
sampling times. (The Working Group noted that cytogenetic changes in lymphocytes
disappeared six days after poisoning.)

4.4.2 Experimel1tal systems

No studies have demonstrated bacterial DNA damage or mutagenicity due to

diazepam itself.
No genetic effects were observed in single studies for mitotic recombination and gene
conversion, in two studies for aneuploidy (no dose-dependent increase) with Saccharo-
myces cerevisiae or in a single study for chromosome malsegregation with Aspergillus
11 idula 11 s .

Diazepam did not induce unscheduled DNA synthesis in primary cultures of rat
hepatocytes in two studies or mutation at the hprt locus of Chinese hamster V79 cells or
rat primary hepatocytes il1 vitro. Micronuclei were induced in vitro in Chinese hamster
cell lines in four studies but the increase in chromosomal aberrations was judged to be
 78 rARC MONOGRAPHS VOLUME 66

inconclusive in two studies (one in the presence of an exogenous metabolic activation

system) and negative in another study.
AlI studies with Chinese hamster cells aimed at the detection of aneuploidy il1 vitro
were positive: moreover, in aIl of the micronucleus tests in which they were examined,
the micronucIei contained kinetochore(s). Two studies out of four which scored for
chromosome numbers detected a significant increase in hypodiploidy but not in hyper-
diploidy. One study which tested the induction of polyploidy in Chinese hamster cells
was positive. Meiotic delay has been observed in mou
se oocytes (Stenchever & Smith,
1981) and mitotic arrest has been demonstrated in Chinese hamster Don cells (Hsu et aL.,
1983) and human fibroblasts (Andersson et al., 1981).
ln a study at very low doses with human fibroblasts il1 vitro, diazepam did not induce
sister chromatid exchange, whereas a significant increase in micronuclei was observed at
higher dose levels. Two studies in human lymphocytes gave contradictory results. AlI the
studies in either human fibroblasts or lymphocytes aimed at the detection of chromo-
somal aberrations il1 vitro were negative. c-Mitosis and hypodiploidy (but not poly-
ploidy) were observed in single studies with human lymphocytes treated il1 vitro with
diazepam. One study reported the induction of large-sized micronucIei in human lym-
phocytes il1 vitro.
Diazepam caused inhibition of gap-junctional intercellular communication in two out
of five studies.

Urine of mice exposed il1 vivo to diazepam induced gene mutations in Salmol1ella
typhimurium TA 1 00 or T A98 in one study but not in another; metabolites from dog and
human gastric mucosa incubated il1 vitro with diazepam induced a significant increase in
gene mutations in S. typhimurium T A98.
Two mammalian studies indicated a lack of micronucleus induction in the bone
marrow of mice il1 vivo; however, three other similar studies were positive at lower dose
levels. (The Working Group noted that the authors reported induction of larger micro-
nuclei taking into account neither interanimal variation nor objective criteria for micro-
nucleus scoring.)
No increase in DNA single-strand breaks and/or alkali-Iabile sites was observed in the
liver of rats given a single dose (1 mmol/kg) or 15 successive daily doses (0.2 mmol/kg)
orally. However, predominantly negative results (seven negative studies and two
inconclusive) for the induction of chromosomal aberrations have been obtained in studies
of mouse or rat bone marrow. Aneuploidy, c-mitoses or polyploidy were not observed in
mouse bone marrow (the Working Group noted the inadequate presentation of the
results). ln mou se secondary spermatocytes, meiotic delay and aneuploidy were found at
higher concentrations (150 mg/mL; Miler & Adler, 1992) but not at a lower concen-
tration (30 mg/mL; Leopardi et al., 1993); the positive effect was due to an increase in
hyperploidy. At the same higher concentration, abnormalities of chromosome/spindle
segregation were also reported in male mouse germ ceIls.
As reported in abstracts, treatment of mièe with diazepam increases the proportion of
sperm with ab normal head morphology.
 DIAZEPAM 79

5. Summary of Data Reported and Evaluation

5.1 Exposure data

Diazepam is the most widely used of the benzodiazepine pharmaceuticals. Produced

since the 1960s, it is prescribed for the treatment of anxiety and as a sedative, muscle
relaxant, and anticonvulsant.

5.2 Human carcinogenicity data

Studies investigating unspecified hypnotics or tranquillizers as weil as diazepam
specifically have been incIuded in this monograph because of the dominance of this
benzodiazepine among those prescribed. The risk for a variety of cancers, especially of
the breast, associated with diazepam use has been investigated in two cohort studies and
in six distinct and three related case-control studies.
ln none of the two cohort or five case-control studies on benzodiazepine or diazepam
use in relation to breast cancer was a positive association found. One case-control study
of ovarian cancer reported an increased risk for diazepam use, that was not confirmed by
another study. This latter study reported no association between diazepam use and the
risk of several other types of cancer.

5.3 Animal carcinogenicity data

Diazepam was tested for carcinogenicity in one experiment in mice, in one experi-
ment in rats and in one experiment in hamsters by oral administration in the diet and also
in one limited study in gerbils. An increase in the incidence of hepatocellular tumours
occurred in male mice. No significant increase in the incidence of tumours was observed
in rats, hamsters or gerbils.
ln one study in mice, oral administration of diazepam enhanced the occurrence of
hepatocellular tumours induced by N-nitrosodiethylamine. ln two studies in rats initiated
with 2-acetylaminofluorene or 3'-methyl-4-(dimethylamino)azobenzene, there was no
promoting effect of diazepam. ln gerbils initiated with N-nitrosodiethylamine, simulta-
neous administration of diazepam decreased the incidence of cholangiocarcinomas.

5.4 Other relevant data

Diazepam is absorbed rapidly and extensively in humans. A 30-fold range of peak

plasma concentrations is obtained when the sa
me dose is given to different subjects.
Diazepam is metabolized initially to N-desmethyldiazepam (nordiazeparn) and tema-
zepam, both of which may be converted to oxazepam. Diazepam clearance shows
marked inter-subject variability. The me
an elimination half-life is about 32 h.
There is wide inter-species variability in diazepam metabolism. While formation of
N-desmethyldiazepam and temazepam occurs to some extent in all species studied,
hydroxylation in the 5-phenyl ring is the major pathway in rats.
 80 IARC MONOGRAPHS VOLUME 66

Diazepam has low acute and chronic toxicity for humans at therapeutic concen-
trations. The main adverse effects of chronic administration are psychological and
physical dependence and withdrawal phenomena. Specifie organ toxicity of diazepam to
humans has not been observed.
The acute toxicity of diazepam to experimental animais can be considered as low. ln
subchronic toxicity assays in dogs, high doses of diazepam induced mild toxic effects in
ver and gonads, while in rats, slight chemical-related histopathological
the blood, Ii

changes were observed in the kidneys and thyroid gland.

The effects of diazepam on the immune system have been investigated mainly in in-
vitro experiments with conflicting results: both stimulatory and inhibitory effects have
been demonstrated. There are no data on immunosuppressing or immunomodulating
effects in humans.
ln several cultured cell systems, diazepam inhibits cell proliferation.
No consistent association between orofacial clefts and diazepam has been identified
in humans. No increase in the prevalence at birth of congenital abnormalities has been
found associated with attempted maternaI suicide using high doses of diazepam, in some
instances during the first trimester. While excesses of anomalies associated with regular
psychotherapeutic benzodiazepine use have been observed, the types of developmental
defects involved have not been consistent between studies.
High doses of diazepam induce cleft palate in mice, but not in rats. ln hamsters, exen-
cephaly and limb defects are seen, as weil as deft palate.
ln general, diazepam did not induce gene or chromosome mutations in bacteria, yeast
or cultured mammalian cells. ln cultured mammalian cells, it induced micronuc1ei and
aneuploidy, and inhibited gap-junctional intercellular communication. There are contra-
dictory results on the induction of gene mutation in bacteria by the urinary metabolites of
treated mice.
ln general, diazepam did not induce micronuclei, chromosomal aberrations, aneu-
ploidy, c-mitoses or polyploidy in bone marrow of mice il1 vivo. ln rats il1 vivo, neither
chromosomal aberrations in bone marrow, nor DNA strand breaks or alkali-labile sites in
liver were found. ln mouse spermatocytes, but not in oocytes, diazepam induced aneu-
ploidy.

Mechal1istic cOl1sideratiol1s

Diazepam does not cause gene mutations or chromosomal aberrations. One of its
metabolites, oxazepam, increased the incidence of liver tumours (benign and malignant)
(see Monograph on oxazepam, pp. 119- 123). However, it is not dear that levels of oxa-
zepam sufficient to induce hepatic effects are achieved in mice treated with diazepam.
 D IAZEP AM 81

5.5 Evaluation i

There is evidel1ce suggestil1g lack of carcil1ogel1Icity of diazepam to the breast and

il1adequate evidel1ce for carcinogenicity at other sites in humans.
There is il1adequate evidel1ce in experimental animais for the carcinogenicity of
diazepam.

Ove rail evaluation

Diazepam is l10t classifable as to its ca
rcil1ogel1 icit y to humal1s (Croup 3).

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