PHOSPHOROUS CONTAINING ALKYLATIKTG AGENTS;
CAKCINOGENICITY AND STRUCTURE-ACTIVITY
RELATIONSHIPS. OTHER BIOLOGICAL PROPERTIES.
ACTIVATING METABOLISM. ENVIRONMENTAL SIGNIFICANCE,
Yin-tax Woo, Ph. D.,
Joseph C. Arcos, 0,. Sc., -.arid
Mary F. Argus, Ph..;D«
Prepared for the Chemical Hazard
Identification Branch "Current
Awareness" Program
-------
Ta&le of Contents;
/
1.1.4 Phosphorus-Containing Alkylating Agents
5."2.1.4.1 Organophosphorus Compounds (Excluding Cyclophosphamide)
5.2.1.4.1.1 Physico-Chemical and Biological Properties'
5.2.1.4.1.2 Carcinogenicity and Structure-Activity Relationships
5.2.1.4.1.3 Metabolism and Possible Mechanism Of Action
5.2.1.4.1.4 Environmental Significance
5.2.1.4.2 Cyclophosphamide and Related Compounds
5.2.1.4.2.1 Physico-Chemical and Biological Properties
5.2.1.4.2.2 Carcinogenicity
5.2.1.4.2.3 Metabolism and Possible Mechanism1 of Action
''5*2.1.4.2.4 Environmental Significance /
^References
-------
482
5.2.1.4 Phosphor us-Containing A Iky la ting Agents
Synthetic organophosphorus compounds are of great economic impor-
tance. They have been extensively used for various practical purposes -and C.
are invaluable, if not indispensable, as pesticides, flame retardants and can-
cer chemotherapeutic agents. Thus, human exposure to these compounds may
occur in a vast section of the population. Because of their wide use as pesti-
cides, the toxicology of organophosphorus compounds has been extensively
studied; there is, however, a relative scarcity of information on their carcin-
ogenicity. Although organophosphorus compounds are generally regarded as
inactive or as weak carcinogens, recent investigations revealed that some of
them are potent agents. This section discusses the literature of organophos-
phorus compounds with special emphasis on their carcinogenicity. Because
of the voluminous literature on the well-known chemotherapeutic agent, cyclo-
phosphamide, a cyclic organophosphorus compound with a mustard functional
group, this agent is reviewed separately in Section 5.2.1. 4. 2. Phosphora-
mides containing ethyleneimino groups have been previously discussed in Sec-
tion 5. 2. 1. 1. 4.
5.2.1.4.1. Organophosphorus Compounds (Excluding Cyclophospha-
mide). Most of the compounds discussed in this subsection are derivatives of
phosphoric acid and are often collectively termed/organophosphates. The no-
A
menclature of organophosphorus compounds is confusing; there is no simple,
logical or generally accepted system. At least five or six different nomencla-
ture systems have" been used (see 1, 2). For the purpose of discussion in this
subsection, the most common or generic names and abbreviations are used.
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483
The structures of these compounds are depicted in Fig. 20 and their full chem-
ical names following the Chemical Abstract or IUPAC system are listed in Ta.-
ble CVIII. Essentially the compounds discussed include (a) phosphoric acid
triesters, (RO) P=O; (b) thiophosphoric acid triesters containing one thio group
(phosphorothioate) or two thio groups (phosphorodithioate) [the term, phosr
phorothioate is a general term for phosphorothionate, (RO) P=S, and phosphoro-
thiolate, (RO) P=O(SR)':]) (c) dialkyl phosphorofluoridate, (RO) P=O(F), (d)
L* t*
phosphoric triamide, (R N) P=O, (e) dialkyl alkylphosphonate (RO)_P=O(R),
Ls J , £,
® B
and (f) tetraalkyl phosphonium chloride, R P Cl . Most of these compounds
have been used as pesticides, whereas some have been employed as flame re-
tardants (Tris-BP, THPC), nerve gas (DFP), chemos terilant (HMPA) and sol-
vents or interm.ediates in organic synthesis of industrial chemicals (HMPA,
TMP). . !
Biplogical_
5.2.1.4.1.1 Physico-Chemical. and '\^'. Properties. The physical and chem-
ical properties of organophosphorus compounds have been extensively discussed
in several excellent reviews (3-5) and monographs (1, 2, 6). Dichlorvos, in
particular, has received great attention (3, 4) because of its extensive agri-
cultural use. The physical constants of some of the widely, used organophos-
CVt
phorus compounds are summarized in Table CVI. The chemical reactivity of
organophosphorus compounds is dependent on the chemical structure of the
compound and reaction conditions such as the pH, the temperature, the solvent
involved, and the presence of catalytic additives. Trimethyl phosphate is quite
stable in neutral aqueous solution. Substitution of one of the methoxy groups
-------
CH'°\/
CH30// OCH3
IMP
' \,
Dimethoote
CH30 S
CH,0 ,0
CH30 S-CH-COOC^5 CH30 S-CH-COOC2H5
Molathion
Molooxon
CH0
X
Methyl Parathion
C2H50
X
Porathion
/
CH0
X
Fenthion
Diazinon
CH
CH0 0
CH30 OCH=CCI2
Dichlorvos
CH0
Phosphamidon
(CH3)2CH-0 JO
P
^OCHjCHtCH^CHj (CH3)2CH-0 F
2-Ethylhexyl-diphenyl-phosphate
DFP
0
CH30 0-C=CHCI
Tetrachlorvinphos .
Cl
Cl
-.0
(CH3)2N
HMPA
CH3°v
X
CH30 H-CCI,
3 j 3
OH
Trichlorfen
HOH2C CH2OH
THPC
©
Cl
Br
BrCH2CHCH2-0 .0
e ^p^
,/ V
Tris-BP
Br
Br
Figure 20
-------
LEGEND TO FIGURE 20
Fig. 20. Structural formulas of organophosphorus compounds that have
been tested for carcinogenicity. (See Table CVIII for complete chemical
names.)
-------
Table CVI
Physical Constants of Some Widely Used Organophosphorus Compounds'
p. 1 of 2 pp.
Compound
Trimethylphosphate
(TMP)
Dimethoate
Malathion
Methyl Parathion
Parathion
Fenthion
Diazinon
b. p. at
m. p. / TT \
(mm Hg)
197. 2°C
52° C 107°C at
0. 05 mm
2.9CC 156-7°Cat ,
0. 7 mm .
•
37-8°C 109°C at
0. 05 mm
6°C 375°C
87°C at
0. 01 mm
83-4° C at '
, 0. 002mm
r> r L- Vapor
_ . Refractive
Density . , pressure
index
(mmHg)
d19'5=1.197 — —
8.5xlO"6
at 20°C
d^5 = 1.23 n25= 1.4985 4. OxlO"5
4 Tj n
at 30°C
20 25 -5
d =1.358 n' =1.5307 9.7x10
D at20°C
•
d25=1.26 n25= 1.5370 0.003
4 at24°C
20 20 -5
d, =1.25 n =1.5698 3.0x10
4 D at20°C
20 20 -4
d =1.116-8 n =1.4978 4.1x10
at 20 *C
Solubility
Freely soluble in water.
Slightly soluble in water
(3-4%); soluble in
polar organic
solvents.
Sparingly soluble in
water (145 ppm);
miscible with many
organic solvents.
Sparingly soluble in
water (50 ppm);
soluble in most
organic solvents.
Sparingly soluble in
water (20 ppm);
soluble in alcohols,
esteis, katones.
Sparingly soluble in
water (55 ppm);
soluble in alcohols
and organic solvents.
Sparingly soluble in
water (40 ppm);
mlscible with alcohols
and organic solvents.
-------
Table CVI continued
p. 2 of 2 pp.
Dichlorvos
Phosphamidon -45 C
Tetrachlorvinphos 97-8 C
Diisopropyl -82 C
fluorophosphate
(DFP)
Hexamethylphosphorami.de 7. 2 C
(HMPA)
Trichlorfon 83-4°C
Tris (2, 3-dibromopropyl)
phosphate
(Tris-BP)
140°Cat
20 mm
162°C at
1 . 5 mm
____
62°C at
9 mm
i05-7°C at -i
1 1 mm
100°Cat .
0. 1 mm
— —
d =1.415 n =1.451 Slightly soluble in water
(1%); miscible with
organic solvents.
d45 = 1.2132 n^5 = 1.4718 2.5xlO~5 Miscible with water and.
at 20°C most organic solvents.
-8
— — — 4.2x10 Sparingly soluble in
at 20°C water (11 ppm);
soluble in most
organic solvents.
d = 1.0'35 n =1.383 0.579 Slightly soluble in water
at 20°C (1. 54%); soluble in
vegetable oils.
d =1.03 . n =1.4572 — Soluble in water, polar
and nonpolar solvents.
d = 1.73 ; \ : ' , Soluble in water (1^. 4%),
ether, chloroform
and benzene.
25 -4
d =2.27 1.9x10 Practically insoluble in
at 25°C water (1.6 ppm);
soluble in acetone.
dimethylformamide
and organic solvents.
Summarized from M. Eto, "Organophosphorus Pesticides. Organic and Biological Chemistry", CRC Press,
Cleveland, 1974; "The Merck Index, " 9th edn., Merck & Co., Rahway, NJ, 1976; J.A. Dean, "Lange's Handbook of
Chemistry",.l2th edn., McGraw-Hill, New York, 1979.
See Fig. 20 for structural formulas and Table CVIII for complete chemical names.
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484
by an electron-withdrawing ester group may increase susceptibility to hydrolr™-.
ysis, especially under alkaline conditions. The rate of hydrolysis of a num-
ber of organophosphorus pesticides (in a 1:4 mixture of ethanol and buffer, pH
6, at 70°C) follows the order: Dichlorvos (ti =1.35 hr) >Trichlorfon (3.2
2
hr)^Malaoxon (7. 0 hr)yMa-lathion (7. 8 hr)>Methyl Parathion (8. 4 hr) >Phos-
phamidon (10. 5-14 hr)>Dimethoate (12 hr)>Fenthion (22. 4 hr) >Paraoxon (28
hr) )>Diazinon (37 hr) ^>Parathion (43 hr) (2). Amidophosphates-(phosphora-
mides), o,n the other hand, have quite different hydrolytic properties; HMPA,
for example, is quite stable in aqueous solution.
It I
••Organophosphorus compounds with the general structure — P-O-C — have
two electrophilic sites where reaction with nucleophiles may take place. Nu-.
cleophilic attack at the phosphorus atom with subsequent cleavage of the P-O
bond results in phosphorylation of the nucleophile, whereas a similar attack
at the carbon atom brings about alkylation. The phosphorylation of serine res-
idue at the "esteratic site" of cholinesterase, with subsequent irreversible .in-
hibition, is universally regarded to be the mode of action of insecticidal or-
ganophosphorus compounds. This topic has been amply discussed by several
authors (1, 2). The alkylating activity of organophosphorus compounds is be-
lieved to be the basis of their mutagenic, carcinogenic and other deleterious
biological activities. The relationship between chemical structure and alkyl-
ating properties has been reviewed (1, 2). Methylester organophosphates are
stronger alkylating agents than ethyl or higher n_-alkyl ester derivatives. Phos
phate esters are generally more reactive than the corresponding phosphoro-
thionate esters. Substituents, R1 and R" play an important role in modifying
-------
485
R' O
\ 9
the alkylating activity of organophosphates of the type P . The al-
R" O-R
kylating activity of the R group positively correlates with the electron attract-
ing capability of the substituents. Thus, alkylating activity decreases in the
order: R' and/or R" =£-NO-C6H4~O-> C,H-O-> CH3~S-> CHO-> CH-.
A variety of organophosphorus compounds have been shown to exhibit alkylat-
ing activity as measured by the reaction with 4-(p-nitrobenzyl)pyridine (ab-
breviated as NBP). Preussmann _et aj^ (7) semi-quantitatively rated Methyl
Parathion, Malathion and Trichlorfon as strongly positive (+++) and Parathion
as weakly positive (-f-f) alkylating agents. A more detailed study by Bedford
>
and Robinson (3) listed the relative alkylating activity of several organophos-
phorus compounds in the following decreasing order: Dichlorvos} Methyl Para
oxon> Tetrachlorvinphos^ Methyl Parathion^>Malathion^-Trichlorfon^> Trimeth
ylphosphate ^Diethyl analog of Dichlorvos. Dichlorvos is about 70 times less
active than dimethyl sulfate in the NBP reaction. Demethylated Dichlorvos
(!:.£•' 2, 2-dichlorovinyl monomethyl phosphate), dimethyl phosphate and a num
ber of dichloro metabolites of Dichlorvos are all inactive as alkylating agents.
The alkylating activity of Dichlorvos, Methyl Parathion and Trichlorfon was
confirmed by the study of Fischer and Lohs (8); also Dimethoate was shown to
be an active alkylating agent by the NBP reaction.
The toxicity of organophosphorus compounds used as insecticides has
•«- TSWfi C
been extensively investigated; Table CVII summarizes the acute toxicity data.
In general, the principal toxic actions of these compounds are due to the inhib-
ition of cholines terase,thus increasing the neurotransmitter, acetylcholine,
-------
Table CVII p. 1 of 4 pp.
Acute Toxicity of Several Widely Used Organophosphorus Compounds
Compound
Trimethylphosphate
(TMP)
Dimethoate
Malathion
Malaoxon
Species and route
Mouse, oral
Rat, oral
Guinea pig, oral
Rabbit, oral
Rat, oral
Rat, oral
Rat, topical
Wild bird, oral
Mouse, oral
Rat, oral
Rat, oral
Rat, i. p.
Guinea pig, i. p.
Dog, i. p.
Rat, i. p.
LD5Q (mg/kg)
1,470;3,610
840; 2, 000
1,142
1,275
215
147(LP), 152 (HP)
400
' 7
3, 321
1,375(M), 1,000(F)
599 (LP), 140 (HP)
750, 900
500
1,400
25
Reference
22, 128
128, 129
129
129
130
131
130
132
45
130
131
133, 134
135
136
134
-------
Table CVII continued
p. 2 of 4 pp
Methyl Parathion
Parathion
Fenthion
Mouse, oral
Mouse, i. v.
Rat, oral
Rat, topical
Rat, inhalation
Guinea pig, oral
Mouse, oral
Mouse, i. p.
Rat, oral
Rat, oral
Rat, topical
Rat, inhalation
Mouse, oral
Rat, oral
Rat, topical
Domestic bird, oral
17
13
14 (M), 24 (F)
67
LC =200mg/m or
19. 5 ppm for 1 hr.,
120mg/m or
c
11.4 ppm for 4 hr.
417
6, 25
9-10
5-30 (M), 1. 75-6 (F)
4. 9(LP), 37.1 (HP)
21 (M), 6. 8(F)
LC50 = 18 ppm or 214
mg/m for 1 hr.
125 (M), 150 (F),
200
260 (M), 325 (F)
330
6; 1.1
137
137
138
138
139
137
52, 140
141
52, 130, 140, 141
131
130
142
143, 144
143
130
145
-------
Table CVII continued
p. 3 of 4 pp
Diazinon
Dichlorvos
Phospharmdon
T et rachlo r ovinpho s
Dusopropyl
fluorophosphate
(DFP)
Rat, oral
Rat, topical
Chicken, s. c.
Wild bird, oral
Mouse, oral
Rat, oral
Rat, topical
Rat, s. c.
Chicken, s. c.
Dog, oral
Rat, oral
Rat, topical
Wild bird, oral
Mouse, oral
Rat, oral
Chicken, oral
Wild bird, oral
Mouse, topical
Rat, oral or topical
Monkey, i. v.
108 (M), 76 (F)
900 (M), 455 (F)
15
2
140
80 (M), 56 (F)
107 (M), 75 (F)
52
20
483 (newborn), 1.090
(adult)
23.5
143 (M), 107 (F)
2
•.
1,600
1,100
2, 528
100
72
8
0.28
130
130
146
132
147
148
148
149
146
150
130
130
132
151
152
153
132
151
154
151
-------
Table CVII continued
p. 4 of 4 pp.
Hexamethyl-
phosphoramide
(HMPA)
Trichlorfon
Tetrakis(hydroxy-
methyl)phosphon-
lum chloride
(THPC)
Tris (2, 3-dibromo-
propyl) phosphate
(Tris-BPorTRIS)
Rat, oral
Rabbit, topical
Chicken, oral
Mouse, i. p.
Rat, oral
Rat, i. p.
Guinea pig, i. p.
Wild bird, oral
Mouse, oral
Rat, oral
Rabbit, topical
2,650(M), 3, 360 (F)
2,600
835
500
400, 450
225
300
40
400
5,400
>8,000
65
155
156
157
132, 157
157
157
132
158
159
9
See Table CVIII for complete chemical names and Fig. 20 for chemical structures.
Except where specified, the values shown are lethal doses for 50% kill (LD n ) in mg/kg body weight.
(M) =male, (F) = female, (L.P) = low protein diet; (HP) = high protein diet.
LC = lethal concentration for 50% kill within the exposure time period specified.
-------
486
with resultant excessive stimulation of the central and peripheral nervous sys-
tems and somatic motor nerves. The clinical toxic symptoms include, in the
order of appearance, cramping abdominal pain, lacrimation, salivation, mus-.
cle fasciculation, excessive bronchial secretion, extreme respiratory distress,
coma, convulsion and death. Phosphoramides tend to have more selective ac-
tion on peripheral tissues. The toxic effects of insecticidal organophosphorus
compounds can be alleviated by treatment with the muscarinic blocker, atrop-
ine and related compounds. The toxicity of some organophosphorus compounds
shows variations according to the species, sex or age of the animal and may <
be modified by the diet (see Table CVII). The toxicology of the flame retard-
ant, Tris-BP, has been investigated by Kerst (9) and was found to have a rel-
atively low acute toxicity.
The mutagenicity of organophosphorus compounds was investigated in a
variety of test systems. Trimethylphosphate is definitely mutagenic in micro-
organisms, Drosophila, cultured mammalian cells and laboratory rodents, al-
though its potency is very low, and high dosages or concentrations are usual-
ly required (rev., 10). In microbial systems, TMP was found to be weakly
mutagenic in several mutant strains of Salmonella typhimurium (11-14), Es-
cherichia coli (12, 15), Klebsiella pneumoniae (16), Serratia marcescens (15),
Pseudomonas aeruginosa (11), Neurospora eras sa (.17), and Saccharomyces
cerevisiae (18). In some of these studies, a mammalian hepatic activation
system is included; however, it has not been thoroughly investigated whether
the inclusion actually enhances or diminishes the mutagenic effect of TMP.
-------
487
It is important to note that TMP is capable of inducing dominant lethal muta-
tions in several strains of mice (19-22). Although the effect is observed only
at high doses (around 1 g/kg), it can be clearly demonstrated and appears to
be dose-dependent The literature on mutagenicity of organophosphorus in-
secticides has been reviewed by Wild (23) The induction of mutation in mi-
croorganisms in j_n _v_Ur_o systems has been unequivocally shown with Dichlor-
vos (11, 15, 16, 24-29), Dimethoate (23, 26-28), and Methyl Parathion (27, 28)
at millimolar or higher concentrations Malathion, Parathion and Diazinon,
however, appear to be inactive in several tests (15, 23, 24, 27, 28). In gener-
al, the _m vitro mutagenic activity of these and several other organophosphor-
us compounds appears to parallel their alkylating activity as measured by the
NBP reaction (18, 25). In contrast to its in vitro activity, Dichlorvos has been
consistently found to be inactive in various in vivo tests including host-medi-
ated assays (25, 30), chromosome aberration, micronucleus, and dominant leth-
al tests in experimental animals (19, 31-33). The apparent lack of activity in
in vivo systems is believed to be due to the extremely rapid breakdown of the
compound to nonalkylating metabolites and the impossibility to administer high
doses because of toxicity The mutagenic effect of other organophosphorus in-
secticides in in vivo systems has not been experimentally investigated. Cyto-
genetic analysis of humans exposed to organophosphorus compounds such as
Malathion, Methyl Parathion and Trichlorfon, did not indicate any significant
or clear-cut increase in chromosomal aberration (34, 35). The mutagenicity
of HMPA has also been studied, in Ames1 Salmonella test, the compound gave
erratic responses in a manner similar to that of dimethylnitrosamine (36). In
-------
488
a cell transformation assay, however, HMPA consistently increased the trans-
formation frequency of cultured mammalian cells after metabolic activation
(36, 37) The recent discovery of the highly potent mutagenicity of Tns-BP
has stirred great concern about its safety as the main flame retardant in child-
ren's sleepwear Tris-BP has been found to be mutagenic in the Salmonella
test by several laboratories (38-41). The compound is active as such, howev-
er, the inclusion of metabolic activating system (S-9 mix) considerably en-
hances the mutagenic effect Actually, Tris-BP is a more potent mutagen than
such well known human carcinogens as benzidme and /3-naphthylamme (40)
Structure-^
5 2 1 4 1 2 CaEcinogenicity and Y^ -Activity Relationships. Despite extensive
studies of the acute toxicity of organophosphorus compounds, their potential
carcinogenic activity has largely been neglected until recently Close to 20
organophosphorus compounds have been tested or are being tested,'at the time
of this writing, in the Carcinogenes is Testing Program of the National Toxi-
cology Program The results of the completed studies and those of various
other investigators are summarized in Table CV1II. *f£f ** /&Pa£. C V//|
The unsubstituted trialkyl phosphate, TMP, has been found to be a weak
carcinogen in rodents Oral administration of the compound (3 times/week for
2 years) to Fischer 344 rats (50 or 100 mg/kg/dose) or B6C3F mice (250 or
500 mg/kg/dose) led to the induction of benign fibro^nas in the subcutaneous
tissue in male rats and malignant adenocarcinomas of the uterus/endometn-
um in female mice (18% in the low-dose and 38% in the high-dose group) The
effects were statistically significant However, no evidence of carcinogenicity
of the compound in female rats or male mice was found (42)
-------
Table CVIII
p. 1 of'4 pp.
Carcinogenicity of Organophosphorus Compounds
Compound
Species and strain
Principal organs affected (Route)
Reference
Trimethylphosphate
(TMP)
O, O-Dimethyl S-(N-meth-
yl-carbamoylmethyl)
phosphorothioate
(Dimethoate)
O, O-Dimethyl S-(l,2-di-
carboxy ethyl)
phosphorothioate
(Malathion)
O, O-Dimethyl S-(l, 2-di-
carboxy ethyl)
phosphate
(Malaoxon)
Mouse, B6C3F
Rat. F344
Mouse, AB
Mouse, B6C3F
Rat, Wistar
1
1
Rat, Osborne-Mendel
Mouse, B6C3F
Rat, unspecified
Rat, Sprague-Dawley
Rat, Osborne-Mendel
Rat, F344
Mouse, B6C3F
Rat, F344
Uterus (oral)
Subcutaneous tissue (oral)
Hematopoietic system, mammary
gland (topical)
None (oral)
Spleen, forestomach, lung
(oral or i. m.)
None (oral)
No significant effect (oral)
None (oral)
Mammary gland (oral)
(inconclusive)
No significant effect (oral)
None (oral)
None (oral)
None (oral)
42
75
43
44
43
44
48
45'
46
48
49
50
50
-------
Table CVIII continued
p. 2 of 4 pp
O, O-Dimethyl O-(4-nitro-
phenyl) phosphorothioate
(Methyl Parathion)
O, O-Diethyl O-(4-nitro-
phenyl) phosphorothioate
(Parathion)
Mouse, B6C3F
Rat, F344
O, O-Dimethyl O-[4-(meth-
ylthio)-m-tolyl ~]
phosphorothioate
(Fenthion)
O, O-Diethyl O-(2-iso-
p ropy 1-6-me thy 1-
-4-pyrimidyl)
phosphorothioate
(Diazinon)
O, O-Dimethyl 2, 2-di-
chlorovinyl phosphate
(Dichlorvos, DDVP)
Mouse, B6C3F
1
Rat, albino
Rat, unspecified
Rat, Osborne-Mendel
Mouse, B6C3F
Rat, unspecified
Rat, F344
Mouse, B6C3F
Rat, Carworth Farm albino
Rat, F344
Mouse, B6C3F
1
Rat, unspecified
Rat, Carworth Farm E
Rat, Osborne-Mendel
None (oral)
None (oral)
None (oral)
None (oral)
None (oral)
Adrenal gland (oral)
(marginal)
Skin, subcutaneous tissue (oral)
None (oral)
None (oral)
None (oral)
None (oral)
None (oral)
None (oral)
None (oral)
None (inhalation)
None (oral)
51
51
54
52, 53
Lehman, 1965 cited
jji ref. 54
54
55
Douell ejt al., cited
ui ref. 55
55
160
56
160
60
58
59
60
-------
Table CVIII continued
p. 3 of 4 pp.
O, O-Dimethyl 2-chloro-2-
-diethyl-carbamoyl-1 -
-methyl-vinyl phosphate
(Phosphamidon)
O, O-Dimethyl 2-chloro-
-l-[(2, 4, 5-trichloro)-
vinyl 3 phosphate
(Tetrachlorvinphos)
2-Ethylhexyl diphenyl
phosphate
Diisopropyl fluoro-
phosphate
(DFP)
Hexamethylphos -
phoramide
(HMPA, Hexamethyl-
phoric triamide)
O, O-Dimethyl 1 -hydroxy-
-2, 2, 2-trichloroethyl
phosphate
(Trichlorfon, Dipterex)
Mouse, B6C3F
Rat, Carworth Farm N
Rat, Osborne-Mendel
Mouse, B6C3F
Rat, Osborne-Mendel
Rat, Carworth Farm albino
Rat, Wistar
Rat, Sherman
Rat,1 Charles River CD
Mouse, AB
Rat, BD
Rat, Wistar
None (oral)
None (oral)
Spleen (oral) (marginal)
Liver (oral)
Thyroid, adrenal glands (oral)
No significant effect (oral)
Pituitary gland (i. m. )
None (oral)
Nasal cavity (inhalation)
Forestomach, liver, hematopoietic
system (topical)
Local sarcoma (s.c.)
Forestomach (oral or s. c.)
Spleen, forestomach, lung, ovary
(oral or i. m. )
57
61
57
62
62
63
64
65
66, 67
43, 70
69
70
43
-------
Table CVUI continued
p. 4 of 4 pp.
Tet rakis(hydroxy methyl) •
phosphonium chloride
(THPC)
Tris-(2, 3-dibromo-
p ropy 1)-phosphate
(Tris-BP, THIS)
Mouse, Swiss ICR/Ha
Mouse, Swiss ICR/Ha
Mouse, B6C3F
Rat, F344
1
No significant effect (topical)
Luwg, forestomach, oral cavity,
skin (topical)
Kidney, lung, liver, stomach (oral)
Kidney (oral)
72
72
75
75
-------
489
Close to a dozen organophosphorus Insecticides have been tested for car-
cinogenicity. In general, they have been found to be inactive or weakly active
carcinogens. Dimethoate is one of the few organophosphorus insecticides that
exhibit carcinogenic activity. Gibel et^ al. (43) reported that 15 of 101 Wistar
rats of either sex given Dimethoate (5, 15 or 30 mg/kg, p. o. or 15 mg/kg, i. m.,
twice weekly for 10 weeks) developed malignant tumors (compared to none in
controls). The malignancies were mainly reticulum cell sarcomas of the spleen
and malignant reticulosis. In addition, substantial increases in the incidences
of benign tumors, particularly in the forestomach and the lung, were observed.
By topical application to AB mice, Dimethoate induced malignancies in 5 of 1 9
mice 4 leukemia and 1 mammary adenocarcinoma (43). In contrast to the
above finding, however, a more recent study reported by the National Cancer
Institute (44) did not confirm the carcinogenicity of the compound. In this study,
Osborne-Mendel rats and B6C3F mice were fed Dimethoate at doses ranging
from 155 to 500 ppm for up to 115 weeks.* no increases in tumor incidence at-
tributable to the pesticide were observed (44). .„ /--.
The chronic toxicity of Malathion was investigated by Hazleton and Hol-
land (45). Rats of an unspecified strain were fed diets containing 100, 1000 or
5000 ppm of either 65, 90 or 99% pure preparation of the compound. No tu-
mors associated with the administration of the compound were observed. On-
ly indirect and inconclusive evidence of possible carcinogenicity of Malathion
has been provided (46); Sprague-Dawley rats were given a dose of 15 mg di-
methylbenzanthracene, a mammary carcinogen, and kept on diets containing
either 250 ppm or no Malathion. The Malathion-treated rats showed a higher
-------
490
incidence of mammary tumors and shorter induction time (1.31 0.18
tumors/rat; 20 days to the appearance of the first tumor) than controls
(1. 07 0. 15 tumors/rat; 25 days to first tumor). It is not known whether the
enhancement of the carcinogenic effect reflected the modifying role of the pes-
ticide or its carcinogenic effect acting synergistically or additively with di-
methylbenzanthracene. In several industry-sponsored studies reported to the
National Institute of Occupational Safety and Health (47) Malathion was found
to be noncarcinogenic in albino rats after feeding for 2 years. The lack of car-
cinogenicity of Malathion has been confirmed in several recent National Can-
cer Institute studies. No clear evidence of association of tumor induction with
Malathion could be demonstrated in Osborne-Mendel rats (fed diets containing
4700 or 81 50 ppm of the compound for 80 weeks), F3.44 rats (2, 000 or 4, 000
ppm for 105 weeks), or B6C3F mice (8, 000 o'r 16, 000 ppm for 80 weeks) (48,
49). Malaoxon, the activated metabolite of Malathion, has also been tested
and found to be noncarcinogenic after feeding for 103 weeks to F344 rats and
B6C3F mice. The doses administered (500 or 1,000 ppm for both rodents)
were considerably lower than those of Malathion because of the substantially
higher acute toxicity of the metabolite (50).
Four dialkyl aryl phosphorothiates, Methyl Parathion, Parathion, Fen-
thionand Diazinon, have been tested for carcinogen.icity. Among these, Meth-
yl Parathion has been found to be noncarcinogenic in F344 rats and B6C3F
mice. The doses administered were 20 or 40 ppm in the diet for 105 weeks
for the rats and 62.5 or 125 ppm for 102 weeks for the mice (51). Thecarcin-
ogenicity of Parathion has been evaluated by several groups of investigators.
-------
491
In a 2-year chronic toxicity study of the compound, Hazleton and Holland (52)
did not observe, in male albino rats, any increase in tumor incidence attribut-
able to the administration of up to 100 ppm of the compound in the diet. A sim-
ilar observation was reported by Barnes and Denz (53) who fed albino rats for
one year.diets containing 10-100 ppm of the compound. A 2-year study by the
Food and Drug Administration also indicated no carcinogenic effect in an un-
specified strain of rats (Lehman, 1965, cited in Ref. 54). In the recent'NCI
study, B6C3F. mice also did not develop tumors after receiving 80 or 160 ppm
of the pesticide in the diet for 71 weeks. In the Osborne-Mendel rat, how-
ever, there was a higher incidence of cortical tumors (mostly adenomas) of
the adrenal gland after administration of diets containing 23-63 ppm of Para-
thion for 80 weeks. The increase in tumor incidence was s tatistically .signi-
ficant and dose-dependent when compared to pooled or historical controls, but
insignificant when compared to matched controls, probably because of the small
number of rats used (males: pooled control 3/80, matched control 0/9, low-
-dose 7/49, high-dose 11/46; females: pooled control 4/78, matched control
1/10, low-dose 6/47, high-dose 13/42). The results are considered to suggest
a possible (but not conclusive) association between tumor induction in the adren-
al gland and Parathion administration, in this particular strain of rats (54).
The carcinogenicity of Fenthion has also been tested at the National Cancer In-
stitute by feeding F344 rats and B6C3F mice for 103 weeks, diets containing
10 or 20 ppm of the pesticide. No increase in tumor incidence was observed
in the rat. Female mice also did not develop tumors related to the pesticide
treatment. In male mice, however, significant increases in the incidence
-------
492
of sarcomas, fibrosarcomas and especially rhabdomyosarcomas of the inte-
gumentary system were observed, suggesting carcinogenicity of this compound
(55). In a previously unreported study, Doull and associates did not detect any
increase in tumor incidence after feeding rats for 1 year diets containing up
to 100 ppm of Fenthion (Doull _e_t aj^, cited in ref. 55). The substitution of the
£-nitrophenyl group of Parathion by a substituted pyrimidine ring (yielding Di-
azinon) does not seem to confer any carcinogenicity. Bruce e_t al. (56) ob-
served no tumors in Carworth Farm albino rats after feeding for 72 weeks di —
ets containing 10-1, 000 ppm Diazinon. The lack of carcinogenicity of Diazin-
on has been confirmed by a recent National Cancer Institute study in which
F344 rats and B6C3F mice were fed diets containing 400 or 800 ppm (for rats)
and 100 or 200 ppm (for mice) of the compound, for 103 weeks (57). Thus,
with a few exceptional cases, dialkyl aryl phosphorothioates are mostly inac-
tive as carcinogens.
Among the organophosphorus compounds, dialkyl vinyl phosphates are
considered active alkylating agents and are therefore suspected to be potenti-
al carcinogens. The carcinogenicity of Dichlorvos has been extensively inves-
tigated; it appears, however, that there is no evidence that the compound is
carcinogenic in experimental animals. The lack of carcinogenicity of Dichlor-
vos was reported in a Food and Agriculture Organization/World Health Organ-
ization Joint Meeting in 1966. Rats of an unspecified strain were fed nominal
concentrations of 0. 1, 1, 10, 100 and 500 ppm Dichlorvos in food for 2 years;
there was no indication that the compound led to any increase in the tumor in-
cidence (58). A 2-year inhalation carcinogenes is study has been conducted for
-------
493
Dichlorvos using Carworth Farm E strain rats The animals were exposed to
atmospheres containing 0 05, 0 5 and 5 0 mg/m Dichlorvos continuously for
up to 2 years There was no compound-related increase in tumor incidence
in rats of either sex (59) In a National Cancer Institute study, Osborne-Men-
del rats and B6C3F mice were given technical grade Dichlorvos in the diet
at two dose levels 1 50 ppm for 80 weeks or 1, 000 ppm for 3 weeks followed
by 300 ppm for 77 weeks There was no statistically significant increase in
the incidence of tumors attributable to exposure to Dichlorvos in either animal
species (60). Phosphamidon, another dialkyl vinyl phosphate, was also shown
to have no carcinogenic effect in Carworth Farm N strain rats The animals
received, in the diet, 01-5 mg/kg Phosphamidon daily for two years (61). In
the National Cancer Institute study, Osborne-Mendel rats and B6C3F mice
i -i
received 80 or 160 ppm Phosphamidon in the feed for 62-80 weeks There was
no increase in tumor incidence in the mouse of either sex In the male rats,
the combined incidence of hemangiomas and hemangiosarcomas in the spleen
showed a dose-related increase In the female rats, increases in C-cell ade-
noma and carcinomas of thyroid were observed These increases were stat-
istically significant compared to pooled controls but insignificant compared to
matched controls The data are considered marginal and insufficient to estab-
lish an association between tumor induction and the compound (57) In contrast
to the above two compounds, there are strong indications for the carcinogen-
icity of Tetrachlorvinphos In a National Cancer Institute study, Osborne-
-Mendel rats were fed Tetrachlorvinphos for 80 weeks at the dose of 4, 250 or
8, 500 ppm, wheieas B6C3F mice were given 8, 000 or 16, 000 ppm of the
-------
494
compound for 80 weeks There were significant dose-related increases in
prohferative lesions of the C-cells of the thyroid and cortical adenoma of the
adrenals in female rats. In male mice, Tetrachlorvinphos was clearly car-
cinogenic causing significant increases in the induction of hepatocellular car-
cinomas (0/9 m controls, 36/50 in low-dose group, 40/50 in high-dose group).
In female mice, the increase in hepatocellulai carcinoma was not statistically
significant, although substantial increases in the incidence of neoplastic nod-
ules of the liver were observed (62)
The carcmogenicity of 2-ethylhexyl diphenylphosphate, a monoalkyl di-
aryl phosphate, has been tested by Treon jet a_l_ (63). Carworth albino rats fed
1% or less of the compound in the diet for 2 years did not develop tumors at-
tributable to the administration Limited studies were also carried out using
several dogs, there was no sign of carcjnogenicity (63)
In a study originally designed to investigate the behavioral effect of di-
isopropyl fluorophosphate (DFP), Glow (64) noted the induction of pituitary
tumors in female Wistar rats Out of 100 rats, 16 confirmed cases of pituitary
tumors were recorded This figure did not include those animals that died or
were killed because of other causes The animals were administered an ini-
tial dose of 1 mg/kg followed by booster doses of 0 5 mg/kg every 72 hours
The average induction time was 12-18 months It should be noted that the spon-
•
taneous occurrence of pituitary tumors is extremely Tare in Wistar rats. The
author (64) suggested that DFP could have a specific oncogenic effect on the
pituitary gland of female Wistar rat
-------
495
The carcinogenicity of hexamethylphosphoramide (HMPA) has been tested
by two different routes of administration, with entirely different results. Oral
administration of the compound to Sherman strain rats at the doses of 6. 25,
3. 12, 1. 57 and 0. 78 mg/kg/day for 2 years did not elicit any significant car-
cinogenic effect (65). By the inhalation route, however, HMPA has been found
to be an extremely potent carcinogen in the Sprague-Dawley rat (66, 67). The
induction of tumors in the nasal cavity (a site with extremely low spontaneous
tumor incidence) was observed after exposure (5 hr/day, 5 days/week for 1
year; to HMPA in concentrations as low as 50 ppb (67). The carcinogenic ef-
fect was clearly dose-dependent. The time to the appearance of the first tu-
mor was 6-7 months for the 4, 000- and 400-ppb groups, 9 months for the
100-ppb group, and 13 months for the 50-ppb group; nasal tumors were not ob-
served in the 10-ppb group (67). The nasal tumors were predominantly squam-
ous cell carcinomas and occasionally, squamoadenocarcinomas or papillomas.
Some tumors metastasized to the cervical lymph nodes and lungs, or invaded
directly into the brain (66, 67).
The carcinogenicity of phosphoramides is probably not uniquely associ-
ated with HMPA; other members of the class may also possess carcinogenic
activity. Ashby et al. (37) have evaluated the carcinogenic potential of vari-
ous derivatives of HMPA by in vitro cell transformation assay of Styles (68).
The results are summarized in Fig. 21. Hexamethylphosphoramide (!)• has
been found to substantially increase the transformation frequency of cultured
BHK 21/CIS cells after metabolic activation by the standard rat liver homogen-
ate activation system (S-9 mix). Among the derivatives tested, hexamethyl-
phosphorus triamide (ii) and .hexamethylphosphorothioic triamide (iii) were
-------
3\
\±p
H3C
(M) (+)
HC
X
/'
N4P=S
(in) (+,m)
H3C
N4P = 0
(l) (+)
H'
-"3
(vn) (-)
N+P=0
3
dv)
(vi)
Figure 21
-------
LEGEND TO FIGURE 21
Fig. 21. Structural formulas of the results of cell transformation
assays obtained with hexamethylphosphoramide (HMPA) derivatives. The chemical
names of the compounds are: "L = hexamethylphosphoramide; 'li- = hexamethyl-
phosphorous triamide; iii = hexamethylphosphorothioic triamide; iv = tri-
piperidinophosphine oxide; v = phosphorothioic trimorpholide; vi = diethoxy-
morpholinophosphine oxide; vii = phosphoric trianilide. The results of the
cell transformation assays are designated as follows: "+" = positive after
addition of standard S-9 mix; "+, m" = positive after addition of modified
S-9 mix; "-" negative.
-------
496
I I I
also active, suggesting that the —P=O, —P and —P=S groups may be considered
metabolically equivalent because of the ready oxidation of the latter two groups
to the former. Three alicyclic analogs of HMPA, tripiperidinophosphine ox-
ide (iv), phosphorothioic trimorpholide (v), and diethoxymorpholinophosphine
oxide (vi), have been found inactive after incubation with the standard S-9 mix.
However, using a modified S-9 mix (containing higher proportion of S-9 tissue
fraction in the mix) cell-transforming activity has been clearly demonstrated.
Thus, these three compounds are regarded as potentially carcinogenic but pos-
sessing a much lower potency than (i). In contrast to the above compounds,
phosphoric trianilide (vii), which lacks hydrogen on the carbon atom alpha to
the nitrogen, is completely inactive. These results are supportive of a sug- .
gestion made earlier by Ashby e_t al. (36) that there is a structural analogy be-
^ '
tween phosphoramides ( N — P=O) and nitrosamines ( N —N=O) and that the
relative structure-activity relationships of the latter class of carcinogens may
be of help in predicting the potential carcinogenicity of compounds in the form-
er class. Further studies are needed to establish whether the conclusions
reached from cell transformation assays are applicable to ui vivo carcinogen-
esis assays.
Trichlorfon is the only alkyl phosphonate pesticide that has been tested
for carcinogenicity. It has been found carcinogenic in rodents by several in-
vestigators. By s. c. injection to BD rats,a dose of 50 mg/kg induced local
sarcomas in 2/24 animals (69). Topical application to AB mice led to the in-
duction of one liver carcinoma and one forestomach papilloma out of 16 ani-
mals in one study (70), and myeloid leukemia in 5/14 mice in another study
-------
: '. 497
(43). In the Wistar rat, oral or s. c. administration of Trichlorfon elicited the
development of papillomas in the forestomach of 3/52 or 7/6l rats, respective-
ly. Extensive liver necrosis and cirrhosis were also observed (70). In a more
recent study, Wistar rats were given Trichlorfon orally or intramuscularly.
/-'' •.
Eleven of the 55 rats developed a variety of malignant tumors (compared to
zero in controls); close to 50% (26/55) of the animals developed benign tu-
mors. The malignancies were mainly reticulum cell sarcomas of the spleen
and malignant reticulosis, whereas benign tumors were mostly found i'n the
forestomach, the lung and the ovary (43).
-•The carcinogenic potential of chemical flame retardants has been a sub-
ject of great concern because of their extensive use in children's sleepwear and
the discovery of potent mutagenicity of some of these compounds. Two organo-
i
phosphorus flame retardants, THPC and Tris-BP, have been tested. The con-
cern over THPC stems from the fact that the compound may break down into
phosphine (PH ), formaldehyde and hydrochloric acid. The latter two prod-
ucts may in turn combine to form the potent carcinogen, bis (chlormethyl) eth-
er (see Section 5. 2. ] . 1. 2. 7). In a preliminary mouse skin experiment, appli-
cation of THPC (2 mg in 0. 1 ml dimethylsulfoxide, 3 times/week for 400 days)
to 20 female ICR/Ha Swiss mice led to the induction of one squamous carcin-
oma of the skin (71). In a subsequent experiment with a larger group (60) of
animals at the same dose, but with acetonerwater (9:1) as the solvent, no sig-
nificant increase in the tumor incidence could be observed (72). The differ-
ence in the results from these two experiments was attributed to the solvent
-------
498
(72) as dimethylsulfoxide has been reported to have an unusual effect in mouse
skin carcinogenesis studies (73).
Tris-(2, 3-dibromopropyl)phosphate was first reported to be carcinogen-
ic in rodents by the Consumer Product Safety Commission (74) based on the
preliminary results of a National Cancer Institute study. The study has now
been completed and published (75). Fischer 344 rats and B6C3F mice were
given technical grade Tris-BP in the feed for 103 weeks at doses of 50 or 100
ppm for rats and 500 or 1,000 ppm for mice. Among mice, a variety of tu-
mors arose as a result of Tris-BP administration; these included renal tubu-
lar cell adenoma and carcinoma, squamous cell adenoma and .carcinoma of the
forestomach, hepatocellular adenoma and carcinoma, and bronchiolar/alveolar
adenoma and carcinoma. The incidences of renal adenoma, gastric adenoma,
and pulmonary carcinoma were all significantly higher in dosed male .mice than
controls mice, whereas in dosed female mice significant increases in the inci-
dence of hepatocellular adenoma and carcinoma, gastric adenoma and carcin-
oma, and pulmonary tumors were observed. In the rat, renal tubular adeno-
mas were also observed at incidences which were significantly higher in dosed
rats of both sexes than in the controls. For male rats, there was also a pos-
itive association between renal tubular cell adenocarcinoma and Tris-BP treat-
ment. The possibility that the above carcinogenic -effects might be due to
1, 2-dibromo-3-chloropropane, a common carcinogenic contaminant of Tris-BP,
was considered. After comparing the types and incidences of tumors induced
by the contaminant (s ee Section 5. 2.2. 1) and Tris-BP, it was concluded that
the effects observed were principally due to Tris-BP itself (75). In another
-------
499
recent study (72), Tris-BP has been shown to be an active carcinogen induc-
ing a variety of local as well as distant tumors in female ICR/Ha Swiss mice
after skin application (3 times/week for 420-496 days) at two dose levels. In
the high dose group (30 mg/application), the tumor incidences (out of 30 ani-
mals) in various organs were: dorsal skin 5 (3 papillomas, 1 carcinoma, 1
sarcoma), lung 28 (papillary tumors), liver 1 (carcinoma), forestomach 20
(13 papillomas, 7 carcinomas), oral cavity 4 (2 carcinomas each in the tongue
and gingiva), and kidney 1 (tubular adenoma). Essentially the same targets
were affected in the low-dose group (10 mg/application) but with lower inci-
dences. The incidences in all the above targets were significantly higher than
those of control mice. The time,of the appearance of the first stomach car-
cinoma was as short as 21 weeks. The induction of tumors in the oral cavity .,-•
and gingiva was thought to be -related to the grooming habit of the mice (72).
The relationship between the chemical structure and carcinogenic activ-
ity of organophosphorus compounds has not been systematically investigated.
From the available carcinogenicity data, it seems evident that the carcinogen-
icity of an organophosphorus compound is not solely dependent on its chemic-
al structure. Biological factors such as activation and biodegradation play
important limiting roles. To be carcinogenic, an organophosphorus compound
must possess alkylating activity and also be able to-reach target tissues in an
active form. Thus, despite being one of the most active alkylating agents
(among organophosphorus compounds), Dichlorvos is not carcinogenic because
of its extremely short lifetime in biological systems (see "Metabolism"). On
the other hand, TMP, a very weak alkylating agent, exerts a carcinogenic
-------
500
effect at high doses because of its slov metabolism. The structural features
that render several of the organophosphorus compounds carcinogenic possibly
obey the following rationale. Tetrachlorvinphos may be carcinogenic because
the elctron-withdrawing properties of the vinyl and aryl moieties make the
cleavage of the H,C-O bond easier thus enhancing the alkylating activity of
the methyl group. Although the alkylating activity of tetrachlorvmphos is not
as strong as dichlorvos, tetrachlorvmphos may, because of its relatively lower
susceptibility to alkaline hydrolysis (3), have a better chance of reaching the
^
target tissue. For DFP, the presence of the strongly electronegative
fluorine makes the departure of the isopropyl group easier. Stabilization
by the inductive effect of fluroine renders monoisopropyl fluorophosphate
a good leaving group. Trichlorfon may be considered a precursor of
dichlorvos (see "Metabolism"); it is possible that Trichlorfon has a greater
probability of gaining access to the target tissue before being degraded. In
the case of Tris-BP, the presence of the electronegative bromine atoms
should make the compound a good alkyl donor possibly through displacement
type of reaction. Alternatively, the cleavage of either C-O or O-P bond may
generate 2, 3-dibromopropanol which could, under mildly alkaline condition,
generate highly reactive epoxide intermediate-
w
BrCH -CH-CH > BrCH,-CH-CH0 + Br + H.O
L I L i, L iL
Br
-------
501
Possible
~ • -•—..^—-
5.2.1.4.1..3 Metabolism and f. Mechanism of Action. Organophosphorus com-
pounds undergo, in virtually every biological system, extensive metabolic trans-
formations that can bring about either activation or detoxlcation. Extensive ••
reviews of this subject have been undertaken by various authors in recent years
(2, 4-6, 76-79). Essentially, the activating pathways, as depicted in Fig. 22, T~1>4'
include (i) oxidative /des.ulfu'r.aticta..- -.• of the thionophosphorus moiety (P=S) to the.
corresponding oxon (P=O) analogs, (ii) sulfoxidation of a thioether moiety pres-
ent in the ester group to the respective sulfoxides or sulfones, (iii) N-dealkyl-
ation of the amide moiety of phosphoramide or organophosphate-containing
amide structure, and (iv) thiono-thiolo rearrangement (isomerization) which
transforms phosphorothionate to the more reactive phosphorothiolate. The
former three reactions are catalyzed by the microsomal mixed-function oxi-
dase present in mammalian cells, whereas the thiono-thiolo rearrangement
may be triggered enzymatically or may occur nonenzymatically. It is not
known whether the above-mentioned activation pathways, derived mainly from
studies dealing with activation of insecticidal activity and acute toxicity, also
apply to the generation of potential carcinogenic (presumably alkylating) inter-
mediates. However, as discussed in the section on physico-chemical proper-
ties, modification of the chemical structure, known to increase the positive
charge on the phosphorus atom (_£•£•, thiono —5*. oxen, thiono —^ thiolo trans-
formations), can also increase the alkylating activity of the organophosphorus
compound. The detoxication pathways of the organophosphorus compounds are
of great variety in mammals; they include (a) hydrolysis of the phosphoric es-
ter linkage, (b) hydrolys is of carboxyes ter (e_. £., Malathion) or carboxamide
-------
(i) Oxidotive desulfurotion
(RO)3P=S —
•\
'Oj
(RO)3P=0
(ii) Sulfoxidotion
O(orS)
,P(orS)
(R0)2p;
0
\
•(RO)2P
JD(orS)
OtorSMCH^-S-R'
N
or
\
0(orSHCH2)n-S-R'
0
(iii) N-Deolkylotion
P .CH3
/
CHgOH
O
N
?' 0 CH3 — (RO)2P
0-C=C-C-li
/
/
0-C=CH-C-N
CH3
8 CH2OH^(RO)
/ R' n «..«.. .™, r^/ R' o
\ I ii /'
O-C=CH-C-NX
,H
'CH3
(iv) Thiono-thiolo rearrangement (isomerizotion)
,S ,0
(RO)2PN
\
(RO)2PV
OR
\
SR
Figure 22
-------
LEGEND TO FIGURE 22
Fig. 22. Activating pathways of organophosphorus compounds.
-------
502
(j2. £., Dimethoate) groups, (c) O-dealkylation, (d) dechlorination (je.£., Phos-
phamidon), (e) aitro reduction, and (f) conjugation. The hydrolytic reactions
may be catalyzed by a variety of esterases present in virtually all the tissues-
or by microsomal mixed-function oxidases, whereas the other reactions may
be mediated by microsomal mixed-function oxidases, nitro-reductase, gluta-
thione S-transferase or glucuronyl transferase. These pathways can evident-
ly transform alkylating agents into nonalkylating metabolites. The metabol-
ism of several representative organophosphorus compounds is briefly dis-
cussed below.
"' Trimethylphosphate is almost inactive as a phosphorylating agent. Many
of the esterase-catalyzed reactions do not appear to operate on TMP. The on-
ly major metabolic pathway is O-demethylation catalyzed by glutathione-depen-
dent S-alkyl transferase present in the liver. The major urinary metabolites
of TMP in rodents are dimethylphosphate (which is inactive as an alkylating
agent), S-methylcysteime and its N-acetate (which are derived from S-methyl
glutathione) (80, 81). It has been postulated that high doses of TMP are re-
quired to saturate this pathway before it may exert deleterious effects (3). -^ ^
**— F*3
The metabolic pathways of Parathion, shown in Fig. 23, are probably
representative of many phosphothionate insecticides. Oxidative desulfxiar.a-ta6'n.,
catalyzed by microsomal mixed-function oxidase, is the principal activation-
pathway (82-88). The desulfvi:rafci6n> of Parathion (i) is postulated to proceed
via a cyclic intermediate, formed by direct addition of atomic oxygen at the
P=S bond (88). The resulting metabolite, Paraoxon (ii), is more toxic and
more reactive both as phosphorylating and alkylating agent, than the parent
-------
W /(orO)
p
HOX ^OH
/ (viii)
+ HO-C'' VNOz
(iv) \
A
HO
0-deolkylose
I
microsomal
mixed-function oxidase
C2H50X OH
microsomal
mixed-function oxidase
-N02 + CjHg-glutothione + HO'
(vii)
0-deolkylose
I
(oxidative desulfuration)
_NOz
orylesterase
OH
ruminonts "nitroreductose"
•olkylotion phosphorylation
Figure 23
-------
. LEGEND TO FIGURE 23
Fig. 23. Proposed metabolic.,pathways of Parathion. The chemical names
of the compounds are: i = Parathion; ii. = Paraoxon; ii,i = diethylphosphorothionic
acid; iv = £-nitrophenol; v = diethylphosphoric acid; vi = desethylparathion;
vii = desethylparaoxon; viii = monoethylthiophosphoric(phosphoric) acid.
[Adapted from A. deBruin, "Biochemical Toxicology of Environmental Agents".
Elsevies, Amsterdam, 1976, p. 171]
-------
503
compound. On the other hand, Parathion is also detoxified by microsomal
mixed-function oxidase by hydrolytic degradation into diethylphosphorothionic
acid (iii) and £-nitrophenol (iv) (88-92). Dearylation is also postulated to
proceed via a cyclic intermediate (88), although the microsomal mixed-func-
tion oxidase system involved appears to behave differently from that involved
in oxidative de,sulf,uVati6h'.--• (88, 93, 94). The dearylation of Parathion may al-
so proceed via glutathion-S-aryltransferase present in mammalian liver, de-
grading Parathion into S-£-nitrophenylglutathione and diethylphosphorothionic
acid (95). The dearylation of Paraoxon also proceeds via nonspecific aryles-
terase(s) present in blood serum and several tissues (96, 97). Another detox-
ification pathway, although occurring only to a minor extent for both Parathi-
onand Paraoxon, is a glutathione-S-alkyl-transferase catalyzed O-dealkylation
reaction.. This:enzyme is specifically present in the liver and requires NADPH
and oxygen for activity. It deethylates Parathion and Paraoxon to desethylpara-
thion (vi) and desethylparaoxon (vii), which eventually give rise to monoethyl-
phosphorothionic acid and monoethylphosphoric acid, respectively (98, 99).
Dichlorvos undergoes very rapid metabolic changes in various mammal-
ian tissues with the concomitant loss of alkylating as well as phosphorylat-
ing activity (rev., 4). Blair ^_t aj_._ (100) reported that Dichlorvos could not be
detected in any tissues of rodents after exposure to atmospheres containing up
to 0. 5 pg/1 of the compound. Measurable amounts could be detected in sever-
al tissues (with the highest in the kidney) after exposure to a high concentra-
tion of 90 Hg/1. Under these conditions, the half-life of the compound in the
kidney was not more than 13. 5 min (100). Rapid disappearance of Dichlorvos
-------
504
was also observed in pigs receiving the compound as vapor via tracheal can-
«*£- ft$
nuiae (101). As shown in Fig. 24, the metabolism of Dichlorvos (i) proceeds
via two major routes, hydrolysis and demethylation. These pathways appear
to be common to rodents '(102), pigs (101, 103), hamsters and humans (104),
although their relative importance shows variations according to the species
and route of administration. Hydrolysis is a prominent degradative route in
the metabolism of Dichlorvos. The reaction is catalyzed by nonspecific es-
terases (also variously named hydroxylases or phosphatases) present in near-
ly all mammalian tissues tested (see ref. 4 for overview). The resultant prod-
ucts are dimethylphosphate (ii), which is excreted unchanged and 2, 2-dichloro-
acetaldehyde (iii), which may either (a) be reduced to 2, 2-dichloroethanol (iv)
and then conjugated as a glucuronide, or (b) enter the 2-carbon pool and be ox-
idized to CO , transformed to urea, or incorporated into hippuric acid or pro-
tein. The demethylation may proceed either by (a) glutathione-dependent S-al-
kyl transferase-catalyzed transfer of a methyl group to yield S-methyl-gluta-
thione:.and desmethyldichlorvos (v), or (b) microsomal mixed-function oxidase-
-catalyzed oxidation of a methyl group yielding desmethyldichlorvos (v) and
formaldehyde (vii). In each case, desmethyldichlorvos is further hydrolyzed
to monomethylphosphate (vi), phosphate and methanol. The S-alkyl transferase is
a soluble liver enzyme (105-109) demonstrated experimentally to function invivo.(4)
and its existence is further supported by the finding that depletion of glutathione
enhances the acute toxicity of Dichlorvos (78). The oxidative demethylation of
Dichlorvos has been shown to occur hi vitro (106); but its in vivo role is still
undefined.
-------
f^
S-olkyl
transferase
H3C-glutathione -*S-methylcysteine -*S-methylcysteine N-acetate
et/P\
en/\
phosphate + methanol
0 X)-CH=CCI2 ^0 . OH
esterase
CH30X
/ (Jv)
+ CI2CHCHO
CH30' V0-CH=CCI2 CH30' "0" (ijj) \
(i) \ (ii)
-*CI2CHCH20-glucuronide
microsomal mixed-
function oxidose
a-carbon pool
,C02
urea
CH30 Q
\^
^hippuric acid
protein
+ HCHO^CC^
t)' N0-CH=CCI2 (vii)
(v)
Figure
-------
LEGEND TO FIGURE 24
Fig. 24. Proposed metabolic pathways of Dichlorvos. The chemical
names of the compounds are: i, = Dichlorvos; ii = dimethylphosphate; Hi =
2,2-dichloroacetaldehyde; iv = 2,2-dichloroethanol; V = desmethyldichlorvos;
vi = monomethylphosphate; vii = formaldehyde.
-------
505
Hexamethylphosphoramide (HMPA) has been shown to undergo sequential
demethylation in vivo to yield N1, N", N'"-trimethylphosphoramide in rats and
mice (110). The N-demethylation also proceeds in vitro resulting in the re-
lease of formaldehyde using liver slices (110)'or 9,000 g postmitochondnal
supernatant of liver homogenate (S-9 fraction) (36) as the enzyme source The
overall reaction is thought to be detoxifying because the final metabolite lost
its biological activity (anti-spermatogenic effect) (110) Even the loss of one
methyl group may be inactivating as suggested by the lack of chemos terilant
activity of pentamethylphosphoramide in the housefly (111) However, in the
process of demethylation, unstable reactive intermediates are produced These
O
inteimediates, which may be N-oxide [(CHo)?N- ]or>.methylol ( CHo-N -CH2OH),
\
I
have been suggested to be the activated form(s) of phosphorarmde (110, 112,
113) It has been shown than the cell-transforming activity of HMPA requires
metabolic activation by S-9 liver fraction (36, 37)
The metabolic fate of Trichlorfon has not been thoroughly investigated
Tnchlorfon is readily transformed nonenzymatically into Dichlorvos (2) which
may be the actual toxicant. However, this pathway does not seem to operate
to any great extent in some species because the hydrolysis of Trichlorfon it-
self may be faster than that of Dichlorvos in these s.pecies (114) .After injec-
tion of Trichlorfon into dogs, trichloroethyl glucuronide was detected, suggest-
ing the cleavage of the P —C bonding of Irichlorfon In the rabbit, however, a
glucuronide of desmethy Idichlorvos was found instead, suggesting that the me-
tabolic pathway involved transformation of Trichlorfon to Dichlorvos, followed
by demethylation and glucuronidation (115)
-------
506
Information on the role of metabolism in the activation of Tris(2, 3-dibro-
mopropyl)phosphate (Tris-BP) is not available at the time of writing. As pre-
viously discussed, the compound is mutagenic as such; however, inclusion of
S-9 liver fraction substantially enhances its mutagenic activity.
The mechanism of carcinogenic action of organophosphorus compounds
has not been thoroughly investigated. It is generally assumed that they initi-
ate carcinogenesis by alkylating. key cellular macromolecules, although exper-
imental evidence in support of the hypothesis has yet to be obtained. Dichlor-
vos (which is not considered carcinogenic) is the only organophosphorus insec-
ticide whose reaction with macromolecules has been studied. Lofroth (116)
detected first the presence of 7-methylguanine from calf thymus DNA after in-
cubation with Dichlorvos. This in vitro reaction has been confirmed by Lawley
_e_t al. (117) using salmon sperm DNA and _E^ coli DNA. In addition, other mi-
nor DNA -•na-ethylaiiran ..products including 1-methyladenine, 3-methyl- adenine,
6 14
guanine and cytosine, and O -methylguanine have been found. C-Labeled
7-methylguanine could be.detected in the urine of mice given labeled Dichlor-
vos by i. p. injection (19-39mg/kg) or inhalation (5 mg/kg), indicating in vivo
alkylation. However, the possibility that the label arose via the 1-carbon pool
could not be eliminated. A more recent study by Wooder et_ a_l._ (118) could not
demonstrate methylation of nucleic acids in various tissues after exposure of
CFE strain rats to atmospheres containing practical use concentrations of Di-
chlorvos. It has been suggested that the rapid metabolism of Dichlorvos ren-
ders the rate of in vivo alkylation insignificant.
-------
507
Environmental
5.2.1.4_l.4' ^Y"~Significance. Since organophosphorus compounds are ex-
tensively used, human exposure may occur under occupational settings as well
as in the general population consuming products containing these compounds
as residues.
T rime thy Iphosphate is used mainly as a methylating agent in the synthe-
sis of organophosphorus insecticides, as a gasoline additive for controlling
surface ignition and spark plug fouling, and as a catalyst in the manufacture
of polyesters (119). The annual production of the compound in the United States
has been estimated to be not more than 1, 000 Ib in the recent years (120). There
is no'information available on the extent of human exposure and potential health
hazard.
Organophosphorus compounds and chlorinated hydrocarbons are the two
most important :classes of insecticides used in the United States. Compared
to chlorinated hydrocarbons, o rgan
-------
508
122). There is no epidemiologic evidence to suggest any potential carcinogen-
ic risk of human exposure to occupational levels of organophosphorus insecti-
cides. The Time-Weighted Average Threshold Limit Values (TLV-TWA)
adopted by the American Conference of Governmental and Industrial Hygienists
for air exposure are as follows (in mg compound/m air): Diazinon 0. 1, Di-
chlorvos 1.0, Malathion 10, Methyl Parathion 0. 2, and Parathion 0. 1 (123).
A variety of organophosphorus compounds have been used as flame re-
tardants in the textile and plastics industries. Among these, Tris-BP is no.
doubt the most widely publicized compound. An estimated 10 million pounds
of Tri's-BP was produced in 1976 (120) about 65% of which was applied to poly-
ester and polyacetate fabrics (used for manufacturing children's sleepwear) in
compliance of the U.S. Flammable Fabric Act. The remainder was used in
other materials, such as acrylic fibers and urethane foams. However, the re-
cent findings of (a) potent mutagenicity, (b) animal carcinogenicity, (c) leach-
ing from the fabric (124), and (d) skin absorption (125) of Tris-BP have prompted
a reexamination of the safety of the flame retardant. The problem is further
stressed by the fact that children (as all young mammals) are, generally speak-
ing, more susceptible to chemical carcinogenesis than are adults. In early
1977, in view of the potential risk and the nature of the population at risk, the
Consumer Product Safety Commission decided to barn the sale of fabrics con-
taining Tris-BP (74). Tetrakis(hydroxymethyl)phosphonium chloride (THPC)
is another flame retardant that may represent a potential carcinogenic risk.
THPC may break down to its ingredients, phosphine, formaldehyde and hydro-
chloric acid, and the latter-two products may in turn combine to form the potent
-------
509
carcinogen, bis(chloromethyl)e-'ther, THPC has now been largely replaced by
tetrakis(hydroxymethyl)sulfate (1 26).
In addition to the above, hexamethylphosphoramide (HMPA) is an effec-
tive chemosterilant of the housefly and other insects (127). Also, it has in-
creasingly been used as an industrial solvent in the last few years (6,6). The
annual production of the compound surpassed 1, 000 pounds in 1975 (120). How-
ever, in view of its very potent" inhalational carcinogenicity in rodents, HMPA
is regarded to represent a carcinogenic risk for man(123); threshold limit val-
ues for the compound have yet to be developed.
5.2.1.4.2 Cyclophosphamide. Cyclophosphamide (Cytoxan, Endoxan,
or 2- fjBis(2-chloroethyl)amino ]-tetrahydro-2H-l, 3, 2-oxazaphosphorine 2-ox-
ide), the structure of which is depicted below, is no doubt the most extensive-
ly studied phosphorus-containing alkylating agent. It is a derivative of nitrogen
Insert here Text-Fig. 26
mustard and was originally synthesized in 1958 by Arnold and Bourseaux (161)
with the intention of developing a latent cytostatic agent that generates reac-
tive cytotoxic intermediates after activation by the presumed "phosphoridami-
dase" inside tumor cells. Since the first report (162) of its cytostatic activ-
ity, Cyclophosphamide has been developed into one of the most important, if
not indispensable, cancer chemotherapeutic agents. It has been used, either
-------
. ,, r , u u v,
4 V Cyclophosphamide ,.
H,C - N y R2=-H
5H2C 0=P-N Isophosphamid
UV 7 XCH2CH,CI
H^ - 0 Trophosphamide R,=R2=-CH2CH2CI
Text-Figure 26
-------
510
singly or in combination with other agents, as a cytostatic as well as an im-
munosuppressive agent, in the treatment of various malignant diseases and
nonneoplastic disorders. Interest in the drug is reflected by the enormous
number (estimated to be around 10, 000 in 1975 by Hill; ref. 163) of publications
dealing with the biological properties of the compound. A number of search-
ing reviews on the various aspects of cyclophosphamide have been published
(163-166). This section reviews the literature of cyclophosphamide and its
newly developed analogs, isophosphamide (iphosphamide, ifosfamide) and tro-
phosphamide (trofosfamide), with special emphasis on carcinogenicity and me-
tabolis:m.
Biological
5.2.1.4.2.1 Physico-Chemical and ."VProperties. The physical and chemic-.
al properties of cyclophosphamide have been described (163-166). Cyclophos-
phamide (monohydrate) is a white crystalline solid with a m. p. of 49. 3-53°C.
It is soluble in water (40 mg/ml), benzene, chloroform, dioxane and glycols.
In unbuffered aqueous solutions, cyclophosphamide slowly hydrolyzes to nor-
nitrogen mustard, phosphate and n-propanol., At temperatures above 30°C,
spontaneous hydrolysis of the compound takes place with the liberation of chlor-
ine. Acid-catalyzed hydrolysis of cyclophosphamide gives rise to cytoxyl .
amine. Different other breakdown products are generated under various con-
ditions (rev., 164). Isophosphamide and trophosphamide have properties sim-
ilar to cyclophosphamide. Isophosphamide is more soluble (130 mg/ml) where-
as trophosphamide is less soluble (15 mg/ml) in water than cyclophosphamide
(166). In buffered aqueous solution, the relative stability of the three compounds,
-------
511
(the)
as measured byjrate of chlorine liberation, follows the order: trophospha-
mide >isophosphamide>> cyclophosphamide (167, 168).
Cy clophosphamide has a variety of biological activities including toxic,
•«- TaWc CIX
cytostatic, mutagenic and teratogenic effects. Table CIX summarizes the acute
toxicity data. The acute toxicity of the drug in the rat appears to be strongly
age-dependent; the LD by s. c. administration is 35 mg/kg for one-day-old
rats but increases to 320 mg/kg for 30-day-old rats (169). Investigation of the
perinatal toxicity of the drug in the mouse indicates that the drug may exert
toxic effects before the full development of the ability of the animal to metab-
olize the drug to alkylating intermediate(s) (1 70). The predominant toxic ef-
fects inmice, rats and dogs include leucopenia (171) and marked necrosis of
the tubular and pelvic epithelia of the urinary bladder (172^174); there is rel-
atively little damage of the liver (175; 176). A detailed review of the toxicol-
ogical properties of cyclophosphamide has been presented by Hill (163). Iso-
phosphamide and trophosphamide have toxic properties similar to cyclophos-
phamide; the LE> values in the rat are 250 and 110 mg/kg by i. v. route (1 77)
and 350 and 210 mg/kg by s. c. route (169) for isophosphamide and trophospha-
mide, respectively.
Shortly after its introduction as a cytostatic drug, cyclophosphamide was
found to induce mutation (recessive lethals) in Drosophila melanogaster (1 78).
Since then, numerous studies have been carried out on the mutagenic effects
of the drug in a variety of systems including microorganisms, plants, insects
and mammals; a detailed review of its mutagenic effects has been published
^andj
(166). Cyclophosphamide is not mutagenic perse in microbial systemsTthe
-------
Table CIX
Acute Toxicity of Cyclophosphamide
Species
Mouse
Rat
Guinea pig
Rabbit
Dog
Route
i. p.
oral
i. p.
1. V.
s. c.
1. V.
i. V.
1. V.
oral
LD5Q (mg/kg)
400
360
94
190
180
175
160-196
35 (newborn)
320
400
130
40
44
Reference
Schabei cited in ref. 163
271
171
272
273
\
274
177, 273, 275
169
169
273
273
273
171
-------
VfJ
inclusion of [mammalian metabolic activation system.- is required for activity.
Urinary metabolites from cyclophosphamide-treated rats have been shown to
induce gene conversion in Saccharomyces cerevisiae D4 (1 79). The induction
of mutation has also been'observed in several strains of. bacteria (166, 180,
^thej
181) injhost-rnediated assay . Direct evidence of the involvement of the he-
patic mixed-function oxidase system has been recently provided by Ellenberg-
er and Mohn (182) who demonstrated that cyclophosphamide exerted mutagen-
ic effects toward Escherichia coli 343/11 3 only after incubation with 9iOOOxg
supernatant (post-mitochondrial fraction) of mammalian liver homogenates (S-9 rnix).
The in'duction of dominant lethal mutation has been demonstrated in various
strains of mice of both sexes (32, 183-186); there is some preliminary evidence
of the induction of specific locus mutation in germ cells of male mice (187).
In addition to the above effects, there are a variety of reports of chromosomal
aberrations by cyclophosphamide in cultured human or mammalian cells (rev.,
166, 188-190). The mutagenicity of isophosphamide and trophosphamide has
also been reviewed (166). In comparative investigations in Drosophila, yeast,
E. coli and human peripheral leukocytes, the mutagenic potency follows the
general ranking: trophosphamide >-isophosphamide^cyclophosphamide. The
(*J
inclusion of|microsomal activation system is required for all three compounds
for the expression of activity.
Cyclophosphamide has been reported to exert teratogenic effects in rats
(196-200), mice (201, 202), rabbits (203), and chicks (204). In humans, there
tbutj
is some suggestivejinconclusive ->evidence that cyclophosphamide therapy duf-
ing pregnancy may induce teratogenic effect (205-207). In rodents, the fetuses
-------
513
are most susceptible to the teratogenic effect of the drug during the 10th-15th
day of gestation. The principal effects include encephaloceles, malformations
of the extremities and cervical lesions in rats.whereas mice respond with a
high incidence of malformation of the skeleton.
(^Carcinogenicity.J
5.2.1.4.2.2 ^Y~ The carcinogenicity of cyclophosphamide has been, ex-
tensively tested in mice and rats; the major findings of these studies are sum-
-*- 7IWe CX
marized in Table CX. In the strains of mice used, the lung appears to be the
predominant target organ. Tokuoka (208), Duhig (191) and Shimkin et al. (192)
were among the first to study the carcinogenicity of the drug in the-mouse.
Using'strains A and dd mice, Tokuoka (208) observed an apparent increase in
the tumor incidence after 30 i. p. administrations at a dose of 5 mg/kg. The
tumors mainly developed in the lung of strain A mice and in the lung, liver,
i
testis and mammary gland of strain dd mice; however, because of the relative-
ly high spontaneous tumor incidence in these strains, the increases were not
statistically significant. The induction of pulmonary adenomas was also ob-
served in strain A mice after five i. p. injections of 0. 02 mg cyclophosphamide
(191). A more detailed study was 'carried out by Shimkin e^ al. (1 92) us ing
strain A/J mice. Four different dose levels totaling. 449, 114, 36 and 9 mg/kg
were administered intraperitoneally. Increases in pulmonary tumor incidences
were observed; the increase was statistically significant at the second highest
dose level. Compared to uracil mustard on a molar basis, cyclophosphamide
is about 380 times less potent as a pulmonary carcinogen in the mouse (192).
More recently, Kelly £t a_L_ (193) have evaluated the carc.inogenicity of cy-
clophosphamide in newborn Charles River CD-I mice. Intraperitoneal
-------
Table CX
Carcmogemcity of Cyclophospharmde in Rodents
Species
Mouse,
Mouse,
and
strain
A or A/J
dd
Route
i.
i.
P.
P-
Lung
Lu ng
Principal
, liver,
organs affected
Reference
191.
testis, mammary gland
192, 208
208
Mouse, Charles River
CD-I (newborn)
Mouse, Swiss-Webster
Mouse, XVII/Bln or AWD
Mouse, NMRI
Mouse, NZB/NZW
Rat, BR46
Rat, Sprague-Dawley
i. p.
i. p.
oral
s. c.
8. C.
i. v.
i. v.
i. p.
oral
(not statistically significant)
Lung (weak)
\
Lung, bladder ,
Lung, liver, skin
Mammary gland, ovary, lung, local
Lymphoreticular system, local, lung
Various sites
Various sites
Mammary gland
Bladder, leukemia
193
194
195
209
210-212
209, 213
214
194
276
-------
514
administration of the drug (0. 8, 4. 0 or 20. 0 mg/kg) within 24 hours of birth
and at 3 and 6 days of age elicited only slight increases in pulmonary tumor
incidence. More definite evidence of the carcinogenic effect of cyclophospha-
mide has been provided by Weisburger et al. (194) using Swiss-Webster mice.
Intraperitoneal administration of the drug (3x/week, for 6 months; 12-25
mg/kg/dose) led to the induction of lung tumors in 7/30 male and 10/35 female
mice. Four male mice also had papillomas of the urinary bladder. The re-
sults are statistically significant with p <0. 05 (194). Carcinogenic effects of
cyclophosphamide have also been demonstrated by oral administration of ther-
apeutic doses of the drug. Doses of 0. 1 mg/kg/day in the drinking water (up
to a total dose of 55 mg) to XVII/Bln and AWD mice led to significant increases
in-the incidences of predominantly lung tumors, and to a lesser extent, hepa-
tornas and skin tumors (195). The carcinogenicity of cyclophosphamide has
also been tested by s. c. administration. Marked carcinogenic effects were
demonstrated in female NMRI mice after 52 weekly doses of 26 mg/kg (209).
Over 60% (28/46) of the animals developed tumors. The mammary gland was
the most affected site with 13 tumors (12 carcinomas and 1 benign); however,
tumors also developed in the ovary, lung and at the injection site. For com-
parison, only 3/46 control mice developed tumors (all stem-cell leukemia)
(209). Also in NZB/NZW hybrid mice carcinogenic, effects were observed af-
ter daily s. c. administration of 1 or 8 mg/kg cyclophosphamide for up to 93
weeks. The lymphoreticular system was the most affected tissue whereas lo-
cal and pulmonary tumors were occasionally observed (210-212).
-------
515
Two strains have been used to test the carcinogenicity of cyclophospha-
mide in the rat. In male BR46 rats, an increase in tumor incidence was first
reported by Schm'ahl (213) after injecting intravenously 50 weekly doses (15
mg/kg) of the drug. Some 54% (14/26) of the treated animals developed tumors
(9 malignant and 5 benign) compared to only one benign tumor in 50 control
rats (p<0.001). The carcinogenicity of the drug was confirmed in two addition-
al experiments (209). Male BR46 rats receiving 52 weekly i. v. doses of 1 3
mg/kg had incidences of 11% benign and 17% malignant tumor (compared to 5%
benign, 6% malignant in controls); rats receiving 5 biweekly doses of 33 mg/kg
had incidences of 8% benign and 24% malignant tumors. There were no distinct
target tissues; the spectrum of tumors were: pheochromocytoma, meningioma,
sarcoma of the sternum and inguinal region, hemangioendothelioma of abdominal
cavity, subcutaneous fibromas and lymphoangioma (209). In Sprague-Dawley
rats, the carcinogenicity of cyclophosphamide has been established-in three
separate expe iments, and it appears that the organotropism of the drug is de-
pendent on the route of administration. By ; . i. v. route (weekly injections of
13 mg/kg), Schm'ahl (214) observed a significant increase in the tumor inci-
dence (14/32 in treated rats compared to 6/52 in controls). Again, there were
no distinct target organs; the tumors induced ranged from hemangioendotheli-
omas in various organs to neurogenic sarcoma of the mediastinum, sarcoma
of the heart, leukemia, pheochromocytoma, osteosarcoma of the paranasal si-
nus, and angiosarcoma of the abdomen. In contrast to the above study, by
i. p. route (3 times/week for 6 months, 5-10 mg/kg/dose), the mammary gland was
probably the only target organ of cyclophosphamide in Sprague-Dawley-derived
-------
516
Charles River CD rats. Among the 53 treated female rats, 33 had breast tu-
mors including 9 carcinomas. However, among male rats only 1/50 had a
breast carcinoma (194). In a 2-year study, Sprague-Dawley rats receiving
daily doses of cyclophosphamide (2. 5, 1.25, 0.63, and 0. 31 mg/kg) in drinking
water had malignant tumor incidences of 19/51, 20/53, 15/28 and 7/13, respec-
tively (compared to 3/24 in controls). Among the cyclophosphamide-treated,
tumor-bearing rats, interstitial cell carcinomas of the urinary bladder were
found in 8 rats; 5 rats had papillomas of the bladder and 8 had leukemia. These
findings support the evidence emerging from case reports that patients treated
/
with cyclophosphamide have an increased risk of bladder tumor and leukemia
(see further in "Environmental Significance").
The transplacental carcinogenicity of cyclophosphamide has not been ade-
quately studied. Working with mice, Roschlau and Justus (195) reported that
the offspring of mothers treated with cyclophosphamide during pregnancy have
a higher incidence of tumors than the offspring from untreated mothers. How-
ever, the statistical significance of the study could not be evaluated. Cyclo-
phosphamide has also been shown to bring about, following microsomal activ-
ation, in vitro transformation of C3H/10TjCL8 cells (190). The transformed
cells could be scored as transformation foci on tissue culture plates and shown
to produce sarcomas when injected into syngenic hosts (190).
In contrast to cyclophosphamide, there are very few studies of the car- •
cinogenicity of isophosphamide. Using strain A mice, Stoner et al. (215) found
isophosphamide to be a relatively weak pulmonary carcinogen. The total i. p.
dose found to elicit significant carcinogenic response ranged from 0. 45 to 1.3
-------
517
g/kg. On the molar basis, isophosphamide is about 200 times less potent than
uracil mustard (215). A more detailed study has recently .been carried out un-
der the Carcinogenesis Testing Program of the U.S. National Cancer Institute.
Sprague-Dawley. rats and B6C3F. mice were used. Isophosphamide could not
be clearly shown to induce tumors in male animals of either species. In fe-
male animals, however, cancer of the uterus (leiomyosarcoma) as well as
mammary fibroadenoma occurred with high incidence in isophosphamide-treated
rats whereas mice responded with a high incidence of malignant lymphoma of
the hematopoietic system (216). There is no information on the carcinogen-
icity of trophosphamide.
Possible
— ^^^_ -^
5.2.1.4.2..3: Metabolism.aad . I . Mechanism of Action. Cyclophosphamide was
originally designed as a latent cytotoxic agent to be enz.ymatically activated j_n
vivo to alkylating: intermediates ideally inside the neoplastic tissues (161).
Metabolic studies carried out in a wide range of animal species indicate that
the liver is probably the only site where activation of the drug occurs to a sig-
nificant extent. The metabolic fate of Cyclophosphamide in animals has been
thoroughly reviewed in 1974 by Torkelson ej: aJ^ (164); since then, new findings
««- ?>
have been reported. Figure 25 summarizes the proposed metabolic pathways
leading to the formation of major metabolites. The first step of metabolism is
assumed to involve mixed-function oxidase catalyzed -hydroxylation on carbon-4
of the ring. The 4-hydroxy-cyclophosphamide thus formed is very unstable
and readily tautomeriz.es to its open-chain aldehyde form, aldophosphamide
[2-formylethyl N, N-bis (2-chloroethyl)phosphorodiamidatei]i Aldophospha-
mide may, in turn, either be further oxidized by aldehyde oxidase to the
-------
CICH2CH2
Cvclophosphomide
/
CICH2CH2 N-?
N_p=0\ _
CICHjCH;, 0—/
4-KetocYclophosphamide
t
CICH2CH2 NH2
N— PN=0
Corboxyphosphomide
oldehydeoxidase
H
CICH2CH2 ^
N-R-0
OH
CICH2CH2
4-Hvdroxvcyclophosphomide
tautomerirotion CICH2CH2 mz
, N—Px=0
CICH2CH2
Aldophosphomide
v* . NH2"
V—P(=0 + CH2=CH-CHO
OH
Acrolein
Phosphoromide mustard
CICH2CH2
N-H
CICH2CH2
Nornitroqen mustard
Figure 25
-------
LEGEND TO FIGURE 25
Fig. 25. Major metabolic pathways of Cyclophosphamide.
-------
518
carboxyphosphamide [2-car boxy ethyl N, N-bis(2-chloroethyl)phosphorodiami-
date}]or be degraded, either enzymatically or chemically, to phosphoramide
mustard [N, N-bis(2-chloroethyl)phosphorodiamidic acid?]., acrolein and norni-
trogen mustard. 4-Ketocyclophosphamide, another urinary metabolite, may
arise either by direct oxidation of 4-hydroxycyclophosphamide or by cycliza-
tion of carboxyphosphamide.
The involvement of microsomal mixed-function oxidases in the metabol-
ism of cyclophosphamide has been amply demonstrated both by _in vivo and in.
vitro studies. In vivo studies by various investigators (217-221) showed that
pretreatment of animals with phenobarbital (a typical ind.ucer) enhances while
typical inhibitors .such as 2-rdiethylaminoethyl 2, 2-diphenylvalerate (SKF 525A)
and 2, 4-dichloro-6 -phenyl-phenoxyethyl diethylamine (DPEA) inhibit the me-
tabolic activation of cyclophosphamide. In vitro studies indicated that the en-
zyme system capable of metabolizing cyclophosphamide is located almost ex-
clusively in the microsomes, that it requires NADPH and oxygen for activity,
and is inhibited by carbon monoxide, by inhibitors of microsomal electron
transport chain and by typical substrates of mixed-function oxidases; it is en-
hanced by phenobarbital pretreatment (217, 222-225).
Tissue distribution studies showed that only the liver and, to a much less'
er extent, the lung contain cyclophosphamide-activating enzyme activity; oth-
er tissues such as kidney, spleen, muscle-, brain and intestine, and tumor
tissues are devoid of this activity (225). 4-Hydroxycyclophosphamide has been
identified as a metabolite in the incubation mixture (226),whereas aldophospha-
mide has been detected in m. vitro systems as well as in the plasma and urine
-------
519
of rats (225, 227). The involvement of aldehyde oxidase in the oxidation of al-'
dophosphamide to carboxyphosphamide has been clearly demonstrated. The
conversion is catalyzed by an enzyme present in the cytosol (225, 227); the en-
zyme can be replaced by purified preparation of aldehyde oxidase (225). Car-
boxyphosphamide has been detected in the plasma and identified as the princi-
pal urinary metabolite in various animal species (227-233) and in humans (229).
4-Ketocyclophosphamide has been found in hi vitro systems!£34) and as a mi-
nor metabolite in the urine of dogs and humans (228, 229, 235). It could arise
by oxidation of 4-hydroxycyclophosphamide or by cyclization of carboxyphos-
phami'de. However, the latter pathway is not supported by the study of Taka-
mizawa et al. (230) who were unable to detect any interconversion between car-
boxyphosphamide and 4-ketocyclophosphamide in the rabbit. Phos phoramide
mustard and acrolein have been postulated to arise from the degradation of al-
dophosphamide although the nature of the degradation is not clear. Phos- -.
phoramide mustard canbe detected as an in vitro metabolite (226, 236) as well
as in the plasma of patients receiving cyclophosphamide (237). Further degrad-
ation of phosphoramide mustard yields nornitrogen.mustard which has been
identified as an urinary metabolite (238, 239). Acrolein is formed in in vitro
metabolism of in chemical oxidation of cyclophosphamide (233, 240). Kaye and
Young (241) have provided indirect evidence of acroiein as a possible metab-
olite of cyclophosphamide in humans. In fact, 3-hydroxypropylmercapturic
acid (see Fig. 26) a known metabolite of acrolein in the rat (242), has been
detected in the urine of patients receiving the drug.
-------
520
In addition to the above metabolites, a variety of minor metabolites (Fig.
26) have been identified in the urine of various animal species. These are
3 -hydroxypropionic acid and 3-hydroxypropionamide in the dog (229), 2- (j(2-chlor-
oethyl)amino]-tetrahydro-2H-l, 3, 2-oxazaphosphorine-2-oxide and': 3-phos->. .
phorylpropionic acid in the sheep (232), and 3-(2-chloroethyl)-oxazolidone and
1 , 4-di-(2-chloroethyl)piperazine in humans (239). There is also some evi-
dence that N-(2-chloroethyl)-ethyleneimine is a minor metabolite excreted in
the '^expired', air in very small amounts by rats (243). A number of breakdown
products could arise as a result of hydrolysis of cyclophosphamide (rev., 164),
and some of these may be of biological significance. Cytoxyl amine, for ex-
ample, has been detected in the serum of rats and urine of humans given the
parent compound (244).
The metabolic fate of isophosphamide closely resembles that of cyclo-
phosphamide. Metabolites corresponding to those of cyclophosphamide (includ-
ing acrolein) have been detected (226, 245-248). Trophosphamide is expected
to be metabolized in a similar manner (166, 249).
The main thrust of the metabolic studies of cyclophosphamide has been
to identify active intermediates accounting for the cytostatic activity of the
drug. Relatively little effort has been devoted to identify carcinogenic inter-
mediates. However, in view of the often-observed ambivalence of cytostatic
therapy, it is likely that a similar mechanism may operate. A recent study
by Benedict ^t a_l._ (1 90) indicates that the in vitro oncogenic transformation of
CSH/lOTjCLS cells by cyclophosphamide requires microsomal mixed-function
oxidases, NADPH and oxygen for activation.
-------
NH-CO-CH3
CH-CH2-S-CH2CH2CH2OH
COOH
3-Hydroxypropvlmercopturic odd
HO-CH2-CH2-COOH
3-Hydroxypropionic acid
HO-CH2-CH2-CONH2
3-Hvdroxypropionamide
/N
CICH2CH2-NH-P/=0
0
2r [(2-chloroethyl)omino]-tetra-
hydro-2H-1.2.3-oxazophosphorine
2-oxide
/0H
HO-PN=O
CICH2CH2
3-Phosphorylpropionic ocid 3-(2-chloroethyl)-oxazolidone
CICH2CH2-N N-OfeCH£l [>I-CH2CH2CI
1.4-Di-(2-chloroethyl)piperazine N-(2-chloroethyl)ethyleneimine
CICH2C^2 OH
N-P=0
CICH2CH2 (
Cytoxyl omine
Figure 26
-------
LEGEND TO FIGURE 26
Fig. 26. Some minor metabolites of Cyclophosphamide.
-------
521
It appears to be generally accepted that the two principal urinary me-
tabolites, carboxyphosphamide and 4-ketocyclophosphamide are "detoxified"
;:-..-, . metabolites.and do not represent active forms of the drug (225, 229, 234).
The intermediates postulated to be possible active cytostatic intermediates in-
clude 4-hydroxycyclophosphamide, aldophosphamide, . phosphoramide mustard,
and nornitrogen mustard. Although acrolein is believed by some (233) to be
related to the.therapeutic effect of cyclophosphamide, others (250) consider
that it is unlikely to play a major role in the cytostatic action but may contri-
bute to some of the toxic effects of the drug. The potential carcinogenicity of
acrolein will be discussed in Section 5. 2. 1. 7.
The alkylating properties of potentially active intermediates have been
studied. Sladek (227) presented evidence that an aldehyde with alkylating ac-
tivity is the primary in vitro metabolite of cyclophosphamide as well as the
primary in vivo metabolite in the blood and urine of treated rats. The alde-
hyde is capable of alkylating 4-(p_-nitrobenzyl)pyridine, can be trapped as semi-
carbazone by the addition of semicarbazide, and was concluded to be aldophos-
phamide. The ability of 4-hydroxycyclophosphamide, phosphoramide mustard
and nornitrogen mustard to alkylate 4-(jp_-nitrobenzyl)pyridine has been com-
pared by Struck e_t a_L_ (251). Under the conditions used, 4-hydroxycyclophos-
phamide was inactive; both phosphoramide mustard and nornitrogen mustard
were active alkylators at acidic pH but only phosphoramide mustard retained
activity at physiological pH. The authors (251) postulated that phosphoramide
mustard is physiologically the most significant cytostatic intermediate gener-
ated by cyclophosphamide metabolism. This conclusion is strongly supported
-------
522
by the finding of Colvin et al. (250) who confirmed the potent alkylating ability
of phosphoramide mustard at physiological pH and showed in addition, by mass
spectrometry, the binding of the whole molecule (not its degradative product,
nornitrogen mustard) to the nucleophile, ethanethiol. The postulated reactive
<*$— fig • &
intermediates are depicted in Fig. 27. It should be noted, however, that phos-
phoramide mustard alone does not account for all the cytostatic effects of cy-
clophosphamide (252); Colven jst a_L_ (250) pointed out that the assay techniques
used by Struck et al. (251) could not preclude the existence of low but biolog-
ically significant levels of alkylation and implied that either 4-hydroxycyclo-
phosphamide is the key metabolite to enter target cells or that other addition-
al factors are involved. Further studies are needed to elucidate whether any .
of these alkylating intermediates or some other minor metabolites are related
to the carcinogenic effect of the drug.
Envi ronmental
• —~^^^ -—
5.2.1.4.2.4 ^ir Significance. Human exposure to cyclophosphamide is
probably limited exclusively to iatrogenic sources. Cyclophosphamide is one
/•'"••
s* : • '-..-'
of the most extensively used chemotherapeutic agents in the treatment of vari-
ous malignancies such as myeloma, leukemia, Hodgkin's disease, and ovari-
an, mammary and pulmonary tumors. It has also been used in combination with
other drugs in the therapy of lymphoreticular and a variety of other neoplasms
(165, 253, 254). In addition to its antitumor activity, cyclophosphamide .is an
effective immunosuppressive agent and is increasingly used after organ trans-
plantation (255). Other non-neoplastic diseases for which cyclophosphamide
has been used or clinically tested is the treatment of rheumatoid arthritis (256),
-------
H-N
NH
/
\
CH2CH2CI
1© XCH,CH,CI
H-N
,/CH2
0
NH
le
Figure 27
-------
. LEGEND TO FIGURE 27
Fig. 27. Possible reactive .intermediates formed from phospho.ramide
mustard. •
-------
523
chronic hepatitis (257), systemic lupus erythematosus (258), idiopathic neph-
rotic syndrome in children (259) and a number of others (165, 260).
A variety of antineoplastic drugs have been found to possess carcino-
genic activity (rev., 26l, 262). The potential cancer risk of patients receiving
cyclophosphamide therapy has been reviewed and evaluated by LARC (165),, and
by Schmahl ^t al. (262). There have been at least 40 cases of malignant tu-
mors reported in patients treated with cyclophosphamide for various diseases,
and in nearly half of these cases, cyclophosphamide was the only chemother-
apeutic agent administered. There is increasing evidence to suggest that ther-
apeutic use of the drug may present an increased risk of cancer of the urinary
bladder. Seven cases of the development of bladder carcinomas as second pri-
mary tumors have been reported in cancer patients receiving cyclophospha-
!
mide as the sole ;chemotherapeutic agent (263-265). In addition, seven other
cases of bladder tumors have been noted in patients receiving combination che-
motherapy in which cyclophosphamide was one of the drugs (264-267; Laursen,
cited in ref. 262). Schmahl e_t a_l._ (262) have pointed out that the reported and
published cases may represent only a fraction of the total number of cyclophos-
phamide-related bladder cancers. Of the cases in which the total applied dose
of the drug could be estimated, the total doses were found to range from 100-250
g. The mean latent period for bladde'r cancer was 7>years (262). In addition
to bladder cancer, Gy=clophosphamide could be related to the development of 3
cases of leukemia (265, 268), and reticulosarcomas (269, 270) in patients re-
ceiving the drug for therapy of malignant diseases, and 2 cases of reticulum
cell sarcoma and 1 case each of chronic lymphocytic leukemia, Hodgkin's
-------
524
disease, cervical cancer, malignant melanoma, astrocytoma and glioblastoma
in patients treated for nephrotic syndrome and other nonneoplastic disorders
(rev., 165). Whether or not these tumors are directly related to the drug treat-
ment could not be assessed because of the insufficiency of data. Nevertheless,
the increasing number of reports of possible association between bladder can-
." -\
/•''"" "'.
cer and cyclophosphamide is alarming and suggests that the drug should be re-
garded as a human carcinogen.
-------
525
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544b
SOURCE BOOKS AND MAJOR REVIEWS FOR SECTION 5.2.1.4
1. Fest, C., and Schmidt, K.J.: "The Chemistry of Organo-phosphorus
Pesticides - Reactivity, Synthesis, Mode of Action, Toxicology"
Springer, New York, 1973, 339 pp.
2. Eto, M.: "Organophosphorus Pesticides. Organic and Biological
Chemistry" CRC Press, Cleveland,_0hio, 1974, 386 pp.
3. Bedford, C.T., and Robinson, J.: Xenobiotica 2, 307-337 (1972).
4. Wright, A.S., Hutson, D.H., and Wooder, M.F.: Arch. Toxicoi. 42,
1-18 (1979).
5. Bull, D.L.: Residue Rev'.' 43, 1-22 (1972).
6. Wild, D.: Mutation Res. 32, 133-150 (1975).
7. Hill, D.L.: "Review of Cyclophosphamide" C.C. Thomas,
Springfield, Illinois, 1975, 340 pp.
s
8. International Agency for Research on Cancer: "Some Aziridines, N-
, S- & 0-Mustards and Selenium" IARC Monographs on Evaluation of
Carcinogenic Risk of Chemicals to Man, Vol. 9, Lyon, France,
1975, 268 pp.
9. Mohn, G.R., and Ellenberger, J.: Mutation Res. 32, 331-360
(1976).
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NOTES ADDED AFTER COMPLETION OF SECTION 5.2.1.4
The mutagenicity of 11 bis- or tris-(haloalkyl)-phosphates has been
tested by Nakanura _et_ jil_, (1) using the Ames Salmonella test. Most of the
compounds are base-pair substitution mutagens and are more active in the
presence of metabolic activation system (S-9 mix). Several structure-activity
relationships have been noted: (a) bromoalkyl compounds are more nutagenic
than chloroalkyl compounds, (b) haloethyl derivatives are more mutagenic than
V-halopropyl derivatives, (c) compounds with p,J-dihalo-substitutions are
particularly potent, and (d) branching of the alkyl chain reduces the muta-
genicity. These relationships do not all apply to haloalkanols suggesting
that tris-(haloalkyl)-phosphates are not necessarily activated via halo-
alkanols. MacGregor et_ al_. (2) have tested the mutagenicity of 9 organophos-
phorus flame retardants. Only tris-(2,3-dibromopropyl) phosphate was muta-
genic. All the other phosphonium, phosphine, phosphine oxide, and phosphoric
amide derivatives (none of which contain halogenated side-chains) did not
exhibit mutagenic activity. McCoy et al. (3) have recently found that illumi-
nation of tris-(2,3-dibromopropyl)-phosphate xvith visible light in the
presence of riboflavin resulted in the formation of a stable intermediate with
greatly enhanced mutagenic (Ames test) and DMA-modifying activities. Since
illumination of riboflavin is known to generate short-lived singlet oxygen, it
is assumed that the genotoxic intermediate results from a reaction between the
flame retardant and singlet oxygen. This finding may be of environmental
significance because the polluted urban atmosphef*e is conducive to the genera-
tion of singlet oxygen. Two organophosphorus pesticides, dimethoate and
phosphamidon ('see Figure 20), have been found to be highly mutagenic in the
micronucleus test (using mouse bone marrow cells) and in the host-mediated
test (using Salmonella typhimurium GA6). In the same study, two organo-
1
-------
chlorine (aldrin and enddsulfan), a carbamate (carbaryl) and a mercuric
(ceresan) pesticides are not significantly active (4). The mutagenicity
studies of Dichlorvos have recently been reviewed by the International
Commission for Protection against Environmental Mutagens and Carcinogens
(5). A series of derivatives of Diazinon (see Figure 20), pyridyl phosphoro-
thioate and other organophosphorus compounds have been tested for terato-
genicity using chick embryos, and some interesting structure-activity
relationships have been described. The position, size, and branching of
N-heterocyclic ring substituents in the organophosphorus compounds all play a
role in determining the potency and the type of teratogenic effects (6).
No new animal carcinogenicity studies have been found in the literature
since the completion of Section 5.2.1.4. The evidence of possible carcino-
genicity of Dichlorvos has been reviewed by an international study group
(5). Two additional cases of transitional cell carcinoma of the bladder have
been observed among 54 patients treated with oral doses, of cyclophosphamide
for systemic lupus erythematosus and rheumatoid arthritis. The tumors were
detected 28 and 60 months after the withdrawal of the drug (7).
A possible reaction mechanism for carcinogenesis by tetrachlorvinphos
(Rebon) has been pointed out by Burchfield and Storrs (8). Tetrachlorovinphos
may be readily hydrolyzed to yield dimethylphosphate (DMP) and a vinyl
alcohol, which tautomerizes to yield a highly reactive ^-chloroacetophenone
0
HO-C=CH-CI
CH,0 0
C-CH-C1
+H20
Cl
-------
derivative. The u) -chlorine atom of the intermediate is highly reactive due
to its promimity to the electron-rich carbonyl group, which in turn is in
resonance with the "ft electrons of the benzene ring. The intermediate may'
react with nucleophilic sites in macromolecules to initiate carcinogenesis.
The three chlorine atoms on the benzene ring are relatively inactive; they
could enhance the carcinogenicity of the compound by altering its physico-
chemical properties. The in vivo binding of tris-(2,3-dibromopropyl)-
phosphate (TRIS) and tris-(l,3-dichloro-2-propyl)-phosphate (Fyrol FR-2) in
the mouse has been studied by Morales and Matthews (9). The highest level of
binding was the kidney (the main target tissue) for TRIS and the liver for
Fyrol FR-2. The covalent binding to DNA was substantially higher for TRIS
than that for Fyrol FR-2. The in vitro reaction of phosphoramide mustard, a
metabolite of cyclophosphamide, with nucleosides (guanosine or deoxyguanosine)
has been demonstrated by Mehta £t_jil_« (10). The major reaction product was
phosphoramide mustard with one arm having reacted with the nucleoside at the
7-position. In the DNA, such an adduct is expected to be much less stable
than 7-methylguanine and may contribute to cytotoxic, mutagenic or carcino-
genic action by causing damage by strand scission.
References for Section 5.2.1.4 Update
1, Nakamura, A., Tateno, N., Kojima, S., Kaniwa., M., and Kawamura, T.:
Mutat. Res. 66, 373 (1979).
2. MacGregor, J. T., Diamond, M. J., Mazzeno, L. W. Jr., and Friedman, M.
Environ. Mutagenesis 2, 405 (1980).
-------
3. McCoy, E., Haynan, J., and Rosenkranz, H. S.: Mutat. Res. 77, 209
(1980).
4. Usha Rani, M. V., Reddi, 0. So, and Reddy, P. P.: Bull. Environ. Contain.
Toxicol. 25, 277 (1980).
5. Ramel, C., Drake, J., and Sugimura, T.: Mutat. Res. 76, 297 (1980).
6. Eto, M., Seifert, J., Engel, J. L., and Casida, J. E.: Toxicol. Appl.
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