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

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                          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

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                                                                          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

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                           LEGEND TO FIGURE 20






      Fig. 20.  Structural formulas of organophosphorus compounds that have




been tested for carcinogenicity.  (See Table CVIII for complete chemical




names.)

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                             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.

-------
                                                                       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

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                                                                          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,

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                            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

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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

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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

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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.

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                                                                         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.

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                                                                        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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-------
                                                                            526
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                        » *
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-------
                                                                         527
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                                                                          540
211.  Walker, S. E., and Bole, G.G., Jr.;  J. Lab. Clin.  Med. 82, 619(1973).





212.  Walker, S. E. , and Bole, G.G., Jr.:  A rthritis-Rheum.  16.  137(1973).





213.  Schmahl,  D. :  Deutsch Med.  Wschr. 92, 1150(1967).





214.  Schmahl,  D. :  Z. Krebsforsch. 81, 211 (1974).





215.  Stoner,  G. D. , Shimkin, M. B. , Kniazeff,  A.J., Weisburger,  J. H. ,





      Weisburger,  E. K. , and Gori, G. B. :  Cancer Res. 33, 3069(1973).





216.  NCI:  "Bioassay of Isophosphamide for Possible Carcinogenicity. "  NCI





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217. -'Brock, N. , and Hohorst. H.-J. : Arzneim.-Forsch.  13,  1021  (1963).





218.  Brock, N. :  Cancer Chemotherap. Rep. 51, 315(1967).





219.  Rauen, H. M. , and Kramer, K. P. : A rzneim. -Forsch. 14, 1066(1964).





220.  Dixon, R. L. :  Proc. Soc. Expbl.  Biol. Med.  .127, 1151 (1968).





221.  Hart,  L.G.,  andAdamson, R.H.: Arch.  Intern. Pharmaeodyn. :1-80.  39.1





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222.  Brock, N. , and Hohorst, H.-J.:  Cancer 20.  900(1967).





223.  Cohen, J. L. , and Jao, J.Y.:  J^ Pharmacol.  Exptl.  Ther.  174,  206





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224.  Sladek,  N. E. :  Cancer Res. 31, 901 (1971).





225.  Hill, D. L. ,  Laster,  W. R. ,  Jr.,  and Struck, 'R. F. :   Cancer Res. 32,





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226.  Connors,  T.A.,  Cox, P.'J.,  Farmer,  P. B. , Foster,  A.B., and Jarman,




      M. : Biochem. Pharmacol. 23, 115(1974).




227.  Sladek,  N. E. :  Cancer Res. 33, 651 (1973).

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                                                                             541
228.  Hill, D. L. ,  Kirk, M. C. , and Struck, R. F. :  J.  Am.  Chem. Soc. 92,





      3207 (1970).





229.  Struck,  R.F., Kirk, M. C. ,  Mellett, L. B. ,  Dareer,  S. E. ,  and Hill,





      D. L. :  Mol. Pharmacol.  7.  519(1971).





230.  Takamizawa, A. ,,. Tochino,  Y. , Hamashima, Y., and Iwata, T. :





      Chem.-Pharm. Bull. 20,  1612(1972).





231.  Norpoth, J. K. ,  Knippschild, J., Witting,  U. , and Rauen, H. M. : Ex-




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232.  Bakke,  I.E.,  Feil,  V. J. , Fjelstul,  C. E.  , and Thacker,  E. J. :   J. Agr.





     -•Food Chem. 20,  384 (1972).





233.  Alarcon, R.A.,  and Meienhofer, J. :  Nature New Biol. 233, 250(1971).





234.  Hohorst, H.-J. ,  Ziemann, A.,  and Brock, N. :  Arzneim.-Forsch. 21,
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      1254 (1971').




235.  Mellett, L. B. , El Dareer,  S. M. ,  Struck, R. F.  , and Hill,  D. L. :  Univ.





      Mich. Med.  Ctr. J.  3_6_, 245 (1970).





236.  Colvin,  M. , Padgett, C.A., and Fenselau,  C. :   Cancer Res. 33, 915





      (1973).





237.  Fenselau, C. , Kan,  M.-N. N. ,  Billets,  S. ,  and  Colvin, M.  : Cancer Res.





      35,  1453 (1975).




238.  Hohorst, H.-J.,  Ziemann, A.,  and Brock, N: :  Arzneim.-Forsch. 15,





      432 (1965).                                                 .    /:;;;,    •'."




239.  Cox, P. J.,  and  Levin,  L. :   Eiochem. Pharmacol.  24,  1233(1975).





240.  Thomson, M. ,  and Colvin,  M. :  Cancer Res.  34, 981 (1974).





241.  Kaye, C. M. , and Young,  L. :  Biochem. Soc.  Trans.  2, 308(1974).

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                                                                          542
242.  Kaye, C. M. :  Biochem.  J.  134.  1093(1973).





243.  Rauen, H. M. , Golvinsky, E. ,  and Norpoth,  K. :  Suvremenna Med. 19.





      106 (1968).





244.  Brock, N. ,  andHohorst, H.-J. :  Oncol. Radiol.  (Bucharest) 6,  49(1967).





245.  Alarcon,  R.A.,  Meienhofer, J. , and Altherton,  E. :  Cancer Res. 32,





      2519 (1972).





246.  Allen,  L. M. , and Creaven, P. J. :  Cancer Chemother.  Rep. 56,  603





      (1972).





247.  Brock, N. ,  Hoefer-Janker, H. ,  Hohorst,  H.-J., Scheef,  W. ,  Schneid-





      er, B. , and Wolf, H. C. : A rzneim.-Forsch.  23.,  1 (1973).





248.  Hill, D. L. ,  Laster, W. R. , Jr., Kirk, M. C. , El Dareer,  S. ,  and Struck,





      R.F.:  Cancer Res. 33,  1016(1973).





249.  Wheeler,  G. P. :  Transpl. Proc. 3, 1167(1973).




250.  Colvin, M. ,  Brundrett,  R. B. ,  Kan, M.-N.N., Jardine, I. , and Fense-





      lau,  C. :  Cancer Res. 36, 1121 (1976).





251.  Struck, R. F. , Kirk,  M. C. ,  Witt, M. H. ,  and Laster, W. R. , Jr.:	.•--,





      Biomed.  Mass Spectrometiy 2_, 46 (1975).





252.  Colvin, M. :  Proc. Am. Assoc.  Cancer Res.  15, 70(1974).





253.  Livingston,  R. B. ,  and Carter, S. K. :  Iri "Single Agents in Cancer Che-





      motherapy. "  Plenum Press, New York,  1970; p.  25.





254.  Greenwald,  E. S. :  In "Cancer  Chemotherapy."  Hans Huber, Bern,  1973,





      2nd edn. , p.  121.





255.  Starzl, T. E., Groth, C. G., Putnam,  C. W., Gorman, J., Halgrimson,  C. G.,





      Penn, I.,  Husberg, B., Gustafsson, A.,  Cascardo, S., Geis, P., and Iwat-





      suki, S.:  Transpl. Proc. 5, 511  (1973).

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                                                                            543
256.  Cooperating Clinics Committee of the American Rheumatism Association:


      New Engl.  J.  Med. 283, 883 (1970).


257.  Naccarato, R. ,  Farini,  R. ,  Chiaramonte,  M. ,  Fagiolo, .iU. ,  and Stur-


      niolo,  G. C. :  Postgrad. Med.  J. 50,  16(1974).


258.  Mackay, I. R. , Mathews, J.D.,  Toth,  T.B.H. ,  Baker, H. W. G. ,  and


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260.  Steinberg,  A.D. ,  Plotz, P. H. , Wolff, S. M. ,  Wong,  V. G. , Agus,  S. G. ,


      and Decker, J. L. :  Ann. Int.  Med. 76, 619(1972).


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      Therapy, "  Recent Results Cancer Res. , vol.  52.  Springer-Verlag, Ber-


      lin-Heidelberg,  1975,' 238 pp.
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262.  Schma'hl, D. ,  Thomas,  C. , and Auer, R. :  "latrogenic Carcinogenesis. "


      Springer-Verlag,  Berlin-Heidelberg,  1977, 120pp.
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263.  Worth,  P. H. L. :  Brit.  Med.  J.  3, 182(1971).


264.  Dale, G.A., and Smith, R. B. : J.  Urol.  112, 603(1974).


265.  Wall, R. L. , and Clausen, K. P. : New Engl. J.  Med.  293.  271  (1975).


266. .Reis, H. E. , Hossfeld,  D. K. ,  and Stier,  H. W. :  Med. Welt 2, 2411


      (1969).


267.  Rupprecht, L. ,  and Blessing,  M. H. :  DeutschMed.  Wschr. 98, 1663


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268.  Karchmer, R. K. , Amare, M. , Larsen, W. E. ,  Mallouk, A.G., and


      Caldwell,  G.G. :  Cancer 33,  1103(1974).                .          .


269.  Holt, J.M. , and Robb-Smith, A.H. T. :  J.  Clin.  Path. 26,  649(1973).

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                                                                            544a
270.  Mundy, G.R., and Baikie, A. G. :  Med. J.  Austr.  I,  1240(1973).




271.  VonArdenne, M. ,  Reitnauer,  P. G. , and Rohde, K. :  Arch. Geschwulst-




      forsch. 38, 15 (1971).




272.  Syrkin, A.B., and Zaitzeva,  L.A.: .Neoplasma 17, 377(1970).




273.  Brock, N. , and Wilmanns,  H. :  Deutsch med. Wschr.  83, 453(1958).




274.  Sakurai,  Y. :  NCI Monograph 16.  207(1964).




275.  Scherf, H. R. , Kruger,  C. , and Karsten, C. :  A rzneim.-Forsch. 20,




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276.  Habs, M. , and Schmahl, D. :   Proc. Am. Assoc.  Cancer Res. 19, 14




      (1978).

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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.




     Pharmacol.  54, 20 (1980).








 7.   Plotz,  P. H.,  Klippel,  J. H., Decker, J. L., Grauman, D., Wolff, B.,




     Brown,  B. C.,  and Rutt, G.:  Ann. Intern.  Med.91, 221 (1979).









 8.   Burchfield, H. P., and Storrs, E. E.:  Organohalogen Carcinogens.   In




     "Environmental Cancer"  (H. F. Kraybill and M.  A. Mehlman, eds.) Chapter




     10,  Wiley,  New York, 1977, p. 319.









 9.   Morales,  N. M., and Matthews, H. B.:  Bull. Environ. Contain. Toxicol. 25,




     34 (1980).








10.   Mehta,  J. R.,  Przybylski, M., and Ludlum, D. B.:  Cancer Res. 40, 4183




     (1980).                     .                ^

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