Document GmeEBqZgRmdn6Q9wvoq4MDXm

6- - A summary of the results obtained is shown in the Table. Phosphoramide (II) and HMPA (I) gave positive results on each occasion and the trianilide (V) and the thioamide (VI) gave negative results. The results shown in Fig. ii for dimethyInitrosamine and diphenylnitrosamine were obtained under similar conditions and are recorded here for comparison. Full details of these experiments will appear elsewhere (Styles, in preparation). DISCUSSION The object of the present study was to define an appropriate short-term test with which to evaluate the potential carcinogenicity of chemical relatives of HMPA, and further, to attempt to find a chemical explanation for its carcinogenicity. The results shown in Table I and Table II clearly establish that only the cell transformation test meets the test selection criteria described earlier and it is, therefore, the preferred assay for this class of compounds. Further, the data obtained provides a basis for understanding the in vivo and in vitro effects so far observed. Consideration will first be given to the possible ways by which the carcinogenicity of the HMPA could be mediated. First, an intermediate alkylating species might arise from the dimethylamino group which is present in this compound. This functional group has been directly implicated in the carcinogenic activation of several other carcinogens such as dimethyInitrosamine, dimethylcarbamoyl chloride and 4-dimethylaminoazobenzene. Alternatively, the in vivo effect may derive from purely physical interactions associated with the unusual lipid-aqueous solvent properties of HMPA (Lloyd 1975) which is a liquid. In this connection, it may be significant that the primary tumours observed in the rat inhalation study occurred in the immediate nasal region. Both of these possible methods of action are prevented in phosphoric trianilide (V) which is a solid (mp 212 - 215) and which is also devoid of potential alkylating groups. The positive effects given by HMPA in these two short-term tests together with the fact that it produces mutagenic effects in Drosophila (Banes and Sram 1969) supports the view that this compound, or an active metabolite derived from it, reacts chemically with genetic material. Further, the negative response observed in the present study for the phenyl analogue (V)fwhich is devoid of methyl groups, implicates the methyl groups ucc 033252 II -7- in this process. These results equally make the non-specific physical hypothesis less plausible as the functionally related dipolar solvents dimethylformamide (DMF) and dimethylsulphoxide (DMSO) are without effect in both tests (Purchase et al 1976, 1977 in preparation). The difference in test response observed when comparing HMPA with the phenyl analogue (V) (Figs. Ia and Ic) is similar to that found when comparing the test response for the carcinogenic biological alkylating agent, dimethylnitrosamine (VII) with its non-carcinogenic, phenylated analogue, diphenylnitrosamine (VIII) (Fig. II>. The analogy between these two classes of carcinogen is further strengthened by the fact that the erratic response given by the Ames test for HMPA has also been observed for dimethylnitrosamine (Bartsch et al 1976, Purchase et al 1977, in preparation) when using the same plate incorporation assay technique. It may be that both of these erratic responses are due to variations in the rate of formation or effective half life of a common alkylating species. Such variations could result from changes in the enzyme profile or balance between different batches of microsomes or from slight changes in the chemical environment in the test medium. There is, therefore, sufficient evidence to initially assume that the broad structural requirements for carcinogenicity observed for the nitrosamine carcinogens apply equally to phosphoric amides. The structure-activity relationships observed for nitrosamines have been reviewed (Druckrey 1975) and the major requirement for activity is that the amine nitrogen atom should carry at least one alkyl group having a free a-position potentially capable of undergoing metabolic Ct-hydroxylation. On this basis, it is possible to predict that hexaethylphosphoramide (IV) and phosphoric trimorpholide (X), for example, would both have carcinogenic potential by analogy with the carcinogens diethylnitrosamine and nitrosomorpholine (X) respectively. Clearly, the first step in the evaluation of such compounds would be to submit them to the cell transformation test with HMPA (I) as the positive control. UCC 033253 -8- The validity of the above chemical class analogy is partially confirmed by the observation that HMPA (I), and two of its alkylated analogues, undergo in vivo and in vitro a-hydroxylation leading to formaldehyde formation. (Jones and Jackson 1968). We have confirmed this observation for HMPA (I) using the S-9 liver fraction described above, a dose-dependent relationship between the concentration of HMPA (I) and formaldehyde formation being observed. These experiments, which mirror those described for dimethyInitrosamine (VII)(McLean and Day 1974), together with others aimed at trapping the postulated intermediate alkylating species formed from HMPA (I) will be described in a subsequent publication (Ashby, Lefevre and Penman in preparation). The usefulness of a tellable test is illustrated by the problem posed when attempting to evaluate a compound such as the thioamide (VI). This com.a'M'.nd is superficially related to HMPA yet it has no counterpart in nitrosamine chemistry. The negative result obtained (Fig. Id) clearly indicates that this compound can be dissociated from the HMPA type in vivo carcinogenic activity. Finally, it must be mentioned that the in vitro test response and the in vivo leukaemogenicity observed for phosphoramide (II) require an explanation not involving alkyl groups (since none is present). Any separate hypothesis might, of course, be additionally involved in explaining the carcinogenic activity of HMPA itself. Curiously, a similar situation is encountered when considering the carcinogenicity of derivatives of hydrazine (NHjNHj) (Toth 1975). In this case the activity of a variety of alkylated hydrazines can be explained in terms of derived alkyl carbonium ions yet this leaves the activity observed for hydrazine itself unexplained (Biancifiori and Ribacchi 1962). JA/AJD 14 June 77. ucc 033254 9 REFERENCES Ames BN., McCann J. and Yamasaki E (1975). Methods for detecting carcinogens and mutagens with the Salmonella/rnammalian-microsome mutagenicity test. Mutation Res 31_ 347. Arceneaux RL., Frick JG., Leonard E.K., and Reid J.D (1959). N-methyl amides of phosphorus (T) acid. J. Org.Chem 24 1419. Autenrieth W and Rudolph F. (1900). Die phosphorylung der aromatischen aminbasen. Chem. Ber. 33 2099. Bartsch H., Camus A. and Malaveille (1976) . Comparative mutagenicity of N-nitrosamines in a semi-solid and in a liquid incubation system in the presence of rat or human tissue fractions. Mutation Res. 37^ 149. Biancifiori C. and Ribacchi R. (1962) Pulmonary tumours in mice induced by oral isoniazid and its metabolites. Nature 194 488. Benes V and Sram R.J. (1969). Mutagenic activity of some pesticides in the Drosophila melanogaster. Indus. Med. Surg. 38 442. Brookes F. and de Serres F. (1976). Report on the workshop on the mutagenicity of chemical carcinogens, Honolulu, Dec 9-11 1974. Mutation Res. 38^ 155. Druckey H. (1975). Chemical carcinogenesis on N-nitroso compounds. Gann Monograph in Cancer Research 17. 107 . ucc 033255 10 Hadidian Z, Fredrickson TN., Weisburger EK., Weisburger JH., Glass R.M., and Mantel N. Tests for chemical carcinogens. Report on the activity of derivatives of somatic amines, nitrosamines, quinolines, nitroalkanes, amides, epoxides, aziridines, and purine anti-metabolites. J. Nat. Can. Inst. 41 985-1036 (1968) Kimbrough R.D., and Gaines T.B., (1973). The chronic toxicity of hexamethylphosphoramide in rats. Bull. Environ. Contain, Toxicol lO 225. Element R,, and Koch 0., (1954). Phospnoroxy-triamiu and phosphorthiotriamid. Chem. Ber. 87 333. Lloyd J.W. (1975). Hexamethylphosphoric triamide (HMPA) Am. Ind. Hyg. Ass, J. 36 917. McCann J and Ames BN., (1976). Detection of carcinogens as mutagens in the Salmonella/microsome test : Assay of 300 chemicals : Fart II Discussion. Proc. Natl. Acad. Sci. U.S.A. 73^ 950. McCann J., Choi E., Yamasaki E., and Ames B.N. (1975). Detection of carcinogens as mutagens in the Salmonella/microsome test. Part I, assay of 300 chemicals. Proc. Natl. Acad. Sci. (U.S.A.) 72 5135 Purchase IFH., Longstaff E., Ashby J., Styles J.A., Anderson D., Lefevre P.A., and Westwood F.R. (1976). Evaluation of six short-term tests for detecting organic chemical carcinogens and recommendations for their use. Nature 264 624. ucc 033256 11 Purchase IFH., Longstaff E., Ashby J., Styles J.A., Anderson D., Lefevre P.A., and Westwood F.R (1977). Evaluation of six short-term tests for detecting organic chemical carcinogens and recommendations for their use. Brit. J. Cancer (in preparation). Toth B., (1975) Synthetic and naturally occuring hydrazines as possible cancer causative agents. Cancer Res 15 3693. VeBela H. (1962). Induction of leukemia with phosphoramide in C-57 61 mice. Neoplasma 9^ 75. Zapp J.A. (1975). Am. Ind. Hyg. Ass. J. 36 916. Inhalation toxicity of hexamethylphosphoramide. I ucc 033257 - .12 - .Legend TABLE I Results given by the Ames test and the cell transformation test for hexamethylphosphoramide (I), phosphoramide (II), phosphoric trianilide (V) and N,N,N"-trimethylphosphorothioic triamide (TI). ""''-^COMPOUND (I) (ID (VI) EXPERIMENT I+ve control) Ames test 1 + - (V) (-ve control) - 2 Cell transformation 1 + 2 - + - - ucc 033258 13 Fig 1 (Legend) Survival and transformation dose response curves of BHK cells treated with compound (I)(Fig la), (IX) (Fig lb), (V)(Fig Ic), (VI)(Fig Id), benzidine (Fig le) (- * ) and DMSO (Fig Ie) (4--A--A-) Fig la. I ucc 033259 14 Fig lb. ucc 033260 15 Fig Ic. ucc 033261 16 Fig Id. UCC 033262 17 Benzidine DMSO Fig Ie. --*---------- -------4---------4 -- ucc 033263 18 Fig II . (Legend) Survival and transformation dose response curves of BHK cells treated with diphenylnitrosamine (--A--A--) and dimethylnitrosamine i ucc 033264 c\ /="> N CV I ^Cl CM/' J )i O ^CH, 1 (I) 19 MU,. I o (II) o *(III) tv ./ Et O (IV) NHPV, I PHHN -- ^ -- NHPV, O (V) NHCH, I C+tJH N--NHCM, s (VI) C.H i VW--M= o CH, (VII) 0 O-s-O fV \ M--N--O tv (VIII) C) N I w O (X) ucc 033265