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The relationship between mutagenicity and carcinogenicity of some nitrosamines
319
Mutation Research, 51 (1978) 319--326 © Elsevier/North-Holland Biomedical Press
THE R E L A T I O N S H I P BETWEEN MUTAGENICITY AND CARCINOGENICITY O F SOME NITROSAMINES
A.W. ANDREWS, LENITA H. THIBAULT and W. LIJINSKY
Chemical Carcinogenesis Program, Frederick Cancer Research Center, Frederick, MD 21701 (U.S.A.) (Received 6 December 1977) (Revision received 9 March 1978) (Accepted 17 March 1978)
Summary The carcinogenicity and mutagenicity of 26 nitrosamines were compared to help validate the predictability of a short-term in vitro test. 80% of the compounds showed agreement between the t w o characteristics, while 20% did not. Of the latter group, 8% were false positives and 12% were false negatives.
Introduction
As mentioned in the accompanying paper [3], the search for an abbreviated test of the carcinogenic potential of chemicals has yielded one which has achieved widespread application. This is the test proposed b y Ames et al. [1,2] using mutants of Salmonella typhimurium, which respond to chemical mutagens either b y themselves or upon activation by a crude enzyme preparation from rat-liver cells. The usefulness of the test depends on h o w precisely it predicts the carcinogenicity of a chemical from the mutagenic response. In an initial series [4], the correlation of mutagenicity with carcinogenicity was quite good. Most of the c o m p o u n d s evaluated are well k n o w n carcinogens or well known non-carcinogens. A further evaluation of the system is to test a large group of chemicals of similar structure (including both carcinogens and non-carcinogens), which have been tested for carcinogenic activity under similar conditions. One such group, in which great interest is presently shown, is the nitrosamines, which have varied carcinogenic activity and a wide variation of target organs. Research sponsored by the L i t t o n B i o n e t i c s , Inc.
National Cancer
Institute under Contract
No. NO1-CO-75380 w i t h
320 Three categories of nitrosamines were chosen: (1) aliphatic nitrosamines: dimethylnitrosamine; diethylnitrosamine; nitrosomethylundecylamine; nitrosomethyldodecylamine; nitrosodioctylamine; nitrosomethoxymethylamine; nitrosomethylaniline and nitrosomethylcyclohexylamine, (2) cyclic nitrosamines: nitrosomorpholine; 2,6-dimethylnitrosomorpholine; 2-phenyl-3-methylnitrosomorpholine; dinitrosopiperazine; dinitrosohomopiperazine; 2-methyldinitrisopiperazine; 2,5-dimethyldinitrosopiperazine; 2,6-dimethyldinitrosopiperazine; nitrosomethylphenidate; nitrosotetrahydropyridine; nitrosonornicotine; nitrosoazetidine; nitrosopyrroline; and nitrosododecamethyleneimine; and (3) nitrosotrialkylureas: nitrosodimethylethylurea, nitrosodiethylmethylurea~ nitrosotrimethylurea and nitrosotriethylurea. Within these groups it should be possible to discern parallels between mutagenic and carcinogenic activity. Materials and Methods
Chemicals The preparation and characterization of the nitrosamines has been previously described [7--13] together with a description of the results of their carcinogenicity testing. Those nitrosamines that were carcinogenic in Sprague--Dawley rats showed a variety of target organ specificities, including liver, esophagus, bladder, nasal cavity and central nervous system. Mutagenesis assay The SalmoneUa/mammalian-microsome Mutagenicity Test [1] was performed essentially as described by Ames et al. Instead of 0.1 ml of the tester strain, 0.2 ml was used. The volume of VBE agar was 20 ml per plate. The final concentrations of the $9 mixes were 150 #1 for the phenobarbital-stimulated enzymes and 75 pl for those stimulated with Aroclor 1254. Slightly more than
TA1535 + Phenobarbital S9 Control Mean Value Based on 51 Tests 1000
DMNMe NTHP m DNHP ~
.=_ 750 o
t,.) c
500
2,6DMDNPe
MDNPO
o ,,Q
E=
Z 95%
'1 500
102
I 103
~g Fig. 1. The c o m p a r a t i v e m u t a g e n i c i t y o f n i t r o s a m i n e s using t e s t e r s t r a i n T A 1 5 3 5 w i t h a p h e n o b a r b i t a l i n d u c e d m i c r o s o m e p r e p a r a t i o n s h o w i n g the c o n t r o l m e a n v a l u e ( X ) and the u p p e r 9 5 % t o l e r a n c e l i m i t .
321 TA1535 + Aroclor S 9 Control M e a n V a l u e B a s e d on 74 T e s t s 2000 I NMUDA 2,5DMDNPo
1000 500"
o
NMDDA
300
,.Q E z NDDME
• NDOA
100.
ODNP NMCHAo ~NMA
95% 102
250
500
103
~g
Fig. 2. The c o m p a r a t i v e m u t a g e n i c i t y of n i t r o s a m i n e s u s i n g t e s t e r s t r a i n T A 1 5 3 5 w i t h a n A r o c l o r 1 2 5 4 i n d u c e d m i e r o s o m a l p r e p a r a t i o n s h o w i n g the c o n t r o l m e a n value (X) a n d the u p p e r 95% t o l e r a n c e limit.
half of the test compounds were shown to be mutagenic in the standard plate incorporation method. The remainder were tested using the liquid test. The suspensions were incubated without shaking at 37°C. We report the intervals which show maximum mutagenesis: 10 min for nitrosodiethylmethylurea, nitrosotriethylurea, 2-phenyl-3-methyl-nitrosomorpholine; 15 min for nitrosomethylphenidate; 20 rain for nitrosomethylaniline, nitrosotrimethylurea, and nitrosodimethylethylurea; and 30 min for dimethylnitrosamine and diethylnitrosamine. Top agar was then added to the incubation tubes and the entire contents poured onto the surface of the VBE agar. All testing was performed
TA1530 + Phenobarbital S 9 Control Mean Value Based on 15 T e s t s
NMO
300 250 200 fz: o
150 --
E
= 100 -z 95% 50-
100
NMPDO I 5
I 101
I 102
I 103
~g Fig. 3. The c o m p a r a t i v e m u t a g e r d c i t y of n i t r o s a m i n e s using t e s t e r s t r a i n T A 1 5 3 0 w i t h a p h e n o b a r b i t a l i n d u c e d m i c r o s o m a l p r e p a r a t i o n also s h o w i n g the c o n t r o l m e a n value (X') a n d the u p p e r 95% t o l e r a n c e level.
322 TA1530 + Aroclor S9 Control Mean Value Based on 9 T e s t s 210 NMMAO 180 m --
150 NAZe
g: ~o
120 90
NNNe
E 7= 9 5 %
N%
3oI 102
103
~g Fig. 4. T h e c o m p a r a t i v e m u t a g e n i c i t y o f n i t r o s a m i n e s using t e s t e r s w a i n T A 1 5 3 0 w i t h a n A r o c l o r 1 2 5 4 i n d u c e d m i c r o s o m a l p r e p a r a t i o n s h o w i n g the c o n t r o l m e a n v a l u e (.~) a n d t h e u p p e r 9 5 % t o l e r a n c e level.
under yellow fluorescent lighting (Sylvania F15T8-G0). Colonies were counted using a hand-held tally after incubation at 37°C for 48 h.
Statistical analysis of the data The c o m p o u n d s were tested in dose--response curves over a 1 - - 1 0 0 0 pg range using the method, tester strain and $9 mix which gave m a x i m u m mutagenesis. The detailed description of the methods may be found in the accompanying publication [3]. Most of the c o m p o u n d s listed as mutagenic indicated a dose--response relationship. Those which were mutagenic, but did not have a TA1530 + Phenobarbital S9 Control Mean Value Based on 15 Liquid T e s t s 1000
NTMU •
750
50O
250 NTEU • 95%
PMNM
- ~'~i~J']l
NDEMU
10o
I
|
101
102
,
I
103
~g
Fig. 5. T h e c o m p a r a t i v e m u t a g e n i e i t y o f n i t r o ~ u n i n e s u s i n g t e e t e r s t r a i n T A 1 5 3 0 w i t h a p h e n o b a r b i t a l i n d u c e d m i c r o s o m a l p r e p a r a t i o n in t h e liquid t e s t also s h o w i n g the c o n t r o l m e a n v a l u e ( ~ ) and t h e u p p e r 9 5 % t o l e r a n c e level.
323 TA1530 + Phenobarbital S9
Control Mean Value Based on 11 Liquid Test (saline as solvent)
120 DMN • u)
100
co "c
~
80
6O ~ 95°/° E 40 z=
DEN •
20
102
I
I
500
103
~g Fig. 6. T h e c o m p a r a t i v e m u t a g e n i c i t y o f n i t r 0 s a m i n e s u s i n g t e s t e r s t r a i n T A 1 5 3 0 w i t h a p h e n o b a r b i t a l i n d u c e d r n i c r o s o m a l p r e p a r a t i o n in the l i q u i d t e s t w i t h saline as t h e s o l v e n t and s h o w i n g t h e c o n t r o l m e a n v a l u e (X) and t h e u p p e r 9 5 % t o l e r a n c e level.
dose--response which demonstrated linear regression, were nitrosomethylundecylamine, nitrosomethoxymethylamine, nitrosopyrroline, nitrosotrimethylurea, and nitrosotriethylurea. The values in Figs. 1--6 are the number of revertants at the upper end of the dose--response curves at the corresponding dose level. When no dose-response was indicated (nitrosomethylaniline, nitrosomethylphenidate, nitrosodimethylethylurea and nitrosodiethylmethylurea), the values are the greatest number of revertants observed at the corresponding dose. The control mean and the 95% tolerance limits are also indicated. Results
The results of the mutagenesis tests are given in Table 1. It can readily be seen that most of the nitrosamines tested were mutagenic. The majority had to be activated by rat-liver microsomes to show mutagenic activity. The direct acting mutagens were nitrosomethoxymethylamine, dinitrosohomopiperazine, nitrosotetrahydropyridine and nitrosoazetidine. Dimethylnitrosamine and diethylnitrosamine were activated only with phenobarbital-stimulated $9 mix and only if saline and not dimethylsulfoxide was the solvent. This supplements previously published studies of the mutagenesis of nitrosamines in which most of the compounds tested were mutagenic [4,6,16]. It seems that the correlation of mutagenic activity with carcinogenic activity is very good among the 14 cyclic nitrosamines examined, there being 12 compounds both mutagenic and carcinogenic and 2 both non-mutagenic and noncarcinogenic. Among the 8 aliphatic nitrosamines 5 of the 6 carcinogens were also mutagenic. Both nitrosomethoxymethylamine and nitrosodioctylamine, which were not carcinogenic, were mutagenic.
324 Table 1. The C o m p a r i s o n of the Chemical Structure, Carcinogenicitv and M u t a g e n i c i l y of A l i p h a t i c and Cyclic Nitrosamines and Nilrosoalkylureas Abt)revi;lhofl
(;~=r
~ ~ ~ ~
~' 4 + ÷
nitrosodioet y l a m i n e nitrosomelhoxymethylamine
DMN DEN NMUDA NMDDA NDOA NMMA
¢~itiosow~e thyiat~dit~e nittosomethylcyclohexylamine
NMA NMCHA
,~ ~
4
NM DMNM PMNM
+
+
2.phenyl.3-mel hyl.nitr o s o m o r p h o l i n e
dinitrosopiperazine dinit rosohomopiperazine 2-met h y l d i n i t rosopiperazine 2,5-dimet h y l d i n i t rosopiperazine 2,6-dimetbyldinitrosopiperazine
DNP DNHP MDNP 2,5-DMDNP 2,6-DMDNP
Tesl C o m p o u n d
MuI
Aliphatic Nitrosamines dimethylnitrosamine diethylnitrosamine nitrosomethylundecylamine
nilrosomelhyldodecylamine
NO
I
R--N
"'
-R ~
CH3-N 2
Cyclic N i t r o s a m i n e s nit r o s o m o r p h o l i n e
2,6.dimethylnitrosomorpholine
• *
--N--
NO +
+
543 I NO
nitrosomethylphenidate
NMPD
nitrosotet rahydropyridine
NTHP
nitrosonornicotine
NNN
. .
NO I
NO
I nitrosoazetidine
NAZ
nilrosopyrroline
NP
nit r o s o d o d e c a m e t h y l e n e i m i n e
NDDME
+
NDMEU N DEM U NTMU NTEU
+ + + +
NO I
N--NO
Nitrosotrialkylureas nit r o s o d i m e t h y l e t h y l u r e a nitrosodiethylmethylurea nitrosotrimethylurea nitrosotriethylurea
° M u t a g e n i c o n l y in l i q u i d test. • * Not m u t a g e n i c in l i q u i d or plate test, all strains tested.
-•* _o.
++"
R O \ II N--C--N--NO
#
,...
325 The 4 nitrosoalkylureas are all carcinogens for the nervous system of rats [ 11 ]. Nitrosodiethylmethylurea, the most p o t e n t carcinogen of the four tested [11], is n o t mutagenic. Neither is nitrosodimethylethylurea. Nitrosotrimethylurea, the least p o t e n t carcinogen, is mutagenic as is nitrosotriethylurea. Discussion
Qualitatively, the cyclic nitrosamines showed excellent agreement between the carcinogenic and mutagenic activities. The aliphatic nitrosamine, nitrosomethylaniline, was carcinogenic b u t n o t mutagenic. Both nitrosomethylaniline and nitrosomethylcyclohexylamine are esophageal carcinogens of equal p o t e n c y in rats [8] and do n o t indice liver tumors. Nitrosomethylcyclohexylamine is toxic to the rat liver and causes extensive methylation of rat-liver DNA [14]. Nitrosomethylaniline, which is similar in structure, has little effect on rat liver and does not lead to methylation of DNA in the rat organ. The reason for the sharp difference in mutagenicity among the nitrosotrialkylureas is n o t understood. Neither is it clear w h y these and other nitrosamines, which are carcinogenic in rats b u t do not induce tumors in rat liver, are activated by rat liver to bacterial mutagens. This does not make the test any less useful, b u t it does imply that one must interpret the results with caution. It might be useful to test the c o m p o u n d s giving false negative mutagenicity results with $9 preparations made from the organs in which they produce tumors. The test failed to detect 3 of the 22 carcinogens among the 26 nitrosamines tested. While it is most desirable to find a test to replace the cumbersome long-term animal bioassay for carcinogenicity, it is plain, from the results presented here and elsewhere [3], that the Salmonella mutagenicity test, while useful, is n o t reliable. Until this assay, or some other test, demonstrates a quantitative and qualitative correlation between mutagenic p o t e n c y and carcinogenic potency, it will be very difficult to establish a hierarchy of carcinogenic risk among a group of c o m p o u n d s such as the nitrosamines. Acknowledgements The authors wish to thank Carl Valentine, Corinthia Brown, Jan Reichard and James Fornwald for their expert technical assistance. We are also indebted to Charles W. Riggs for aid in the statistical analyses. References 1 A m e s , B . N . , W.E. D u r s t o n , E. Y a m a s a k i a n d F . D . Lee, C a r c i n o g e n s are m u t a g e n s : A s i m p l e t e s t syst e m c o m b i n i n g liver h o m o g e n a t e s for a c t i v a t i o n and b a c t e r i a f o r d e t e c t i o n , P r o c . N a t l . A e a d . Sci. (U.S.A.) 70 (1973) 2281--2285. 2 A m e s , B.N., J . M c C a n n a n d E. Y a m a s a k i , M e t h o d s for d e t e c t i n g c a r c i n o g e n s a n d m u t a g e n s w i t h the S a l m o n e l l a / m a m m a l i a n - m i c r o s o m e m u t a g e n i c i t y test, M u t a t i o n Res., 3 1 ( 1 9 7 5 ) 3 4 7 - - 3 6 4 . 3 A n d r e w s , A . W . , L . H . T h i b a u l t a n d W. Lijinsky, T h e r e l a t i o n s h i p b e t w e e n c a r c i n o g e n i c i t y and m u t a genicity of some Dolynuclear hydrocarbons. Mutation Res., 51 (1978) 311--318. 4 B a r t s c h , H., C. Malaveille a n d R . M o n t e s a n o , T h e p r e d i c t i v e v a l u e o f tissue m e d i a t e d m u t a g e n i c i t y assays t o assess the c a r c i n o g e n i c r i s k o f c h e m i c a l s , in S c r e e n i n g t e s t s in c h e m i c a l c a r c i n o g e n e s i s , I A R C Scientific Publications, No. 12, Lyon, 1976 pp. 467--481.
326 5 D r u c k r e y , H., R. P r e u s s m a n n , S. I v a n k o v i c a n d D. Schm~thl, O r g a n o t r o p e c a r c i n o g e n e W i r k u n g e n bei 6 5 v e r s c h i e d e n e n N - N i t r o s o - V e r b i n d u n g e n a n B D - R a t t e n , Z. K r e b s f o r s c h . , 6 9 ( 1 9 6 7 ) 1 0 3 - - 2 0 1 . 6 E l e s p u r u , R . K . , a n d W. L i j i n s k y , M u t a g e n i c i t y o f c y c l i c n i t r o s a m i n e s in E. coli f o l l o w i n g a c t i v a t i o n w i t h r a t fiver m i c r o s o m e s , C a n c e r Res., 36 ( 1 9 7 6 ) 4 0 9 9 - - 4 1 0 1 . 7 G a r e i a , H., a n d W. L i j i n s k y , T u m o r i g e n i c i t y o f five c y c l i c n i t r o s a m i n e s in M R C r a t s , Z. K r e b s f o r s c h . , 77 ( 1 9 7 2 ) 2 5 7 - - 2 6 1 . 8 G o o d a U , C.M., W. L i j i n s k y , L. T o m a t i s a n d C.E.M. W e n y o n , T o x i c i t y a n d o n c o g e n i c i t y o f n i t r o s o m e t h y l a n i l i n e a n d n i t r o s o m e t h y l c y c l o h e x y l a m i n e , T o x i e o l . A p p l . P h a r m a c o l . , 17 ( 1 9 7 0 ) 4 2 6 - - 4 3 2 . 9 G r e e n b l a t t , M., a n d W. L i j i n s k y , N i t r o s a m i n e s t u d i e s : N e o p l a s m s o f liver a n d g e n i t a l m e s o t h e f i u m in n i t r o s o p y r r o f i d i n e t r e a t e d M R C r a t s , J. N a t l . C a n c e r Inst., 4 8 ( 1 9 7 2 ) 1 6 8 7 - - 1 6 9 6 . 1 0 H o f f m a n n , D., R. R a i n e r i , S.S. H e c h t , R. M a r o n p o t a n d E.L. W y n d e r , A s t u d y o f t o b a c c o c a r c i n o genesis, XIV. E f f e c t s o f N ' - n i t r o s o n o r n i c o t i n e in r a t s , J. N a t l . C a n c e r Inst., 5 5 ( 1 9 7 5 ) 9 7 7 - - 9 7 9 . 11 L i j i n s k y , W., a n d H.W. T a y l o r , T u m o r s o f n e r v o u s o r i g i n i n d u c e d in r a t s b y n i t r o s o t r i a l k y l u r e a s , Z. Krebsforsch., 83 (1975) 315--321. 1 2 L i j i n s k y , W., a n d H.W. T a y l o r , C a r e i n o g e n i c i t y o f t w o u n s a t u r a t e d d e r i v a t i v e s o f n i t r o s o p i p e r i d i n e in S p r a g u e - - D a w l e y r a t s , J. N a t l . C a n c e r Inst., 57 ( 1 9 7 6 ) 1 3 1 5 - - 1 3 1 7 . 13 L i i i n s k y , W., a n d H.W. T a y l o r , C a r c i n o g e n e s i s t e s t o f n i t r o s o - N - m e t h y l p i p e r a z i n e , 2 , 3 , 5 , 6 - t e t r a m e t h y l dinitrosopiperazine, nitrosoisonipecotic acid and nitrosomethoxymethylamine in r a t s , Z. K r e b s forsch., 89 (1977) 31--36. 1 4 L i j i n s k y , W., L. K e e f e r , J. L o o a n d A . E . R o s s , S t u d i e s o f a l k y l a t i o n o f n u c l e i c a c i d s in r a t s b y c y c l i c nitrosamines, Cancer Res., 33 (1973) 1634--1641. 1 5 M c C a n n , J., E. C h o i , E. Y a m a s a k i a n d B.N. A m e s , D e t e c t i o n o f c a r c i n o g e n s as m u t a g e n s in t h e SaN m o n e l l a / m i e r o s o m e test: A s s a y o f 3 0 0 c h e m i c a l s , P r o e . N a t l . A c a d . Sei. ( U . S . A . ) 7 2 ( 1 9 7 5 ) 5 1 3 5 - 5139. 16 R a o , T . K . , A . A . H a r d i g r e e , J . A . Y o u n g , W. L i j i n s k y a n d J . L . E p l e r , M u t a g e n i c i t y o f N - n i t r o s o p i p e r i dines with Salmonella typhimurium/microsomal a c t i v a t i o n s y s t e m , M u t a t i o n Res., 56 ( 1 9 7 7 ) 1 3 1 - 145.
Mutation Research, 51 (1978) 319--326 © Elsevier/North-Holland Biomedical Press
THE R E L A T I O N S H I P BETWEEN MUTAGENICITY AND CARCINOGENICITY O F SOME NITROSAMINES
A.W. ANDREWS, LENITA H. THIBAULT and W. LIJINSKY
Chemical Carcinogenesis Program, Frederick Cancer Research Center, Frederick, MD 21701 (U.S.A.) (Received 6 December 1977) (Revision received 9 March 1978) (Accepted 17 March 1978)
Summary The carcinogenicity and mutagenicity of 26 nitrosamines were compared to help validate the predictability of a short-term in vitro test. 80% of the compounds showed agreement between the t w o characteristics, while 20% did not. Of the latter group, 8% were false positives and 12% were false negatives.
Introduction
As mentioned in the accompanying paper [3], the search for an abbreviated test of the carcinogenic potential of chemicals has yielded one which has achieved widespread application. This is the test proposed b y Ames et al. [1,2] using mutants of Salmonella typhimurium, which respond to chemical mutagens either b y themselves or upon activation by a crude enzyme preparation from rat-liver cells. The usefulness of the test depends on h o w precisely it predicts the carcinogenicity of a chemical from the mutagenic response. In an initial series [4], the correlation of mutagenicity with carcinogenicity was quite good. Most of the c o m p o u n d s evaluated are well k n o w n carcinogens or well known non-carcinogens. A further evaluation of the system is to test a large group of chemicals of similar structure (including both carcinogens and non-carcinogens), which have been tested for carcinogenic activity under similar conditions. One such group, in which great interest is presently shown, is the nitrosamines, which have varied carcinogenic activity and a wide variation of target organs. Research sponsored by the L i t t o n B i o n e t i c s , Inc.
National Cancer
Institute under Contract
No. NO1-CO-75380 w i t h
320 Three categories of nitrosamines were chosen: (1) aliphatic nitrosamines: dimethylnitrosamine; diethylnitrosamine; nitrosomethylundecylamine; nitrosomethyldodecylamine; nitrosodioctylamine; nitrosomethoxymethylamine; nitrosomethylaniline and nitrosomethylcyclohexylamine, (2) cyclic nitrosamines: nitrosomorpholine; 2,6-dimethylnitrosomorpholine; 2-phenyl-3-methylnitrosomorpholine; dinitrosopiperazine; dinitrosohomopiperazine; 2-methyldinitrisopiperazine; 2,5-dimethyldinitrosopiperazine; 2,6-dimethyldinitrosopiperazine; nitrosomethylphenidate; nitrosotetrahydropyridine; nitrosonornicotine; nitrosoazetidine; nitrosopyrroline; and nitrosododecamethyleneimine; and (3) nitrosotrialkylureas: nitrosodimethylethylurea, nitrosodiethylmethylurea~ nitrosotrimethylurea and nitrosotriethylurea. Within these groups it should be possible to discern parallels between mutagenic and carcinogenic activity. Materials and Methods
Chemicals The preparation and characterization of the nitrosamines has been previously described [7--13] together with a description of the results of their carcinogenicity testing. Those nitrosamines that were carcinogenic in Sprague--Dawley rats showed a variety of target organ specificities, including liver, esophagus, bladder, nasal cavity and central nervous system. Mutagenesis assay The SalmoneUa/mammalian-microsome Mutagenicity Test [1] was performed essentially as described by Ames et al. Instead of 0.1 ml of the tester strain, 0.2 ml was used. The volume of VBE agar was 20 ml per plate. The final concentrations of the $9 mixes were 150 #1 for the phenobarbital-stimulated enzymes and 75 pl for those stimulated with Aroclor 1254. Slightly more than
TA1535 + Phenobarbital S9 Control Mean Value Based on 51 Tests 1000
DMNMe NTHP m DNHP ~
.=_ 750 o
t,.) c
500
2,6DMDNPe
MDNPO
o ,,Q
E=
Z 95%
'1 500
102
I 103
~g Fig. 1. The c o m p a r a t i v e m u t a g e n i c i t y o f n i t r o s a m i n e s using t e s t e r s t r a i n T A 1 5 3 5 w i t h a p h e n o b a r b i t a l i n d u c e d m i c r o s o m e p r e p a r a t i o n s h o w i n g the c o n t r o l m e a n v a l u e ( X ) and the u p p e r 9 5 % t o l e r a n c e l i m i t .
321 TA1535 + Aroclor S 9 Control M e a n V a l u e B a s e d on 74 T e s t s 2000 I NMUDA 2,5DMDNPo
1000 500"
o
NMDDA
300
,.Q E z NDDME
• NDOA
100.
ODNP NMCHAo ~NMA
95% 102
250
500
103
~g
Fig. 2. The c o m p a r a t i v e m u t a g e n i c i t y of n i t r o s a m i n e s u s i n g t e s t e r s t r a i n T A 1 5 3 5 w i t h a n A r o c l o r 1 2 5 4 i n d u c e d m i e r o s o m a l p r e p a r a t i o n s h o w i n g the c o n t r o l m e a n value (X) a n d the u p p e r 95% t o l e r a n c e limit.
half of the test compounds were shown to be mutagenic in the standard plate incorporation method. The remainder were tested using the liquid test. The suspensions were incubated without shaking at 37°C. We report the intervals which show maximum mutagenesis: 10 min for nitrosodiethylmethylurea, nitrosotriethylurea, 2-phenyl-3-methyl-nitrosomorpholine; 15 min for nitrosomethylphenidate; 20 rain for nitrosomethylaniline, nitrosotrimethylurea, and nitrosodimethylethylurea; and 30 min for dimethylnitrosamine and diethylnitrosamine. Top agar was then added to the incubation tubes and the entire contents poured onto the surface of the VBE agar. All testing was performed
TA1530 + Phenobarbital S 9 Control Mean Value Based on 15 T e s t s
NMO
300 250 200 fz: o
150 --
E
= 100 -z 95% 50-
100
NMPDO I 5
I 101
I 102
I 103
~g Fig. 3. The c o m p a r a t i v e m u t a g e r d c i t y of n i t r o s a m i n e s using t e s t e r s t r a i n T A 1 5 3 0 w i t h a p h e n o b a r b i t a l i n d u c e d m i c r o s o m a l p r e p a r a t i o n also s h o w i n g the c o n t r o l m e a n value (X') a n d the u p p e r 95% t o l e r a n c e level.
322 TA1530 + Aroclor S9 Control Mean Value Based on 9 T e s t s 210 NMMAO 180 m --
150 NAZe
g: ~o
120 90
NNNe
E 7= 9 5 %
N%
3oI 102
103
~g Fig. 4. T h e c o m p a r a t i v e m u t a g e n i c i t y o f n i t r o s a m i n e s using t e s t e r s w a i n T A 1 5 3 0 w i t h a n A r o c l o r 1 2 5 4 i n d u c e d m i c r o s o m a l p r e p a r a t i o n s h o w i n g the c o n t r o l m e a n v a l u e (.~) a n d t h e u p p e r 9 5 % t o l e r a n c e level.
under yellow fluorescent lighting (Sylvania F15T8-G0). Colonies were counted using a hand-held tally after incubation at 37°C for 48 h.
Statistical analysis of the data The c o m p o u n d s were tested in dose--response curves over a 1 - - 1 0 0 0 pg range using the method, tester strain and $9 mix which gave m a x i m u m mutagenesis. The detailed description of the methods may be found in the accompanying publication [3]. Most of the c o m p o u n d s listed as mutagenic indicated a dose--response relationship. Those which were mutagenic, but did not have a TA1530 + Phenobarbital S9 Control Mean Value Based on 15 Liquid T e s t s 1000
NTMU •
750
50O
250 NTEU • 95%
PMNM
- ~'~i~J']l
NDEMU
10o
I
|
101
102
,
I
103
~g
Fig. 5. T h e c o m p a r a t i v e m u t a g e n i e i t y o f n i t r o ~ u n i n e s u s i n g t e e t e r s t r a i n T A 1 5 3 0 w i t h a p h e n o b a r b i t a l i n d u c e d m i c r o s o m a l p r e p a r a t i o n in t h e liquid t e s t also s h o w i n g the c o n t r o l m e a n v a l u e ( ~ ) and t h e u p p e r 9 5 % t o l e r a n c e level.
323 TA1530 + Phenobarbital S9
Control Mean Value Based on 11 Liquid Test (saline as solvent)
120 DMN • u)
100
co "c
~
80
6O ~ 95°/° E 40 z=
DEN •
20
102
I
I
500
103
~g Fig. 6. T h e c o m p a r a t i v e m u t a g e n i c i t y o f n i t r 0 s a m i n e s u s i n g t e s t e r s t r a i n T A 1 5 3 0 w i t h a p h e n o b a r b i t a l i n d u c e d r n i c r o s o m a l p r e p a r a t i o n in the l i q u i d t e s t w i t h saline as t h e s o l v e n t and s h o w i n g t h e c o n t r o l m e a n v a l u e (X) and t h e u p p e r 9 5 % t o l e r a n c e level.
dose--response which demonstrated linear regression, were nitrosomethylundecylamine, nitrosomethoxymethylamine, nitrosopyrroline, nitrosotrimethylurea, and nitrosotriethylurea. The values in Figs. 1--6 are the number of revertants at the upper end of the dose--response curves at the corresponding dose level. When no dose-response was indicated (nitrosomethylaniline, nitrosomethylphenidate, nitrosodimethylethylurea and nitrosodiethylmethylurea), the values are the greatest number of revertants observed at the corresponding dose. The control mean and the 95% tolerance limits are also indicated. Results
The results of the mutagenesis tests are given in Table 1. It can readily be seen that most of the nitrosamines tested were mutagenic. The majority had to be activated by rat-liver microsomes to show mutagenic activity. The direct acting mutagens were nitrosomethoxymethylamine, dinitrosohomopiperazine, nitrosotetrahydropyridine and nitrosoazetidine. Dimethylnitrosamine and diethylnitrosamine were activated only with phenobarbital-stimulated $9 mix and only if saline and not dimethylsulfoxide was the solvent. This supplements previously published studies of the mutagenesis of nitrosamines in which most of the compounds tested were mutagenic [4,6,16]. It seems that the correlation of mutagenic activity with carcinogenic activity is very good among the 14 cyclic nitrosamines examined, there being 12 compounds both mutagenic and carcinogenic and 2 both non-mutagenic and noncarcinogenic. Among the 8 aliphatic nitrosamines 5 of the 6 carcinogens were also mutagenic. Both nitrosomethoxymethylamine and nitrosodioctylamine, which were not carcinogenic, were mutagenic.
324 Table 1. The C o m p a r i s o n of the Chemical Structure, Carcinogenicitv and M u t a g e n i c i l y of A l i p h a t i c and Cyclic Nitrosamines and Nilrosoalkylureas Abt)revi;lhofl
(;~=r
~ ~ ~ ~
~' 4 + ÷
nitrosodioet y l a m i n e nitrosomelhoxymethylamine
DMN DEN NMUDA NMDDA NDOA NMMA
¢~itiosow~e thyiat~dit~e nittosomethylcyclohexylamine
NMA NMCHA
,~ ~
4
NM DMNM PMNM
+
+
2.phenyl.3-mel hyl.nitr o s o m o r p h o l i n e
dinitrosopiperazine dinit rosohomopiperazine 2-met h y l d i n i t rosopiperazine 2,5-dimet h y l d i n i t rosopiperazine 2,6-dimetbyldinitrosopiperazine
DNP DNHP MDNP 2,5-DMDNP 2,6-DMDNP
Tesl C o m p o u n d
MuI
Aliphatic Nitrosamines dimethylnitrosamine diethylnitrosamine nitrosomethylundecylamine
nilrosomelhyldodecylamine
NO
I
R--N
"'
-R ~
CH3-N 2
Cyclic N i t r o s a m i n e s nit r o s o m o r p h o l i n e
2,6.dimethylnitrosomorpholine
• *
--N--
NO +
+
543 I NO
nitrosomethylphenidate
NMPD
nitrosotet rahydropyridine
NTHP
nitrosonornicotine
NNN
. .
NO I
NO
I nitrosoazetidine
NAZ
nilrosopyrroline
NP
nit r o s o d o d e c a m e t h y l e n e i m i n e
NDDME
+
NDMEU N DEM U NTMU NTEU
+ + + +
NO I
N--NO
Nitrosotrialkylureas nit r o s o d i m e t h y l e t h y l u r e a nitrosodiethylmethylurea nitrosotrimethylurea nitrosotriethylurea
° M u t a g e n i c o n l y in l i q u i d test. • * Not m u t a g e n i c in l i q u i d or plate test, all strains tested.
-•* _o.
++"
R O \ II N--C--N--NO
#
,...
325 The 4 nitrosoalkylureas are all carcinogens for the nervous system of rats [ 11 ]. Nitrosodiethylmethylurea, the most p o t e n t carcinogen of the four tested [11], is n o t mutagenic. Neither is nitrosodimethylethylurea. Nitrosotrimethylurea, the least p o t e n t carcinogen, is mutagenic as is nitrosotriethylurea. Discussion
Qualitatively, the cyclic nitrosamines showed excellent agreement between the carcinogenic and mutagenic activities. The aliphatic nitrosamine, nitrosomethylaniline, was carcinogenic b u t n o t mutagenic. Both nitrosomethylaniline and nitrosomethylcyclohexylamine are esophageal carcinogens of equal p o t e n c y in rats [8] and do n o t indice liver tumors. Nitrosomethylcyclohexylamine is toxic to the rat liver and causes extensive methylation of rat-liver DNA [14]. Nitrosomethylaniline, which is similar in structure, has little effect on rat liver and does not lead to methylation of DNA in the rat organ. The reason for the sharp difference in mutagenicity among the nitrosotrialkylureas is n o t understood. Neither is it clear w h y these and other nitrosamines, which are carcinogenic in rats b u t do not induce tumors in rat liver, are activated by rat liver to bacterial mutagens. This does not make the test any less useful, b u t it does imply that one must interpret the results with caution. It might be useful to test the c o m p o u n d s giving false negative mutagenicity results with $9 preparations made from the organs in which they produce tumors. The test failed to detect 3 of the 22 carcinogens among the 26 nitrosamines tested. While it is most desirable to find a test to replace the cumbersome long-term animal bioassay for carcinogenicity, it is plain, from the results presented here and elsewhere [3], that the Salmonella mutagenicity test, while useful, is n o t reliable. Until this assay, or some other test, demonstrates a quantitative and qualitative correlation between mutagenic p o t e n c y and carcinogenic potency, it will be very difficult to establish a hierarchy of carcinogenic risk among a group of c o m p o u n d s such as the nitrosamines. Acknowledgements The authors wish to thank Carl Valentine, Corinthia Brown, Jan Reichard and James Fornwald for their expert technical assistance. We are also indebted to Charles W. Riggs for aid in the statistical analyses. References 1 A m e s , B . N . , W.E. D u r s t o n , E. Y a m a s a k i a n d F . D . Lee, C a r c i n o g e n s are m u t a g e n s : A s i m p l e t e s t syst e m c o m b i n i n g liver h o m o g e n a t e s for a c t i v a t i o n and b a c t e r i a f o r d e t e c t i o n , P r o c . N a t l . A e a d . Sci. (U.S.A.) 70 (1973) 2281--2285. 2 A m e s , B.N., J . M c C a n n a n d E. Y a m a s a k i , M e t h o d s for d e t e c t i n g c a r c i n o g e n s a n d m u t a g e n s w i t h the S a l m o n e l l a / m a m m a l i a n - m i c r o s o m e m u t a g e n i c i t y test, M u t a t i o n Res., 3 1 ( 1 9 7 5 ) 3 4 7 - - 3 6 4 . 3 A n d r e w s , A . W . , L . H . T h i b a u l t a n d W. Lijinsky, T h e r e l a t i o n s h i p b e t w e e n c a r c i n o g e n i c i t y and m u t a genicity of some Dolynuclear hydrocarbons. Mutation Res., 51 (1978) 311--318. 4 B a r t s c h , H., C. Malaveille a n d R . M o n t e s a n o , T h e p r e d i c t i v e v a l u e o f tissue m e d i a t e d m u t a g e n i c i t y assays t o assess the c a r c i n o g e n i c r i s k o f c h e m i c a l s , in S c r e e n i n g t e s t s in c h e m i c a l c a r c i n o g e n e s i s , I A R C Scientific Publications, No. 12, Lyon, 1976 pp. 467--481.
326 5 D r u c k r e y , H., R. P r e u s s m a n n , S. I v a n k o v i c a n d D. Schm~thl, O r g a n o t r o p e c a r c i n o g e n e W i r k u n g e n bei 6 5 v e r s c h i e d e n e n N - N i t r o s o - V e r b i n d u n g e n a n B D - R a t t e n , Z. K r e b s f o r s c h . , 6 9 ( 1 9 6 7 ) 1 0 3 - - 2 0 1 . 6 E l e s p u r u , R . K . , a n d W. L i j i n s k y , M u t a g e n i c i t y o f c y c l i c n i t r o s a m i n e s in E. coli f o l l o w i n g a c t i v a t i o n w i t h r a t fiver m i c r o s o m e s , C a n c e r Res., 36 ( 1 9 7 6 ) 4 0 9 9 - - 4 1 0 1 . 7 G a r e i a , H., a n d W. L i j i n s k y , T u m o r i g e n i c i t y o f five c y c l i c n i t r o s a m i n e s in M R C r a t s , Z. K r e b s f o r s c h . , 77 ( 1 9 7 2 ) 2 5 7 - - 2 6 1 . 8 G o o d a U , C.M., W. L i j i n s k y , L. T o m a t i s a n d C.E.M. W e n y o n , T o x i c i t y a n d o n c o g e n i c i t y o f n i t r o s o m e t h y l a n i l i n e a n d n i t r o s o m e t h y l c y c l o h e x y l a m i n e , T o x i e o l . A p p l . P h a r m a c o l . , 17 ( 1 9 7 0 ) 4 2 6 - - 4 3 2 . 9 G r e e n b l a t t , M., a n d W. L i j i n s k y , N i t r o s a m i n e s t u d i e s : N e o p l a s m s o f liver a n d g e n i t a l m e s o t h e f i u m in n i t r o s o p y r r o f i d i n e t r e a t e d M R C r a t s , J. N a t l . C a n c e r Inst., 4 8 ( 1 9 7 2 ) 1 6 8 7 - - 1 6 9 6 . 1 0 H o f f m a n n , D., R. R a i n e r i , S.S. H e c h t , R. M a r o n p o t a n d E.L. W y n d e r , A s t u d y o f t o b a c c o c a r c i n o genesis, XIV. E f f e c t s o f N ' - n i t r o s o n o r n i c o t i n e in r a t s , J. N a t l . C a n c e r Inst., 5 5 ( 1 9 7 5 ) 9 7 7 - - 9 7 9 . 11 L i j i n s k y , W., a n d H.W. T a y l o r , T u m o r s o f n e r v o u s o r i g i n i n d u c e d in r a t s b y n i t r o s o t r i a l k y l u r e a s , Z. Krebsforsch., 83 (1975) 315--321. 1 2 L i j i n s k y , W., a n d H.W. T a y l o r , C a r e i n o g e n i c i t y o f t w o u n s a t u r a t e d d e r i v a t i v e s o f n i t r o s o p i p e r i d i n e in S p r a g u e - - D a w l e y r a t s , J. N a t l . C a n c e r Inst., 57 ( 1 9 7 6 ) 1 3 1 5 - - 1 3 1 7 . 13 L i i i n s k y , W., a n d H.W. T a y l o r , C a r c i n o g e n e s i s t e s t o f n i t r o s o - N - m e t h y l p i p e r a z i n e , 2 , 3 , 5 , 6 - t e t r a m e t h y l dinitrosopiperazine, nitrosoisonipecotic acid and nitrosomethoxymethylamine in r a t s , Z. K r e b s forsch., 89 (1977) 31--36. 1 4 L i j i n s k y , W., L. K e e f e r , J. L o o a n d A . E . R o s s , S t u d i e s o f a l k y l a t i o n o f n u c l e i c a c i d s in r a t s b y c y c l i c nitrosamines, Cancer Res., 33 (1973) 1634--1641. 1 5 M c C a n n , J., E. C h o i , E. Y a m a s a k i a n d B.N. A m e s , D e t e c t i o n o f c a r c i n o g e n s as m u t a g e n s in t h e SaN m o n e l l a / m i e r o s o m e test: A s s a y o f 3 0 0 c h e m i c a l s , P r o e . N a t l . A c a d . Sei. ( U . S . A . ) 7 2 ( 1 9 7 5 ) 5 1 3 5 - 5139. 16 R a o , T . K . , A . A . H a r d i g r e e , J . A . Y o u n g , W. L i j i n s k y a n d J . L . E p l e r , M u t a g e n i c i t y o f N - n i t r o s o p i p e r i dines with Salmonella typhimurium/microsomal a c t i v a t i o n s y s t e m , M u t a t i o n Res., 56 ( 1 9 7 7 ) 1 3 1 - 145.