131

Mutation Research, 48 (1977) 131--138 © Elsevier/North-Holland Biomedical Press

MUTAGENICITY OF 22 N-NITROSAMIDES AND RELATED COMPOUNDS FOR S A L M O N E L L A TYPHIMURIUM TA1535

KYU LEE, BARRY GOLD and SIDNEY S. MIRVISH

Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha, Nebraska 68105 (U.S.A.)

(Received August 6th, 1976} (Revision received October 18th, 1976) (Accepted October 28th, 1976)

Summary

Twenty- two N-nitrosamides and related compounds, including 14 nitrosoure- as, 5 nitrosocarbamates, and one nitrosocyanamide, were tested at various concentrations for mutagenic activity towards Salmonella typhimurium TA1535 wi thout the use of microsomes. The ether-water partition coefficient, solubility in water, and half-life in aqueous solution were also measured. Twen- ty compounds were mutagenic, with "standard mutagenic concentrat ions" (i.e. those producing 100 mutants/dish) of 0 .0024--6500 pM. Standard mutagenic concentration was negatively correlated with the partition coefficient. Three compounds (ethyl 2-acetoxyethylnitrosocarbamate, nitrosocarbaryl, and me- thylnitrosobenzamide) were more active than the classic mutagen methylnitro- sonitroguanidine. Nitrosocarbamates were at least 50 times more mutagenic than the corresponding nitrosoureas. Nitrosodihydrouracil and propylene-nitro- sourea were more active than related compounds. Ethylni trosocyanamide was 730 times more mutagenic than ethylnitrosourea. Fifteen of the test com- pounds (of which 14 were mutagenic) had previously been assayed in rats for carcinogenicity, all with positive results.

Introduct ion

We are currently testing nitrosamides for carcinogenic activity in rats [15,19, 20] and have determined their ease of formation from the corresponding amides [14]. As an adjunct to these studies, we tested 22 N-nitrosamides and related compounds for their ability to cause mutations in Salmonella typhimurium strain TA1535 [1,2]. The test compounds are listed and assigned numbers in Table I and Fig. 1. Unlike nitrosamines, most nitrosamides do not require enzymic activation to convert them to alkylating agents, which are the pre-

132

T A B L E I

M U T A G E N I C I T Y , P H Y S I C A L P R O P E R T I E S , A N D C A R C I N O G E N I C I T Y OF 22 N - N I T R O S O COM PO U N D S

No. C o m p o u n d Mutagenic i ty

Name (Chem. Abs. access ion No.) Highest Muta t ions /d i sh a concen- (mean ± S.D.) t m t i o n used 0~M)

Ni t rosoureas

1. Met hy in i t rosouxea (684-93-5) 4 8 0 0

2. E thy ln i t ro sou rea (759-73-9) 4 3 0 0

3. n -Hexyln i t rosou~ea (18774-85-1) 290

4. 2 - H y d r o x y e t h y l n i t r o s o u r e a (13743-07-2) 380

5. N i t roso -hydan to ic acid 3400

6. Nitroso-L-ci t rul l ine 2500

7. Ni t roso-DL-ci t ru l l ine 2500

8. 1 , 3 - D i m e t h y i n i t r o s o u r e a (13256-32-1 ) 4300

9. T r i m e t h y l n i t r o s o u r e a (3475-63-6) 1 9 , 0 0 0

10. E thy lene -n i t ro sourea (3844-63-1) 440

11. Pr opy lene -n i t ro sou rea 39

12. 1 - N i t r o s o h y d a n t o i n 390

13. 1 -Ni t roso-5 ,6 -d ihydrourac i l (16813-36-8 ) 35

14. 1-Nitroso- 5 , 6 - d i h y d r o t h y m i n e 3 2 0 0

N i t ro soca rbam a t e s 15. E thy l N - m e t h y l n i t r o s o c a r b a m a t e

(615-53-2) 38

16. E thy l N-e thy l rd t rosocaxbamate ( 614-9 5-9 ) 34

17. E thy l N - 2 - h y d r o x y e t h y l n i t r o s o - c a r b a m a t e 31

18. E thy l N-2-acet o xye thy in i t ro so - c a r b a m a t e

19. 1 - N a p h t h y l N - m e t h yini t r os oca rba ma te (n i t roso-carbary l ) 25

Other N-ni t roso c o m p o u n d s 20. N-Methy l -N-n i t rosobenzamide 30

21. 1 -Methyl-1 -nitros o- 3-nitr o guanidine (70-25-7) 34

22. E t h y i n i t r o s o c y a n a m i d e (38434-77-4) 51

0 .24

760 ± I I 0 , 650 ± 20 , 41 ± 20

9 1 0 ± 4 0 , 4 6 ± 2 2 , 1 1 ± 3

4 4 ± I 0 , 2 3 ± 9, 1 6 ± 4

890 -+ 60, 92 ± 33, 20 ± 1 1

1 6 2 5 , 8 + 3 , 4 + - 3

1 3 0 ± 2 0 , 3 6 ± 4, 20 ± 5

1 2 0 ± 30, 74 ± 10, 32 ± 14

8 6 0 ± 2 5 0 , 7 - + 1 ,8 ± 3

1900 ± 300, 520 ± 130, 9 ± 3

1700 -+ 100, 310 ± 50, 24 ± 6

1000 ± 100, 120 -+ 40, 15 ± 5

1100 ± 100, 170 + 50, 60 ± 25

160 ± 50, 120 ± 20, 18 ± 5

2100 ± 200, 820 ± 60, 85 ± 30

2 5 0 0 ± 400 , 350 ± 40 , 5 ± 1

6 5 0 ± I 0 0 , 300 ± 80, 14 ± 4

1 5 0 0 ± i 0 0 , 2 8 0 ± 30, 47 ± 6

1 9 0 0 ± 200, 4 1 0 ± 70 , 98 ± 26

390 ± 170 , 640 +- 160 , 100 -+ 30

120 ± 80 f, 1 5 0 0 ± 100 , 83 ± 44

1 5 0 0 ± 200 f 3 0 0 0 + 1 0 0 0 , 3 4 + 7

0 ± 0 f , 270 +- 150 , 65 +- 20

a Resul ts were ob t a ined a t the " h i g h e s t c o n c e n t r a t i o n t e s t e d " , and 0.1 and 0.01 X t h a t c o n c e n t r a t i o n , in order . Resul ts are r eco rded as m e a n -+ S.D. for 4 - -5 dishes, wi th the m e a n given to 2 s ignif icant figures.

b Co ncen t r a t i on causing 100 mu ta n t s / d i s h . c +, carc inogenic ; -- , nonca rc inogen ic ; n. t . , no t tes ted; u. t . , u n d e r tes t a t this Ins t i tu te . d In this case, each c o n c e n t r a t i o n was 0 .5 X the prev ious one. e No t m e a s u r e d because of insolubi l i ty . f A light " l a w n " ind ica ted general tox ic i ty . g S.S. Mirvish e t al., p ape r in p repa ra t ion .

sumed active mutagens [3,11]. Hence the tests were performed without the use of microsomes. Certain physical properties were also measured.

133

S t a n d a r d

c o n c e n t r a -

t i o n 0zM) b

E t h e r - -H 2 0 S o l u b i l i t y Ha l f - l i f e C a r c i n o g e n i c i t y

p a r t i t i o n i n w a t e r a t p H 7,

c o e f f i c i e n t a t 2 5 ° C 37 ° C (h) + / _ c Refs .

a t 2 3 - - 2 5 ° C ( r aM)

100 1.1 140 0.5 + 3

730 3.4 11 0.5 + 3,19

>300 >500 1.0 0.4 n.t. --

41 0.39 260 1.0 + 20

> 3 4 0 0 < 0 . 0 1 2 1 0 0 I . I + - - g

1 5 0 0 < 0 . 0 1 48 0 . 2 5 n . t . - -

1 1 0 0 < 0 . 0 1 2 .0 0 . 2 3 n . t . - -

1 4 0 0 1 .4 2 5 0 11 + 3

7 0 0 0 1 .6 oo > 5 0 + 3

1 6 0 . 0 2 7 7 0 0 0 . 1 3 + 3

3 .3 0 . 0 1 5 8 9 0 2 .4 n . t . - -

1 2 0 . 4 5 7 7 0 1.1 + 19

2.9 0 . 2 2 77 1 .5 + 1 5

38 0 . 4 8 1 7 0 2 .6 n . t . - -

1 .7 2 4 2 8 0 1 4 + 3

1 .7 8 5 4 5 32 + 3

0 . 8 0 1 . 5 oo 0 . 0 8 n . t . - -

0 . 0 0 2 4 31 77 2 4 n . t . - -

0 . 2 4 > 5 0 0 0 . 2 4 _ e + 4

0 . 3 4 > 5 0 0 5 .0 0 . 8 2 + - - g

0.58 2.8 30 1.5 + 3

1 .0 1 2 1 4 0 4 .4 + 1 9

Materials and methods

Ethyl 2-hydroxyethylcarbamate Ethanolamine (134 g, 2 mol) was dissolved in 600 ml anhydrous ether and

cooled in ice. Ethyl chloroformate (108 g, 1 tool) dissolved in 300 ml ether was slowly added. The mixture was stirred overnight at room temperature and filtered with the aid of Celite. The filtrate was concentrated in vacuo to yield

134

R-?.CONH 2

NO

No. i: R = Me

No. 2: R = Et

No. 3: R=CH3.(CH2) 5

No. 4: R = HO.CH2.CH 2

No. 5: R = HOOC.CH 2

Nos. 6 and 7:

R = HOOC.CH(NH2).(CH2) 3

NO I

/N o~ ~ H 2 HN /CH 2

~cH 2 No. ii

R.N.COOEt I NO

No. 15: R = Me

NO. 16: R=Et

No. 17: R=HOCH2.CH 2

No. 18: R = CH~CO.OCH2.CH 2 j

R' / NO

R.N.CON l s \ N~ ~0 R" o~/ ?H 2

NO. 8: R=R' =Me HN- /

= H ~ C H 2 R"

No. 9: R=R ' =R" =Me No. 10

NO I

Oc/N~CH2

NO. 12

O.CO. N. CI-I 3

No. 19

NO I

/N OC ~CH 2

HN CH.R

~co / NO. 13." R=H

NO. 14: R=Me

~ CO.N.CH 3

NO

NO. 20

NH II

CH3.N.C.NH.NO 2 CH3.CH2.N.C -=N

NO NO

No. 21 No. 22

Fig. 1° Structures o f the 2 2 c o m p o u n d s s h o w n in Table I.

a colorless liquid, which was purified by distillation at 134--136°C (1 mm); yield, 90 g (68%); nmr (CDC13) 6 1.23 (t, 3, OCH2CH3), 3.38 (t, 2, NHC__HH2), 3.68 (t, 2, HOCH2), 4.11 (m, 3, O_CH2CH3 and OH), and 5.86 (br. s, NH).

Ethyl 2-hydroxyethyl-nitrosocarbamate (No. 17) Ethyl 2-hydroxyethylcarbamate (13.3 g, 0.1 mol) was dissolved in 50 ml

water, acidified to pH 0.5 with conc. HC104, and cooled in ice. Sodium nitrite (27.6 g, 0.4 mol) was slowly added while the pH of the reaction mixture was kept at 0.5--1.0 with HC104. The mixture was then stirred for 1 h at 0°C and extracted with 3 × 150 ml CH2C12. The dried CH2C12 solution (Na2SO4) was concentrated in vacuo to yield a pink oil. In one experiment, the nitrite ester of the alcohol was formed, as indicated by absorption peaks at 367, 353 ,341 and 330 nm (methanol); this was removed as methyl nitrite by bubbling nitrogen through a methanol solution at 23°C. Yield, 15 g (93%); uv (CH3OH) 386 (77), 402 (99), and 420 nm (94); nmr (DMSO-d6) 6 1.36 (t, 3, CH2CH3, J = 7 Hz), 4.08 (t, 2, HOCH2, J = 5 Hz), 4.46 (q, 2, OCH2CH3, J = 7 Hz), 4.80 (t, 2, CH2N, J = 5 Hz), and 5.56 (s, 1, OH).

n-Hexylnitrosourea (No. 3) n-Hexylurea was synthesized by boiling n-hexylamine (Eastman Organic

Chemicals) for 3 h with excess urea in water adjusted with HCI to pH 4--5. The precipitate was recrystallized from ethanol, m.p. 108--109°C. The hexylurea (2.5 g, 0.0173 mol) was dissolved in a minimum volume of 50% aqueous acetic

135

acid, NaNO2 (10.3 g, 0.15 mol) was added slowly, and the reaction was stirred for 1.5 h. Upon addition of cold water, a precipitate formed. This was collected by filtration, washed with 150 ml cold water, and dried in vacuo to yield 1.8 g (60%) of pale yel low solid; m.p. 76--78°C; uv (CH2CI2) 239 (5970), 384 (74), 396 (106), and 414 nm (102); nmr (DMSO-d6) 0.86 (t, 3, CH2CH3, J = 6 Hz), 1.24 (m, 8, NCH2(CH2)4CHa), 4.12 (t, 2, NCH2, J = 6 Hz) and-7.80 (br. s., NH2). Elemental analysis was in accord with C7H~sNaO2.

Remaining nitrosamides These were synthesized by known methods as follows; Nos. 1, 2, 6, 7, 15, 16

[13]; Nos. 8, 9, 10 and 21 [3]; No. 11 [9]; No. 13 [10,15]; No. 22 [ 1 7 / ; a n d No. 19 [5]. The syntheses of Nos. 5, 12, 14, 18, and 20 will be described in reports on their carcinogenicity (S.S. Mirvish et al., to be published). All com- pounds were stored at --15°C and their puri ty was checked by the melting point and characteristic absorption at 390--420 nm [14].

Physical properties The visible-ultraviolet absorption was used to determine solubility in water

at 25°C, ether : water partition coefficient at 22--25°C [16], and half-life in 0.2 M Na phosphate buffer at pH 7 and 37°C. For the last 2 parameters, the aqueous nitrosamide concentration corresponded initially to an absorptivity of 0.5--1.0 at the 390--420 nm maximum (about 0.5--1.0 mg/ml).

Mutagenesis tests The procedures of Ames et al. [1,2] were followed, using S. typhimurium

TA1535. Solutions of 10 mg compound/ml water or (for Nos. 3, 13, 20, and 21) dimethylsulfoxide (DMSO) were freshly prepared. No. 7 was handled as an aqueous suspension. Aqueous solutions were adjusted to pH 4--6 and filter- sterilized (except for No. 7). Serial 1/10 dilutions in water were prepared at 22--25°C, except that all dilutions of Nos. 3 and 13 and the first dilution of No. 20 were made in DMSO. Within 1 h from the time the solutions were pre- pared, 0.1 ml culture (with 2 × 108 bacteria) and 0.1 ml solution of test com- pound were mixed with 2.0 ml melted top agar at 45 ° C, poured onto a 60-mm petri dish containing minimal agar, and incubated for 48 h at 37 ° C. The revert- ant colonies (but not the surviving bacteria) were then counted. The pH of each agar layer was 6.8--7.0. For each dilution, 4--5 dishes were examined. For 8 pairs of results where the same dilution of compound was assayed on different days, the mean difference between the 2 quintuplet means was 38%. The mean result wi thout test compound or with 0 .1 'ml DMSO/dish was 6 mutants/dish.

Results and discussion

Nitrosamide stability, as indicated by the half-life, was determined at the pH and temperature of the bacterial incubation, i.e. pH 7 and 37°C (Table I). The nitrosamide solutions were stored less than 1 h at 23--25°C and pH 4--6 before they were plated with the bacteria. Since nitrosamide stability is increased by lowering the temperature and lowering the pH from 7 to 4 [17], the com- pounds should have been much more stable during storage than during incuba-

136

tion. For 0.1% solutions of ethyl N-2-hydroxyethylnitrosocarbamate (No. 17), which was the most unstable compound, mutagenicity was unaffected when the solution was stored at pH 4.3 for l h at 23--25°C before plating. Hence decomposition of the compounds was probably not appreciable before plating, though it could have occurred after plating. The high stability of trimethylni- trosourea (No. 9) is attributed to the lack of an N--H group. Nitrosocarbamates were more stable than the corresponding nitrosoureas, except for ethyl 2-hydroxyethylnitrosocarbamate (No. 17), which was much more unstable than 2-hydroxyethylnitrosourea (No. 4), probably because of a cyclic nucleo- philic attack by the hydroxy group on the carbonyl carbon, facilitated by the electron-withdrawing e thoxy group.

Fifty pg/ml of Nos. 1, 12 and 13 were mutagenic for S. typhimurium strain TA1535 but not for strain TA1538, indicating that the mutations were due to base-pair substitutions [1,2]. Of the 22 compounds tested with strain TA1535, 20 had mutagenic activity, with at least one concentration producing >100 mutants/dish (Table I). For some compounds, e.g. Nos. 13 and 18, activity increased consistently as the concentration rose over a 103--104-fold range. This increase was especially rapid for Nos. 2, 8, 9, 15 and 21. For other compounds, e.g. Nos. 20 and 21, there was an increase followed by a decrease attributed to toxicity. The toxic and mutagenic concentrations of ethylnitrosocyanamide (No. 22) were especially close. To compare different compounds, we recorded the "standard mutagenic concentrat ion", i.e. the minimum molar concentra- tion in the agar causing 100 mutants/dish (Table I). This was obtained from plots of log concentration against log activity, and corresponded to a mutat ion frequency of 5 × 10 -7, if all bacteria survived the treatment. Compound A was said to show C X the activity of B, if the standard concentration of A was I /C × that of B.

Correlations were examined between the logarithms of standard mutagenic concentration and those of the 3 physical properties listed in Table I, for all 22 compounds (after equating values of > or < a number to that number, and infi- nite solubility to 2100 mM). Standard mutagenic concentration and partition coefficient were negatively correlated, with correlation coefficient r = --0.50. The correlation was significant, with P < 0.05. This means that mutagenic activity was positively correlated with the partition coefficient, perhaps because the bacteria absorbed more of the lipid-soluble compounds. Partition coefficient was negatively correlated with solubility (r ='--0.44, P < 0.05), as expected [16], and positively correlated with half-life (r = +0.42, P = 0.05).

Standard mutagenic concentration varied by a factor of 2.9 × 106, from 0.0024 pM (No. 18) to 7000 pM (No. 9). The four most active compounds were (in order) ethyl.2-acetoxyethylnitrosocarbamate (No. 18), nitrosocarbaryl (No. 19), methylnitrosobenzamide (No. 20), and methylnitrosonitroguanidine (No. 21). The inactivity of nitrosohydantoic acid (No. 5) and low activity of the nitrosocitrullines (Nos. 6 and 7) may have been due to the ionized state of these carboxylic acids at neutral pH, which would hinder bacterial transport. (Nos. 6 and 7) may have been due to the ionized state of these carboxylic acids at neutral pH, which would hinder bacterial transport.

With respect to changes in the alkyl or alkylidene groups, ethylnitrosourea (No. 2) was 0.14X as active as the methyl analog No. 1, and the hexyl analog

1 3 7

No. 3 was inactive, possibly because of its low solubility in water. Propylene- nitrosourea (No. 11) was 5X more active than ethylene-nitrosourea (No. 10). Nitrosodihydrouracil (No. 13) was 4X more active than the 5-membered-ring analog nitrosohydantoin (No. 12). Nitrosodihydrothymine (No. 14) was 0.08X as active as its parent compound, No. 13.

With respect to changes in the functional groups, ethyl N-methyl-, N-ethyl-, and N-2-hydroxyethyl-nitrosocarbamates (Nos. 15, 16 and 17) were at least 50X more mutagenic than the corresponding nitrosoureas Nos. 1, 2 and 4. The mutagenicity of ethyl 2-hydroxyethylnitrosocarbamate (No. 17) was increased 330X on acetylation to give No. 18, possibly because No. 18 was more stable. The activity was increased 730X on abstraction of water from ethylnitrosourea (No. 2) to give ethylnitrosocyanamide (No. 22). Activity decreased strongly from methyl- to dimethyl- to trimethyl-nitrosourea (Nos. 1, 8 and 9).

The mutagenicity of Nos. 1, 2, 15, 19 and 21 was previously reported for various bacterial systems [1,5,8,12,18]. Our results for ethylnitrosocyan- amide (No. 22) confirm the reports of Endo and Takahashi [6,7] that nitroso- cyanamides are highly mutagenic. Fifteen of the test compounds had previous- ly been assayed for carcinogenicity in the rat, all with positive results, though nitrosohydantoic acid (No. 5) was only a weak carcinogen (Table I). Of these compounds, all except No. 5 were mutagenic.

Acknowledgements

We thank Dr. D.B. Clayson (of this Institute) for his advice, Dr. B.N. Ames (Biochemistry Dept., University of California at Berkeley) for giving us the bacterial strains; and Mrs. Sonja Morton, Mr. J. Sams and Mr. S. Arnold for technical assistance. This work was supported by Public Health Service contract NO1 CP33278 from the Division of Cancer Cause and Prevention, the National Cancer Institute, and by grant BC-39C from the American Cancer Society.

References

1 A m e s , B.N. , F .D . Lee a n d W.E. D u r s t o n , A n i m p r o v e d b a c t e r i a l t e s t s y s t e m f o r t he d e t e c t i o n a n d clas- s i f i c a t i on o f m u t a g e n s a n d c a r c i n o g e n s , P roc . NaU. A c a d . Sci. (U.S. ) , 7 0 ( 1 9 7 3 ) 7 8 2 - - 7 8 6 .

2 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 imp le tes t s y s t e m c o m b i n i n g l iver h o m o g e n a t e s f o r a c t i v a t i o n a n d b a c t e r i a f o r d e t e c t i o n , P roc . Nat l . A c a d . Sci. (U.S. ) , 7 0 ( 1 9 7 3 ) 2 2 8 1 - - 2 2 8 5 .

3 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~ihl, 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 be i 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 .

4 E i s e n b r a n d , G. , O . U n g e r e r a n d R . P r e u s s m a n n , T h e r e a c t i o n o f n i t r i t e w i t h pes t i c ides . II. F o r m a t i o n , c h e m i c a l p r o p e r t i e s a n d c a r c i n o g e n i c ac t i v i t y o f the N - n i t r o s o de r iva t ive o f N - m e t h y l - l - n a p h t h y l c a r b a m a t e ( c a r b a r y l ) , Fd . C o s m e t . Toxicol .~ 13 ( 1 9 7 5 ) ' 3 6 5 - - 3 6 7 .

5 E l e s p u r u , R. , W. L i j i n k y a n d J .K . S e t l o w , N i t r o s o c a r b a r y l as a p o t e n t m u t a g e n o f e n v i r o n m e n t a l sig- n i f i c a n c e , N a t u r e , 2 4 7 ( 1 9 7 4 ) 3 8 6 - - 3 8 7 .

6 E n d o , H. a n d K. T a k a h a s h i , A n i t r o s a t e d a rg in ine de r iva t ive , a p o w e r f u l m u t a g e n , B i o c h e m . B i o p h y s . Res . C o m m u n . , 52 ( 1 9 7 3 ) 2 5 4 - - 2 6 2 .

7 E n d o , H. a n d K. T a k a h a s h i , I d e n t i f i c a t i o n a n d p r o p e r t y o f t h e m u t a g e n i c p r inc ip l e f o r m e d f r o m a f o o d c o m p o n e n L m e t h y l g u a n i d i n e , a f t e r n i t r o s a t i o n in s i m u l a t e d gas t r i c ju ice , B i o c h e m . B i o p h y s . Res . C o m m . , 54 ( 1 9 7 3 ) 1 3 8 4 - - 1 3 9 2 .

8 H ince , T .A . a n d S. Neale , A c o m p a r i s o n o f t he m u t a g e n i c a c t i o n o f t he m e t h y l a n d e t h y l de r iva t ives o f n i t r o s a m i d e s a n d n i t r o s a m i d i n e s o f Escherichia coli, M u t a t i o n Res . , 2 4 ( 1 9 7 4 ) 3 8 3 - - 3 8 7 .

9 J o h n s t o n , T .P . , G.S . McCa leb a n d J .A . M o n t g o m e r y , T h e s y n t h e s i s o f a n t i n e o p l a s t i e agen t s . N - n i t r o - s o u r e a s , I, J . Med. C h e m . , 6 ( 1 9 6 3 ) 669 - - -681 .

138

10 Johnston, T.P. and P.S. Opliger, The synthesis of potent ia l anticancer agents. N-nitrosoureas III. 1,5- Bis(2-chloroethyl-l-ni trosobiuret and related derivatives of biurets, biu~eas and carboxamides, J. Med. Chem., 10 (1967) 675--681.

11 Magee, P.N. and J.M. Barnes, Carcinogenic nitroso compounds, Adv. Cancer Res., 10 (1967) 163-- 246.

12 McCann, J., E. Choi, E. Yamasaki and B.N. Ames, Detection of carcinogens as mutagens in the Salmo- nel la/microsome test: assay of 300 chemicals, Proc. Natl. Acad. Sci. (U.S.), 72 (1975) 5135--5139.

13 Mirvish, S.S., Kinetics of ni trosamide formation from alkylureas, N-alkylurethans, and alkylguani- dines: possible implicat ions for the etiology of human gastric cancer, J. Natl. Cancer Inst., 46 (1971) 1183--1193.

14 Mirvish, S.S., Format ion of N-nitrnso compounds: chemistry kinetics, and in vivo occurrence, Toxi- col. Appl. Pharmacol., 31 (1975) 325--351.

15 Mirvish, S.S. and H. Gareia, 1-Nitrosoo5,6-dihydrouracil: Induct ion of liver cell carcinomas and kidney adenomas in the rat, Z. Krebsforsch., 79 (1973) 304--308.

16 Mirvish, S.S., P. Issenberg and H.C. Sornson, Air-water and ether-water dis t r ibut ion of N-nitroso compounds: implicat ions for laboratory safety, analytical methodology and carcinogenicity for the rat esophagus, nose and liver, J. Natl. Cancer Inst., 56 (1976) 1125--1129.

17 Mirvish, S.S., D.L. Nagel and J. Sams, Methyl- and ethylni t rosocyanamide. Some properties and reac- tions, J. Org. Chem., 38 (1973) 1325--1329.

18 Neale, S., Effect of pH and temperature on nitrosamide-induced muta t ion in Escherichia co/i, Muta- t ion Res., 14 (1972) 155--164.

19 Pelfrsne, A., S.S. Mirvish and H. Garcia, Carcinogenic action of e thylni t rosocyanamide (ENC), 1-nitro- sohydantoin (NH) and ethylni t rosourea (ENU) in the rat, Proc. Amer. Assoc. Cancer Res., 16 (1975) 117.

20 Pelfrene, A., S.S. Mirvish and B. Gold, Induct ion of malignant bone tumors in rats by 1-(2-hydroxy- ethyl)- l -ni t rosourea, J. Natl. Cancer Inst., 56 (1976) 445--446.