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Mutagenicity studies with nitrilotriacetic acid (NTA) and citrex S-5 in drosophila
277
Mutation Research, 40 (1976) 277--280 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
Short communication MUTAGENICITY STUDIES WITH N I T R I L O T R I A C E T I C ACID (NTA) AND CITREX S-5 IN D R O S O P H I L A
P.G.N. K R A M E R S Department of Radiation Genetics and Chemical Mutagenesis, State Universityof Leiden, Wassenaarseweg 72, Leiden (The Netherlands)
(Received September 22nd, 1975) (Revision received February 18th, 1976) (Accepted March 1st, 1976)
Both NTA and Citrex S-5 are potential substitutes for phosphates in household detergents. NTA [N(CH2COOH)s, CAS registry No. 139-13-9] was proposed in the U.S.A., b u t was suspended in 1970 after the appearance of a report pointing at the possible teratogenic activity of NTA in combination with a heavy metal, by increasing the transmission of these metals across the placental barrier [4]. Citrex S-5 is a mixture of the sodium salts of several sulfo-polycarboxylic acids. More information a b o u t the product can be obtained from the manufacturer: Citrex S.A., 249 Pastorijstraat, B-3300 Tienen, Belgium. It is obvious that c o m p o u n d s in such widespread use require a thorough study of their possible undesirable ecological or toxicological properties. In this report the results are described of mutagenicity studies with Drosophila melanogaster. The relevance of this test system for detecting potential mutagens and for specifying the damage detected has been discussed b y Sobels [11,12]. Both c o m p o u n d s were investigated for their ability to induce sex-linked recessive lethal mutations and dominant lethal mutations. Details on materials and methods are summarized in a concise tabulated form (Tables I and II), which could perhaps serve as an example of h o w an efficient format for presenting routine Drosophila testing data could be devised [2]. An a t t e m p t is made to include all experimental parameters that are relevant, irrespective of whether positive or negative results are involved. Such a format had already been proposed in simpler form in 1952 [9]. The results of the experiments are given in Tables III and IV. Table III shows that these experiments do n o t indicate the ability of NTA or Citrex S-5 to induce sex-linked recessive lethals in Drosophila. The first test with NTA suggested a possible increase of the mutation frequency in broods A and B. This was, however, n o t observed in replicate experiments. Statistically one can estimate [6] the frequency of recessive lethals which would represent a significant in-
278 TABLE I SEX-LINKED RECESSIVE LETHAL TESTS, MATERIALS
AND METHODS
NTA
Source o f the compound Way o f administration Number of experiments Exposure
Toxicity Fertility Genotype treated dd Age o f d d a t b e g i n n i n g of t r e a t m e n t Age o f d d a t m a t i n g G e n o t y p e o f P-99 Age of 99 at mating Mating scheme Brooding scheme Scoring criteria in F 2 Culturing temperature
J.T. Baker Chemicals, lot No. A 0316, 99%. (a) F e e d i n g o f a d u l t m a l e s o n glass filters, p l a c e d o n t h e t e s t s o l u t i o n ; (b) i n j e c t i o n o f a d u l t m a l e flies. F e e d i n g , 3; i n j e c t i o n , 1. F e e d i n g : 3 d a y s ( e x p t 2, 4 d a y s ) o n a s a t u r a t e d s o l u t i o n r e p l a c e d d a i l y ; t h e s o l u t i o n w a s m a d e u p i n b o i l i n g w a t e r a t 5 0 m M ( 9 . 5 5 m g / m l ) w i t h 5% sucrose; after 1 h some precipitate had formed, therefore the concentration a c t u a l l y i n g e s t e d w a s l o w e r . I n j e c t i o n : 0.2/~1 o f a 1 0 m M s o l u t i o n w i t h 0 . 7 % NaC1. Feeding: non-toxic at the conc. used. Injection: within 6 days after treatment 40 out of 97 injected males had died. F e e d i n g : n o t n o t i c e a b l y a f f e c t e d . I n j e c t i o n : 4 6 , 4 9 a n d 2 8 % o f live m a l e s fertile in b r o o d s A , B a n d C, r e s p e c t i v e l y . Wild t y p e ( O r e g o n - K ) . Feeding, 1--2 days; injection, 4 days. Feeding, 4--5 days; injection, 4 days. Muner-5. 2--6 days. Standard Muller-5 scheme. Two or three 3-day matings; 4 fresh virgins for each mating; dd mated individually. W h e n less t h a n 4 r o u n d - e y e d m a l e s w e r e p r e s e n t in a n F 2 vial, z e t e s t s w e r e made to confirm possible (semi)lethality. 25°C.
C i t r e x S-5; d a t a s.re o n l y g i v e n w h e n d i f f e r e n t f r o m t h e N T A t e s t s Source of the compound Number of experiments Exposure Toxicity Fertility
"De KAok" soap factories, Heerde, The Netherlands 2 (feeding). 3 days feeding on a 1% solution, replaced daily, prepared from the 50% a q u e o u s s o l u t i o n , ( t h e c u r r e n t t r a d e f o r m u l a t i o n ) , w i t h 5% s u c r o s e . Non-toxic at concentration used. Not noticeably affected.
T A B L E II DOMINANT LETHAL TESTS, MATERIALS AND METHODS NTA Way of administration Number of experiments Exposure Genotype treated dd and 99 Age o f d d a t t r e a t m e n t Age o f 9 9 a t m a t i n g Egg-laying scheme
Counting procedure Culturing temperature
Feeding. 2. See T a b l e I. Oregon-K. 1 or 2 days. 5 days. T r e a t e d m a l e s a n d virgin f e m a l e s w e r e m a t e d s i n g l y i n t h e vials o f e g g - l a y i n g units [10]. Eggs were collected from two or three subsequent overnight egg*laying p e r i o d s o f a b o u t 1 8 h. Immediately after each egg-laying period the eggs were counted; unhatched eggs w e r e s c o r e d a b o u t 3 0 h l a t e r . 25°C.
C i t r e x S-5; d a t a a r e o n l y g i v e n w h e n t h e y a r e d i f f e r e n t f r o m t h o s e i n t h e N T A t e s t s Number of experiments
1.
279 T A B L E III FREQUENCIES OF SEX-LINKED O F N T A O R C I T R E X S-5 Compound
Way of administration
Conc.
NTA
feeding
50 mM
NTA
injection
10 mM
C i t r e x S-5
feeding
1%
RECESSIVE
Expt. No.
MUTATIONS
Brood A days 0--3
AFTER
Brood B days 3--6
ADMINISTRATION
Brood C days 6--9
nehr.
% 1 a
cnhr.
% 1a
nchr.
% I. a
1 2 3
727 1058 1042
0.55 0.0 0.0
749 1025 1125
0.53 0.10 0.18
716 1148
0.26 0.0
1--3
2627
0.14
2399
0.24
1864
0.11
~9 5 0
0.11,
911
0.11
290
0.0
1
697
0.14
730
2
388
0.23
868
0.41 0.23
772 874
0.13 0.24
1585
0.19
1598
0.31
1646
0.18
3136
0.13
2997
0.23
3118
0.13
1--2 Control
LETHAL
inj., f e e d i n g
a F o r statistical c o m m e n t , see text. n c h r . , n u m b e r o f c h r o m o s o m e s t e s t e d ; % 1., p e r c e n t l e t h a l s .
crease (at the 5% level), given the size of the present experiments. For the postmeiotic stages (broods A and B taken together) these percentages are 0.40 for NTA and 0.47 for Citrex S-5. (In other words, a 2-fold increase of the spontaneous mutation frequency, for NTA, and a 2.5-fold increase for Citrex S-5, should have been detectable with 95% probability.) As shown in Table IV, the dominant-lethal tests do n o t show decreased egg hatchability after treatment of the flies with NTA or Citrex S-5. With NTA a few other mutagenicity studies have been carried out. Experiments with bacteria, yeast and Neurospora showed neither induction of gene mutations [13--16] nor a modifying effect of NTA on the mutation rate induced by EMS or X-rays [14,15]. In root-tip cells of Vicia faba, as well as in rat kangaroo cells and human leukocytes in culture, NTA induced chromatid aberrations, b u t only at sub-lethal concentrations (2.5--6 mM) over long exposure
TABLE IV PERCENTAGE Expt. No.
UNHATCHED
Compound, series
1
NTA Control
2
NTA Control
1+2
NTA Control C i t r e x S-5 Control
E G G S A F T E R G I V I N G N T A O R C I T R E X S-5 T O D R O S O P H I L A M A L E S
I s t egg-l, p e r i o d
2 n d egg-l, p e r i o d
3 r d egg-l, p e r i o d
all egg-l, p e r i o d s
n. eggs
% unh.
n. eggs
n. eggs
n . eggs
% unh.
1296 1671
22.1 19.9
1057 1360
7.1 7.2
2353 3031
15.3 14.2
2235 2511
12.9 17.6
4979 4905
20.7 22.4
7332 7936
19.0 19.3
8913 8557
21.3 20.9
3162 3033
32.3 31.6
5751 5524
% unh.
15.0 13.2
2694 2394
% unh.
27.3 27.2
egg-L p e r i o d , e g g - l a y i n g p e r i o d ; n. eggs, n u m b e r o f eggs l a i d ; % u n h . , p e r c e n t a g e o f u n h a t c h e d eggs.
280
times (1--5 days) [1,7,8]. In a mouse dominant-lethal test, no effect of NTA was observed when the c o m p o u n d was given i.p. at 125 mg/kg or orally at higher doses [ 3]. Likewise no induction of translocations was found when mice were given a drinking solution containing 0.1% NaCaNTA for seven weeks [5]. In summary, the N T A data make it clear that (1) all tests for gene mutations and for transmitted genetic effects produced negative results, and (2) the only positive data indicate chromosome breakage effects in somatic cells in vitro, at high exposure levels. Consequently, the available information provides no evidence that N T A presents a genetic hazard. Unfortunately, no data are available on mutagenicity of Citrex S-5, with the exception of the Drosophila results. For a proper comparison of the two compounds with respect to mutagenic properties, Citrex should likewise be investigated in other systems. This work was supported by the Netherlands Ministry of Public Health and Environmental Hygiene. I am indebted to Professor F.H. Sobels for his encouragement, and for critically reading the manuscript. I thank Marja de Jong, Leny Hoogteyling and Antoinette Pex for their indispensable technical assistance. References 1 Bora, K.C., Effects of nitrilotriacetic acid (NTA) on c h r o m o s o m e replication and structure in h u m a n cells, 6 t h ann. m e e t i n g American E n v i r o n m e n t a l Mutagen Society, Miami, 1975. 2 De Serres, F.J., H.V. Mailing, F.H. Sobela, E. Vogel and J.S. Waseom, New section: Genetic Toxicology Testing, M u t a t i o n Res., 31 (1975) 273--274. 3 Epstein, S.S., E. Arnold, J. Andrea, W. Bass and Y. Bishop, D e t e c t i o n of chemical m u t a g e n s by the d o m i n a n t l e t h a l assay in the mouse, Toxicol. Appl. Pharmaeol., 23 (1972) 288--325. 4 H a m m o n d , A.L., Phosphate replacements: problems w i t h the washday miracle, Science, 172 (1971) 361--363. 5 Jorgenson, T.A., G.W. Newell, L.G. Seharpf, P. Gribling, M. O'Brien and D. Chu, Study of the mutagenic p o t e n t i a l of nitrilotriacetic acid (NaCaNTA) in mice by the t ra ns l oc a t i on test, 6 t h ann. me e t i ng Am erican E n v i r o n m e n t a l Mutagen Society, Miami, 1975. 6 Kastenbaum, M.A. and K.O. Bowman, Tables for d e t e r m i n i n g the statistical significance of m u t a t i o n frequencies, M u t a t i o n Res., 9 (1970) 527--549. 7 Kihlman, B.A., R o o t tips for s t u d y i n g the effect of chemicals on chromosomes, in A. H o n a e n d e r (ed.) Chemical Mutagens, Principles and Methods for Their Detection, P l e num Press, New York, 1971, pp. 489--514. 8 Kthlman, B.A. and S. Sturelid, Niti'i]otriacetic acid (NTA) and c h r o m o s o m e breakage, EMS Newsletter, 3 (1970) 32--33. 9 0 s t e r , I.I., Chemicals found inactive as mutagens in Drosophila, D.I.S., 26 (1952) 116--117. 10 Sankaranarayanan, K., The effects of nitrogen and oxygen t r e a t m e n t s on the frequencies of X-ray induced d o m i n a n t lethals and on the physiology of the sperm in D r o s o p h i l a m e l a n o g a s t e r , Mut a t i on Res., 4 (1967) 641--661. 11 Sobels, F.H., The role of Drosophila in the field of m u t a t i o n research, Arch. Genetik, 45 (1972) 101-125. 12 Sobels, F.H., The advantages of Drosophila for m u t a t i o n studies, Mut a t i on Res., 26 (1974) 277--284. 13 Stine, G.J. and P. Adams, The effects of nitrilotriacetic acid on d e v e l o p m e n t of N e u r o s p o r u crussa, Microbial Genet. BuLl., 36 (1974) 8--10. 14 Stine, G.J. and A.A. Hardigree, Effects of nitrilotriacetic acid on E s c h e r i c h i u c o l i K-12, EMS Newsletter, 5 (1971) 38--39. 15 Stine, G.J. and A.A. Hardigree, Effect of nitrilotriacetic acid on grow t h and m a t i n g in strains of E s c h e r i c h i u coli K-12, Can. J. MicrobioL, 18 (1972) 1159---1162. 16 Zetterberg, G., Negative results with nitrilotriacetic acid (NTA) as an i n d u c e r of gene m u t a t i o n in some microorganisms, EMS Newsletter, 3 (1970) 31--32.
Mutation Research, 40 (1976) 277--280 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
Short communication MUTAGENICITY STUDIES WITH N I T R I L O T R I A C E T I C ACID (NTA) AND CITREX S-5 IN D R O S O P H I L A
P.G.N. K R A M E R S Department of Radiation Genetics and Chemical Mutagenesis, State Universityof Leiden, Wassenaarseweg 72, Leiden (The Netherlands)
(Received September 22nd, 1975) (Revision received February 18th, 1976) (Accepted March 1st, 1976)
Both NTA and Citrex S-5 are potential substitutes for phosphates in household detergents. NTA [N(CH2COOH)s, CAS registry No. 139-13-9] was proposed in the U.S.A., b u t was suspended in 1970 after the appearance of a report pointing at the possible teratogenic activity of NTA in combination with a heavy metal, by increasing the transmission of these metals across the placental barrier [4]. Citrex S-5 is a mixture of the sodium salts of several sulfo-polycarboxylic acids. More information a b o u t the product can be obtained from the manufacturer: Citrex S.A., 249 Pastorijstraat, B-3300 Tienen, Belgium. It is obvious that c o m p o u n d s in such widespread use require a thorough study of their possible undesirable ecological or toxicological properties. In this report the results are described of mutagenicity studies with Drosophila melanogaster. The relevance of this test system for detecting potential mutagens and for specifying the damage detected has been discussed b y Sobels [11,12]. Both c o m p o u n d s were investigated for their ability to induce sex-linked recessive lethal mutations and dominant lethal mutations. Details on materials and methods are summarized in a concise tabulated form (Tables I and II), which could perhaps serve as an example of h o w an efficient format for presenting routine Drosophila testing data could be devised [2]. An a t t e m p t is made to include all experimental parameters that are relevant, irrespective of whether positive or negative results are involved. Such a format had already been proposed in simpler form in 1952 [9]. The results of the experiments are given in Tables III and IV. Table III shows that these experiments do n o t indicate the ability of NTA or Citrex S-5 to induce sex-linked recessive lethals in Drosophila. The first test with NTA suggested a possible increase of the mutation frequency in broods A and B. This was, however, n o t observed in replicate experiments. Statistically one can estimate [6] the frequency of recessive lethals which would represent a significant in-
278 TABLE I SEX-LINKED RECESSIVE LETHAL TESTS, MATERIALS
AND METHODS
NTA
Source o f the compound Way o f administration Number of experiments Exposure
Toxicity Fertility Genotype treated dd Age o f d d a t b e g i n n i n g of t r e a t m e n t Age o f d d a t m a t i n g G e n o t y p e o f P-99 Age of 99 at mating Mating scheme Brooding scheme Scoring criteria in F 2 Culturing temperature
J.T. Baker Chemicals, lot No. A 0316, 99%. (a) F e e d i n g o f a d u l t m a l e s o n glass filters, p l a c e d o n t h e t e s t s o l u t i o n ; (b) i n j e c t i o n o f a d u l t m a l e flies. F e e d i n g , 3; i n j e c t i o n , 1. F e e d i n g : 3 d a y s ( e x p t 2, 4 d a y s ) o n a s a t u r a t e d s o l u t i o n r e p l a c e d d a i l y ; t h e s o l u t i o n w a s m a d e u p i n b o i l i n g w a t e r a t 5 0 m M ( 9 . 5 5 m g / m l ) w i t h 5% sucrose; after 1 h some precipitate had formed, therefore the concentration a c t u a l l y i n g e s t e d w a s l o w e r . I n j e c t i o n : 0.2/~1 o f a 1 0 m M s o l u t i o n w i t h 0 . 7 % NaC1. Feeding: non-toxic at the conc. used. Injection: within 6 days after treatment 40 out of 97 injected males had died. F e e d i n g : n o t n o t i c e a b l y a f f e c t e d . I n j e c t i o n : 4 6 , 4 9 a n d 2 8 % o f live m a l e s fertile in b r o o d s A , B a n d C, r e s p e c t i v e l y . Wild t y p e ( O r e g o n - K ) . Feeding, 1--2 days; injection, 4 days. Feeding, 4--5 days; injection, 4 days. Muner-5. 2--6 days. Standard Muller-5 scheme. Two or three 3-day matings; 4 fresh virgins for each mating; dd mated individually. W h e n less t h a n 4 r o u n d - e y e d m a l e s w e r e p r e s e n t in a n F 2 vial, z e t e s t s w e r e made to confirm possible (semi)lethality. 25°C.
C i t r e x S-5; d a t a s.re o n l y g i v e n w h e n d i f f e r e n t f r o m t h e N T A t e s t s Source of the compound Number of experiments Exposure Toxicity Fertility
"De KAok" soap factories, Heerde, The Netherlands 2 (feeding). 3 days feeding on a 1% solution, replaced daily, prepared from the 50% a q u e o u s s o l u t i o n , ( t h e c u r r e n t t r a d e f o r m u l a t i o n ) , w i t h 5% s u c r o s e . Non-toxic at concentration used. Not noticeably affected.
T A B L E II DOMINANT LETHAL TESTS, MATERIALS AND METHODS NTA Way of administration Number of experiments Exposure Genotype treated dd and 99 Age o f d d a t t r e a t m e n t Age o f 9 9 a t m a t i n g Egg-laying scheme
Counting procedure Culturing temperature
Feeding. 2. See T a b l e I. Oregon-K. 1 or 2 days. 5 days. T r e a t e d m a l e s a n d virgin f e m a l e s w e r e m a t e d s i n g l y i n t h e vials o f e g g - l a y i n g units [10]. Eggs were collected from two or three subsequent overnight egg*laying p e r i o d s o f a b o u t 1 8 h. Immediately after each egg-laying period the eggs were counted; unhatched eggs w e r e s c o r e d a b o u t 3 0 h l a t e r . 25°C.
C i t r e x S-5; d a t a a r e o n l y g i v e n w h e n t h e y a r e d i f f e r e n t f r o m t h o s e i n t h e N T A t e s t s Number of experiments
1.
279 T A B L E III FREQUENCIES OF SEX-LINKED O F N T A O R C I T R E X S-5 Compound
Way of administration
Conc.
NTA
feeding
50 mM
NTA
injection
10 mM
C i t r e x S-5
feeding
1%
RECESSIVE
Expt. No.
MUTATIONS
Brood A days 0--3
AFTER
Brood B days 3--6
ADMINISTRATION
Brood C days 6--9
nehr.
% 1 a
cnhr.
% 1a
nchr.
% I. a
1 2 3
727 1058 1042
0.55 0.0 0.0
749 1025 1125
0.53 0.10 0.18
716 1148
0.26 0.0
1--3
2627
0.14
2399
0.24
1864
0.11
~9 5 0
0.11,
911
0.11
290
0.0
1
697
0.14
730
2
388
0.23
868
0.41 0.23
772 874
0.13 0.24
1585
0.19
1598
0.31
1646
0.18
3136
0.13
2997
0.23
3118
0.13
1--2 Control
LETHAL
inj., f e e d i n g
a F o r statistical c o m m e n t , see text. n c h r . , n u m b e r o f c h r o m o s o m e s t e s t e d ; % 1., p e r c e n t l e t h a l s .
crease (at the 5% level), given the size of the present experiments. For the postmeiotic stages (broods A and B taken together) these percentages are 0.40 for NTA and 0.47 for Citrex S-5. (In other words, a 2-fold increase of the spontaneous mutation frequency, for NTA, and a 2.5-fold increase for Citrex S-5, should have been detectable with 95% probability.) As shown in Table IV, the dominant-lethal tests do n o t show decreased egg hatchability after treatment of the flies with NTA or Citrex S-5. With NTA a few other mutagenicity studies have been carried out. Experiments with bacteria, yeast and Neurospora showed neither induction of gene mutations [13--16] nor a modifying effect of NTA on the mutation rate induced by EMS or X-rays [14,15]. In root-tip cells of Vicia faba, as well as in rat kangaroo cells and human leukocytes in culture, NTA induced chromatid aberrations, b u t only at sub-lethal concentrations (2.5--6 mM) over long exposure
TABLE IV PERCENTAGE Expt. No.
UNHATCHED
Compound, series
1
NTA Control
2
NTA Control
1+2
NTA Control C i t r e x S-5 Control
E G G S A F T E R G I V I N G N T A O R C I T R E X S-5 T O D R O S O P H I L A M A L E S
I s t egg-l, p e r i o d
2 n d egg-l, p e r i o d
3 r d egg-l, p e r i o d
all egg-l, p e r i o d s
n. eggs
% unh.
n. eggs
n. eggs
n . eggs
% unh.
1296 1671
22.1 19.9
1057 1360
7.1 7.2
2353 3031
15.3 14.2
2235 2511
12.9 17.6
4979 4905
20.7 22.4
7332 7936
19.0 19.3
8913 8557
21.3 20.9
3162 3033
32.3 31.6
5751 5524
% unh.
15.0 13.2
2694 2394
% unh.
27.3 27.2
egg-L p e r i o d , e g g - l a y i n g p e r i o d ; n. eggs, n u m b e r o f eggs l a i d ; % u n h . , p e r c e n t a g e o f u n h a t c h e d eggs.
280
times (1--5 days) [1,7,8]. In a mouse dominant-lethal test, no effect of NTA was observed when the c o m p o u n d was given i.p. at 125 mg/kg or orally at higher doses [ 3]. Likewise no induction of translocations was found when mice were given a drinking solution containing 0.1% NaCaNTA for seven weeks [5]. In summary, the N T A data make it clear that (1) all tests for gene mutations and for transmitted genetic effects produced negative results, and (2) the only positive data indicate chromosome breakage effects in somatic cells in vitro, at high exposure levels. Consequently, the available information provides no evidence that N T A presents a genetic hazard. Unfortunately, no data are available on mutagenicity of Citrex S-5, with the exception of the Drosophila results. For a proper comparison of the two compounds with respect to mutagenic properties, Citrex should likewise be investigated in other systems. This work was supported by the Netherlands Ministry of Public Health and Environmental Hygiene. I am indebted to Professor F.H. Sobels for his encouragement, and for critically reading the manuscript. I thank Marja de Jong, Leny Hoogteyling and Antoinette Pex for their indispensable technical assistance. References 1 Bora, K.C., Effects of nitrilotriacetic acid (NTA) on c h r o m o s o m e replication and structure in h u m a n cells, 6 t h ann. m e e t i n g American E n v i r o n m e n t a l Mutagen Society, Miami, 1975. 2 De Serres, F.J., H.V. Mailing, F.H. Sobela, E. Vogel and J.S. Waseom, New section: Genetic Toxicology Testing, M u t a t i o n Res., 31 (1975) 273--274. 3 Epstein, S.S., E. Arnold, J. Andrea, W. Bass and Y. Bishop, D e t e c t i o n of chemical m u t a g e n s by the d o m i n a n t l e t h a l assay in the mouse, Toxicol. Appl. Pharmaeol., 23 (1972) 288--325. 4 H a m m o n d , A.L., Phosphate replacements: problems w i t h the washday miracle, Science, 172 (1971) 361--363. 5 Jorgenson, T.A., G.W. Newell, L.G. Seharpf, P. Gribling, M. O'Brien and D. Chu, Study of the mutagenic p o t e n t i a l of nitrilotriacetic acid (NaCaNTA) in mice by the t ra ns l oc a t i on test, 6 t h ann. me e t i ng Am erican E n v i r o n m e n t a l Mutagen Society, Miami, 1975. 6 Kastenbaum, M.A. and K.O. Bowman, Tables for d e t e r m i n i n g the statistical significance of m u t a t i o n frequencies, M u t a t i o n Res., 9 (1970) 527--549. 7 Kihlman, B.A., R o o t tips for s t u d y i n g the effect of chemicals on chromosomes, in A. H o n a e n d e r (ed.) Chemical Mutagens, Principles and Methods for Their Detection, P l e num Press, New York, 1971, pp. 489--514. 8 Kthlman, B.A. and S. Sturelid, Niti'i]otriacetic acid (NTA) and c h r o m o s o m e breakage, EMS Newsletter, 3 (1970) 32--33. 9 0 s t e r , I.I., Chemicals found inactive as mutagens in Drosophila, D.I.S., 26 (1952) 116--117. 10 Sankaranarayanan, K., The effects of nitrogen and oxygen t r e a t m e n t s on the frequencies of X-ray induced d o m i n a n t lethals and on the physiology of the sperm in D r o s o p h i l a m e l a n o g a s t e r , Mut a t i on Res., 4 (1967) 641--661. 11 Sobels, F.H., The role of Drosophila in the field of m u t a t i o n research, Arch. Genetik, 45 (1972) 101-125. 12 Sobels, F.H., The advantages of Drosophila for m u t a t i o n studies, Mut a t i on Res., 26 (1974) 277--284. 13 Stine, G.J. and P. Adams, The effects of nitrilotriacetic acid on d e v e l o p m e n t of N e u r o s p o r u crussa, Microbial Genet. BuLl., 36 (1974) 8--10. 14 Stine, G.J. and A.A. Hardigree, Effects of nitrilotriacetic acid on E s c h e r i c h i u c o l i K-12, EMS Newsletter, 5 (1971) 38--39. 15 Stine, G.J. and A.A. Hardigree, Effect of nitrilotriacetic acid on grow t h and m a t i n g in strains of E s c h e r i c h i u coli K-12, Can. J. MicrobioL, 18 (1972) 1159---1162. 16 Zetterberg, G., Negative results with nitrilotriacetic acid (NTA) as an i n d u c e r of gene m u t a t i o n in some microorganisms, EMS Newsletter, 3 (1970) 31--32.