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Genotoxicity of tartrazine studied in two somatic assays of Drosophila melanogaster
Mutation Research, 224 (1989) 479-483
479
Elsevier MUTGEN 01491
Genotoxicity of tartrazine studied in two somatic assays of Drosophila melanogaster Niraj K. Tripathy, K a l y a n i K. Patnaik and Md.J. Na b i Department of Zoology, Berhampur University, Berhampur 760007, Orissa (India) (Received 23 March 1989) (Revision received 13 June 1989) (Accepted 15 June 1989)
Keywords: Tartrazine; Wing mosaic test; Eye mosaic test; Drosophila melanogaster
Data pertaining to the mutagenicity of several food dyes in various test systems are available (Brown et al., 1978; Edwards and Combes, 1983; Garner and Nutman, 1977; Haveland-Smith, 1981; Kada et al., 1972; Venitt and Bushell, 1976). Tartrazine (CAS No. 1934-21-0), a bright orangeyellow powder (4,5-dihydro-5-oxo-l-(4-sulphophenyl)-4-[(4-sulphophenyl)azo]-IH-pyrazole-3carboxylic acid trisodium salt) is approved in many countries as a food, drug and cosmetic colourant. It is also used as a dye for wool and silk. Since tartrazine has not been adequately tested for mutagenicity (Zimmermann et al., 1984), we were interested to study its genotoxic effects, if any, in Drosophila wing and eye mosaic tests. Materials and methods
The larvae used to test the mutagenic potential of tartrazine were obtained from the cross of w~°/wC°; fir 3 s e / T M 2 , Ubx se females and fs(1)Kao w / Y ; mwh se / m w h se males. The alleles w c° (white-coral) and w (white) are recessive markers used for the eye mosaic test in a se (sepia) background (Lindsley and Grell, 1968) and
Correspondence: Dr. N.K. Tripathy, Department of Zoology, Berhampur University, Berhampur 760007, Orissa (India).
the markers mwh (multiple wing hair) and fir 3 (flare) are used as markers for the wing spot test (Garcia-Bellido and Dapena, 1974; Lindsley and Grell, 1968). Larvae aged 48 h and 72 h (corresponding to the 2nd and 3rd larval instars) were exposed to different concentrations of aqueous solutions of tartrazine (manufactured and marketed as Lemon Yellow by Alpana Industries, Calcutta, India, with 16% dye content and 84% of a mixture of sodium chloride and sodium sulphate) in instant food (obtained from Carolina Biological Supplies) for 72 h and 48 h respectively (Graf et al., 1984) to determine the LDso. The LDs0, where 50% of the treated larvae hatched to adult stage, were 0.06% and 0.12% respectively. Thus the larvae were treated with the LDs0 and 50% of this dose. The wings of adult flies were mounted in Faure's solution (Graf et al., 1984) and the distal compartments (Garcia-Bellido et al., 1976) of the mounted wings were screened at 400 × magnification to record the size of each spot (both mwh and fir 3 single spots and m w h / f l r 3 twin spots). The conditional binomial test (Kastenbaum and Bowman, 1970) was performed for statistical evaluation of the wing spot data. One-sided tests were performed at the 5% level of significance. An outcome was considered significantly positive or negative when the induced frequencies of small single spots were more or less than twice the
0165-1218/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
480 control frequency and 5 times for large single and twin spots. An outcome neither significantly positive nor significantly negative was considered inconclusive (Frei and Wiirgler, 1988; Selby and Olson, 1981). The eyes of 2-day-old flies were screened in a solution composed of 90 parts ethanol, 1 part Tween 80 and 9 parts water at 50-70 x magnification under a stereozoom microscope. Only the mosaic light spots (LS) and the twin spots (TS) were taken into consideration since the w ¢° dark spots are supposed to be due to variegation effects (Wiirgler and Vogel, 1985) and their mode of differentiation is different from the normal eye tissue (Becker, 1966; Vogel, 1983). Statistical analysis of the eye mosaic data was done with chi-square tests. Results
therefore either negative or inconclusive. It was decided to increase the duration of treatment to 72 h by exposing the 2nd-instar larvae to the compound. Here, for both the small single and large single spots the frequency of induction was positive. The increase in the frequency of twin spots was positive at the LDs0 and inconclusive at 50% of this dose. It is known that single spots with the m w h phenotype originate due to mitotic recombination in the chromosome segment between the m w h and the fir 3 loci (Garcia-Bellido and Dapena, 1974), gene mutation a n d / o r gene conversion in the wild-type allele (Graf et al., 1984) or the induction of segmental aneuploidy through chromosome breakage (Haynie and Bryant, 1978). Since a large number of such spots have been induced on treatment with tartrazine, they could have originated in any of these events. The twin spots with m w h and fir 3 subclones are formed due to mitotic recombination in the chromosome segment between the fir 3 locus and the centromere (Becker, 1976) or due to a double chromosome non-disjunction in the wing primordial cells
and discussion
The wing spot data are given in Table 1. The treatments of 3rd-instar larvae for 48 h with both the LDs0 and 50% of the LDs0 led to a non-significant increase in the spot frequencies and were
TABLE 1 INDUCTION OF WING MOSAICISM Larval treatment
Concentration (%)
Pooled control
Number of wings tested
Spots
318
s 1-2 s> 2
Type a
Conclusionb N
t
48 h
72 h
76 14
0.24 0.04
1
0.00
0.12
146
s 1-2 s> 2 t
45 11 1
0.31 0.08 0.01
NEG NEG INC
0.06
146
s 1-2 s> 2 t
42 10 1
0.29 0.07 0.01
NEG NEG INC
0.06
146
s 1-2 s> 2 t
102 31 6
0.70 0.21 0.04
POS POS POS
0.03
152
s 1-2 s> 2
62 29
0.41 0.19 0.01
POS POS INC
t
1-2, small singles; s > 2, large singles; t, twins. b Level of significance P < 0.05: POS, positive; INC, inconclusive; NEG, negative. a s
%
2
481 TABLE 2A INDUCTION OF FEMALE EYE MOSAICISM Larval treatment
Concentration (%)
72 h
Spots
Conclusion (x 2)
Type a
N
%
1380
TS LS
8 18
0.58 1.30
0.12
228
TS LS
9 8
3.95 3.51
POS POS
0.06
172
TS LS
4 6
2.33 3.49
NEG NEG
0.06
160
TS LS
2 7
1.25 4.38
NEG POS
0.03
160
TS LS
3 6
1.88 3.75
NEG POS
Pooled control
48 h
Number of eyes tested
b
a TS, twin spot; LS, light spot. b Level of significance P < 0.05; POS, positive; NEG, negative. ( R a m d a n d M a g n u s s o n , 1979), a l t h o u g h the l a t t e r is a rare p h e n o m e n o n . T h u s o u r d a t a reveal that t a r t r a z i n e is b o t h r e c o m b i n o g e n i c a n d m u t a g e n i c to the wing disc cells o f D r o s o p h i l a after p r o l o n g e d treatments. T h e d a t a on the eye m o s a i c assay are given in T a b l e 2. N o significant difference was f o u n d b e tween the frequencies o f m o s a i c spots in the eyes o f T M 2 a n d n o n - T M 2 flies (chi-square test, P > 0.05), i n d i c a t i n g that the genetic c o n s t i t u t i o n of the 3rd c h r o m o s o m e d o e s n o t influence the genetic
a l t e r a t i o n s in the 1st c h r o m o s o m e . It is o b s e r v e d that the f r e q u e n c y o f twin s p o t s with white a n d coral s u b c l o n e s was significantly h i g h e r in the eyes o f female flies d e v e l o p i n g f r o m 72-h l a r v a e t r e a t e d with the LDso a n d negative in all o t h e r treatments. But the increase in the f r e q u e n c y o f light s p o t s in these flies was significantly higher in all the t r e a t m e n t s except for the 72-h larvae t r e a t e d with 50% o f the LDs0. I n the m a l e eyes the increase in light spots was significantly higher in the flies d e v e l o p i n g f r o m b o t h 48-h a n d 72-h larvae
TABLE 2B INDUCTION OF FEMALE EYE MOSAICISM Larval treatment
Concentration (%)
Pooled control
Number of eyes tested
Spots Type a
N
%
1324
LS
16
1.21
Conclusion b (X2)
48 h
0.12 0.06
186 256
LS LS
9 6
4.84 2.34
POS NEG
72 h
0.06 0.03
160 180
LS LS
6 6
3.75 3.33
POS NEG
a LS, light spot. b Level of significance P < 0.05; POS, positive; NEG, negative.
482 treated with the LDso. The increase in the frequency of spots (both twin and light spots) in female eyes is an indication of the induction of both recombination and mutation while in male eyes it indicates the induction of mutation only. In this assay recombination in the c h r o m o s o m e segment between the w locus and the centromere led to the induction o f w / / w c° twin spots. The light spots, on the other hand, originate due to mutation or deletion of the w c° gene or recombination between the homologous X chromosomes (Mollet and Wiirgler, 1974; Vogel and Zijlstra, 1987). It m a y also be the surviving light partner of an original twin spot in which the dark partner has failed to develop (Becker, 1976). O u r results clearly indicate that tartrazine is mutagenic a n d / o r recombinogenic to the eye disc cells of Drosophila. Tests conducted with somatic cells of Drosophila melanogaster clearly reveal that these assays are very suitable for mutagenicity testing. Although the sex-linked recessive-lethal test is the best validated mutagenicity test in Drosophila, it is timeconsuming and hence the fast 1-generation tests involving somatic cells have been adopte d . The white/white-coral eye mosaic assay has certain limitations c o m p a r e d to the wing mosaic test. This eye mosaic test is not suitable for the identification of mutagens which act late in larval development since in such an event only small clones will be induced which will be difficult to identify in the adult eyes. This is because each o m m a t i d i u m contains several pigment ceils and m a n y of them are shared by adjacent ommatidia (Ready et al., 1976) and further the borders of clones and ommatidia coincide very rarely (Benzer, 1973; H o f b a u e r and Campos-Ortega, 1976). On the contrary, even clones with just 1 mutated cell can easily be detected on the wing blade (Garcia-Bellido and Merriam, 1971; Haynie and Bryant, 1978). Thus it m a y be concluded that the mutagenicity test involving the wing primordial cells is more sensitive and accurate than the w h i t e / w h i t e - c o r a l eye mosaic test.
Acknowledgements W e thank Dr. U. G r a f of the Institute of Toxicology, Schwerzenbach (Switzerland) for statistical analysis of the wing data and Dr. J. Szabad
of the Institute of Genetics, BRC, Szeged (Hungary) for providing the Drosophila stocks. The award of a Senior Research Fellowship to K.K.P. by the Council of Scientific and Industrial Research, N e w Delhi, is gratefully acknowledged.
References Becker, H.J. (1966) Genetic and variegation mosaics in the eye of Drosophila, Curr. Topics Develop. Biol., 1, 155-171. Becker, H.J. (1976) Mitotic recombination, in: M. Ashburner and E. Novitski (Eds.), Genetics and Biology of Drosophila, Vol. lc, Academic Press, New York, pp. 1020-1087. Benzer, S. (1973) Genetic dissection of behaviour, Sci. Am., 229, 24-37. Brown, J.P., W.G. Roehm and R.J. Brown (1978) Mutagenicity testing of certified food colours and related azo, xanthene and triphenylmethane dyes with the Salmonella/microsome system, Mutation Res., 56, 249-271. Edwards, C.N., and R.D. Combes (1983) Studies on the mutagenicity of commercial preparations of the food colour Red 2G after purification of oxidation, Mutation Res., 117, 127-134. Frei, H., and F.E. Wiirgler (1988) Statistical methods to decide whether mutagenicity test data for Drosophila assays indicate a positive, negative or inconclusive result, Mutation Res., 203, 297-308. Garcia-Bellido, A., and J. Dapena (1974) Induction, detection and characterization of cell differentiation mutants in Drosophila, Mol. Gen. Genet., 128, 117-130. Garcia-Bellido, A., and J.R. Merriam (1971) Parameters of the wing disc development of Drosophila melanogaster, Dev. Biol., 24, 61-78. Garcia-Bellido, A., P. Ripoll and G. Morata (1976) Developmental compartmentalization in the dorsal mesothoracic disc of Drosophila, Dev. Biol., 48, 132-147. Garner, R.C., and C.A. Nutman (1977) Testing of some azo dyes and their reduction products for mutagenicity using Salmonella typhimurium TA1538, Mutation Res., 44, 9-19. Graf, U., F.E. Wiirgler, H. Frei, H. Huon, C.B. Hall and P.G. Kale (1984) Somatic mutation and recombination test in Drosophila melanogaster, Environ. Mutagen., 6, 153-188. Haveland-Smith, R.B. (1981) Evaluation of the genotoxicity of some natural food colours using bacterial assays, Mutation Res., 91, 285-290. Haynie, J.L., and P.J. Bryant (1978) The effect of X-rays on the population dynamics of the cells in the wing disc of Drosophilamelanogaster,Wilhelm Roux' Arch., 183, 85-100. Hofbauer, A., and J. Campos-Ortega, (1976) Cell clones and pattern formation: genetic eye mosaics in Drosophila melanogaster, Wilhelm Roux' Arch., 179, 257-289. Kada, T., K. Tutikawa and Y. Sadaie (1972) In vitro and host-mediated 'rec-assay' procedures for screening chemical mutagens: phloxine, a mutagenic red dye detected, Mutation Res., 16, 165-174. Kastenbaum, M.A., and K.O. Bowman (1970) Tables for determining the statistical significance of mutation frequencies, Mutation Res., 9, 527-549.
483 Lindsley, D.L., and E.H. Greil (1968) Genetic variations of Drosophila melanogaster, Carnegie Inst. Wash. Publ., 627. Mollet, P., and F.E. Wiirgler (1974) Detection of somatic recombination and mutation in Drosophila, a method for testing genetic activity of chemical compounds, Mutation Res., 25, 421-424. Ramel, C., and J. Magnusson (1979) Chemical induction of nondisjunction in Drosophila, Env. Health Perspect., 31, 59-66. Ready, D.F., T.E. Hanson and S. Benzer (1976) Development of the Drosophila retina, a neurocrystalline lattice, Dev. Biol., 53, 217-240. Selby, P.B., and W.H. Olson (1981) Methods and criteria for deciding whether specific-locus mutation-rate data in mice indicate a positive, negative, or inconclusive result, Mutation Res., 83, 403-418. Venitt, S., and C.T. Bushell (1976) Mutagenicity of the food colour Brown FK and constituents in Salmonella typhimurium, Mutation Res., 40, 309-316.
Vogel, E. (1983) Approaches to comparative mutagenesis in higher eukaryotes: Significance of DNA modifications with alkylating agents in Drosophila melanogaster, Ann. N.Y. Acad. Sci., 407, 208-220. Vogel, E., and J.A. Zijlstra (1987) Mechanistic and methodological aspects of chemically-induced somatic mutation and recombination in Drosophila melanogaster, Mutation Res., 182, 243-264. Wiirgler, F.E., and E. Vogel (1985) In vitro mutagenicity testing using somatic cells of Drosophila melanogaster, in: F.J. de Serres (Ed.), Chemical Mutagens: Principles and Methods for Their Detection, Vol. 10, Plenum, New York, pp. 1-72. Zimmermann, F.K., R.C. von Borstel, E.S. von Halle, J.M. Perry, G. Zetterberg, D. Siebert, R. Barale and N. Loprieno (1984) Testing of chemicals for genetic activity with Saccharomyces cerevisiae: a report of the U.S. EPA Gene-Tox Programme, Mutation Res., 133, 199-244.
479
Elsevier MUTGEN 01491
Genotoxicity of tartrazine studied in two somatic assays of Drosophila melanogaster Niraj K. Tripathy, K a l y a n i K. Patnaik and Md.J. Na b i Department of Zoology, Berhampur University, Berhampur 760007, Orissa (India) (Received 23 March 1989) (Revision received 13 June 1989) (Accepted 15 June 1989)
Keywords: Tartrazine; Wing mosaic test; Eye mosaic test; Drosophila melanogaster
Data pertaining to the mutagenicity of several food dyes in various test systems are available (Brown et al., 1978; Edwards and Combes, 1983; Garner and Nutman, 1977; Haveland-Smith, 1981; Kada et al., 1972; Venitt and Bushell, 1976). Tartrazine (CAS No. 1934-21-0), a bright orangeyellow powder (4,5-dihydro-5-oxo-l-(4-sulphophenyl)-4-[(4-sulphophenyl)azo]-IH-pyrazole-3carboxylic acid trisodium salt) is approved in many countries as a food, drug and cosmetic colourant. It is also used as a dye for wool and silk. Since tartrazine has not been adequately tested for mutagenicity (Zimmermann et al., 1984), we were interested to study its genotoxic effects, if any, in Drosophila wing and eye mosaic tests. Materials and methods
The larvae used to test the mutagenic potential of tartrazine were obtained from the cross of w~°/wC°; fir 3 s e / T M 2 , Ubx se females and fs(1)Kao w / Y ; mwh se / m w h se males. The alleles w c° (white-coral) and w (white) are recessive markers used for the eye mosaic test in a se (sepia) background (Lindsley and Grell, 1968) and
Correspondence: Dr. N.K. Tripathy, Department of Zoology, Berhampur University, Berhampur 760007, Orissa (India).
the markers mwh (multiple wing hair) and fir 3 (flare) are used as markers for the wing spot test (Garcia-Bellido and Dapena, 1974; Lindsley and Grell, 1968). Larvae aged 48 h and 72 h (corresponding to the 2nd and 3rd larval instars) were exposed to different concentrations of aqueous solutions of tartrazine (manufactured and marketed as Lemon Yellow by Alpana Industries, Calcutta, India, with 16% dye content and 84% of a mixture of sodium chloride and sodium sulphate) in instant food (obtained from Carolina Biological Supplies) for 72 h and 48 h respectively (Graf et al., 1984) to determine the LDso. The LDs0, where 50% of the treated larvae hatched to adult stage, were 0.06% and 0.12% respectively. Thus the larvae were treated with the LDs0 and 50% of this dose. The wings of adult flies were mounted in Faure's solution (Graf et al., 1984) and the distal compartments (Garcia-Bellido et al., 1976) of the mounted wings were screened at 400 × magnification to record the size of each spot (both mwh and fir 3 single spots and m w h / f l r 3 twin spots). The conditional binomial test (Kastenbaum and Bowman, 1970) was performed for statistical evaluation of the wing spot data. One-sided tests were performed at the 5% level of significance. An outcome was considered significantly positive or negative when the induced frequencies of small single spots were more or less than twice the
0165-1218/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
480 control frequency and 5 times for large single and twin spots. An outcome neither significantly positive nor significantly negative was considered inconclusive (Frei and Wiirgler, 1988; Selby and Olson, 1981). The eyes of 2-day-old flies were screened in a solution composed of 90 parts ethanol, 1 part Tween 80 and 9 parts water at 50-70 x magnification under a stereozoom microscope. Only the mosaic light spots (LS) and the twin spots (TS) were taken into consideration since the w ¢° dark spots are supposed to be due to variegation effects (Wiirgler and Vogel, 1985) and their mode of differentiation is different from the normal eye tissue (Becker, 1966; Vogel, 1983). Statistical analysis of the eye mosaic data was done with chi-square tests. Results
therefore either negative or inconclusive. It was decided to increase the duration of treatment to 72 h by exposing the 2nd-instar larvae to the compound. Here, for both the small single and large single spots the frequency of induction was positive. The increase in the frequency of twin spots was positive at the LDs0 and inconclusive at 50% of this dose. It is known that single spots with the m w h phenotype originate due to mitotic recombination in the chromosome segment between the m w h and the fir 3 loci (Garcia-Bellido and Dapena, 1974), gene mutation a n d / o r gene conversion in the wild-type allele (Graf et al., 1984) or the induction of segmental aneuploidy through chromosome breakage (Haynie and Bryant, 1978). Since a large number of such spots have been induced on treatment with tartrazine, they could have originated in any of these events. The twin spots with m w h and fir 3 subclones are formed due to mitotic recombination in the chromosome segment between the fir 3 locus and the centromere (Becker, 1976) or due to a double chromosome non-disjunction in the wing primordial cells
and discussion
The wing spot data are given in Table 1. The treatments of 3rd-instar larvae for 48 h with both the LDs0 and 50% of the LDs0 led to a non-significant increase in the spot frequencies and were
TABLE 1 INDUCTION OF WING MOSAICISM Larval treatment
Concentration (%)
Pooled control
Number of wings tested
Spots
318
s 1-2 s> 2
Type a
Conclusionb N
t
48 h
72 h
76 14
0.24 0.04
1
0.00
0.12
146
s 1-2 s> 2 t
45 11 1
0.31 0.08 0.01
NEG NEG INC
0.06
146
s 1-2 s> 2 t
42 10 1
0.29 0.07 0.01
NEG NEG INC
0.06
146
s 1-2 s> 2 t
102 31 6
0.70 0.21 0.04
POS POS POS
0.03
152
s 1-2 s> 2
62 29
0.41 0.19 0.01
POS POS INC
t
1-2, small singles; s > 2, large singles; t, twins. b Level of significance P < 0.05: POS, positive; INC, inconclusive; NEG, negative. a s
%
2
481 TABLE 2A INDUCTION OF FEMALE EYE MOSAICISM Larval treatment
Concentration (%)
72 h
Spots
Conclusion (x 2)
Type a
N
%
1380
TS LS
8 18
0.58 1.30
0.12
228
TS LS
9 8
3.95 3.51
POS POS
0.06
172
TS LS
4 6
2.33 3.49
NEG NEG
0.06
160
TS LS
2 7
1.25 4.38
NEG POS
0.03
160
TS LS
3 6
1.88 3.75
NEG POS
Pooled control
48 h
Number of eyes tested
b
a TS, twin spot; LS, light spot. b Level of significance P < 0.05; POS, positive; NEG, negative. ( R a m d a n d M a g n u s s o n , 1979), a l t h o u g h the l a t t e r is a rare p h e n o m e n o n . T h u s o u r d a t a reveal that t a r t r a z i n e is b o t h r e c o m b i n o g e n i c a n d m u t a g e n i c to the wing disc cells o f D r o s o p h i l a after p r o l o n g e d treatments. T h e d a t a on the eye m o s a i c assay are given in T a b l e 2. N o significant difference was f o u n d b e tween the frequencies o f m o s a i c spots in the eyes o f T M 2 a n d n o n - T M 2 flies (chi-square test, P > 0.05), i n d i c a t i n g that the genetic c o n s t i t u t i o n of the 3rd c h r o m o s o m e d o e s n o t influence the genetic
a l t e r a t i o n s in the 1st c h r o m o s o m e . It is o b s e r v e d that the f r e q u e n c y o f twin s p o t s with white a n d coral s u b c l o n e s was significantly h i g h e r in the eyes o f female flies d e v e l o p i n g f r o m 72-h l a r v a e t r e a t e d with the LDso a n d negative in all o t h e r treatments. But the increase in the f r e q u e n c y o f light s p o t s in these flies was significantly higher in all the t r e a t m e n t s except for the 72-h larvae t r e a t e d with 50% o f the LDs0. I n the m a l e eyes the increase in light spots was significantly higher in the flies d e v e l o p i n g f r o m b o t h 48-h a n d 72-h larvae
TABLE 2B INDUCTION OF FEMALE EYE MOSAICISM Larval treatment
Concentration (%)
Pooled control
Number of eyes tested
Spots Type a
N
%
1324
LS
16
1.21
Conclusion b (X2)
48 h
0.12 0.06
186 256
LS LS
9 6
4.84 2.34
POS NEG
72 h
0.06 0.03
160 180
LS LS
6 6
3.75 3.33
POS NEG
a LS, light spot. b Level of significance P < 0.05; POS, positive; NEG, negative.
482 treated with the LDso. The increase in the frequency of spots (both twin and light spots) in female eyes is an indication of the induction of both recombination and mutation while in male eyes it indicates the induction of mutation only. In this assay recombination in the c h r o m o s o m e segment between the w locus and the centromere led to the induction o f w / / w c° twin spots. The light spots, on the other hand, originate due to mutation or deletion of the w c° gene or recombination between the homologous X chromosomes (Mollet and Wiirgler, 1974; Vogel and Zijlstra, 1987). It m a y also be the surviving light partner of an original twin spot in which the dark partner has failed to develop (Becker, 1976). O u r results clearly indicate that tartrazine is mutagenic a n d / o r recombinogenic to the eye disc cells of Drosophila. Tests conducted with somatic cells of Drosophila melanogaster clearly reveal that these assays are very suitable for mutagenicity testing. Although the sex-linked recessive-lethal test is the best validated mutagenicity test in Drosophila, it is timeconsuming and hence the fast 1-generation tests involving somatic cells have been adopte d . The white/white-coral eye mosaic assay has certain limitations c o m p a r e d to the wing mosaic test. This eye mosaic test is not suitable for the identification of mutagens which act late in larval development since in such an event only small clones will be induced which will be difficult to identify in the adult eyes. This is because each o m m a t i d i u m contains several pigment ceils and m a n y of them are shared by adjacent ommatidia (Ready et al., 1976) and further the borders of clones and ommatidia coincide very rarely (Benzer, 1973; H o f b a u e r and Campos-Ortega, 1976). On the contrary, even clones with just 1 mutated cell can easily be detected on the wing blade (Garcia-Bellido and Merriam, 1971; Haynie and Bryant, 1978). Thus it m a y be concluded that the mutagenicity test involving the wing primordial cells is more sensitive and accurate than the w h i t e / w h i t e - c o r a l eye mosaic test.
Acknowledgements W e thank Dr. U. G r a f of the Institute of Toxicology, Schwerzenbach (Switzerland) for statistical analysis of the wing data and Dr. J. Szabad
of the Institute of Genetics, BRC, Szeged (Hungary) for providing the Drosophila stocks. The award of a Senior Research Fellowship to K.K.P. by the Council of Scientific and Industrial Research, N e w Delhi, is gratefully acknowledged.
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