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The mammalian spot test and its use for testing of mutagenic and carcinogenic potential: Experience with the pesticide chlordimeform, its principal metabolites and the drug lisuride hydrogen maleate
Mutation Research, 135 (1984) 219-224
219
Elsevier MTR 00846
The mammalian spot test and its use for testing of mutagenic and carcinogenic potential: experience with the pesticide chlordimeform, its principal metabolites and the drug lisuride hydrogen maleate R. Lang Department o/Experimental Toxicololo', Sehering, Berlin/Bergkamen (F.R. G.)
(Received 12 April 1983) (Revision received22 July 1983) (Accepted 17 October 1983)
Summa~ The mammalian spot test has recently been demonstrated to be a promising method for the detection of somatic mutations induced by chemicals and is at present under validation as an in vivo screening test for carcinogenic potential. The pesticide chlordimeform, its principal, metabolites N-formyl-4-chloro-o-toluidine and 4-chloro-otoluidine, and the drug lisuride hydrogen maleate, as well as the known mutagens ethyl methanesulfonate (EMS) and N-ethyl-N-nitrosourea (ENU) were tested in the mammalian spot test. Female C57BL/6J mice were mated to T-stock males and treated by gavage with maximal tolerated doses of the test compounds, or by intraperitoneal injection with EMS and ENU at days 8, 9 and 10 of pregnancy. Mutation induction was monitored post-natally by checking the fur of the offspring for color spots that result from expression of a recessive gene involved in coat-color determination. Of the environmental chemicals tested, only 4-chloro-o-toluidine was mutagenic in the spot test. This positive result was in agreement with in vitro experiments and its carcinogenic potential in the mouse. However, the mouse carcinogens chlordimeform and N-formyl-4-chloro-o-toluidine were negative, as was the non-carcinogen lisuride hydrogen maleate. N-Formyl-4-chloro-o-toluidine and lisuride hydrogen maleate have been reported to be weakly positive in the Ames test. The positive controls EMS and ENU showed a clear mutagenic response.
The insecticide and acaracide chlordimeformHCi [N,N'-dimethyl-N'-(2-methyl-4-chlorophenyl)-formamidine-HCI; CDF] and its 2 principal metabolites, N-formyl-4-chloro-o-toluidine (formyl-4-COT) and 4-chloro-o-toluidine. HCI (4COT), were carcinogenic in the mouse but not in rats (FAO, 1979, 1980, 1981). Additionally, the metabolites showed some mutagenic potential in short-term bioassays (FAO, 1979, 1980, 1981); 4-COT was found to be clearly mutagenic after metabolic activation in Salmonella typhimurium strains TA98 and TA100, but the formyl metabo0165-1218/84/$03.00 © 1984 ElsevierScience Publishers B.V.
lite showed a very weak response with TA100. C D F was negative in the Ames test (FAO, 1979, 1980, 1981; unpublished results). None of the 3 compounds showed a clastogenic potential (FAO, 1979, 1980, 1981), and none induced translocation heterozygosity in the mouse heritable translocation test (Lang and Adler, 1982) (Table 1). Lisuride hydrogen maleate [3-(10,10a-didehydro-7-methyl-9a-ergolinyl)-l,l-diethylurea, hydrogen maleate; LHM], an ergot alkaloid with dopamine agonist activity, showed weak mutagenic effects at very high concentrations (mg range
220 TABLE 1 PRINCIPAL RESULTS OF T U M O R I G E N I C I T Y A N D MUTAGENICITY STUDIES WITH CDF. FORMYL-4-COT, 4-COT, A N D LHM
Tumorigenicity Mouse Rat
CDF
Formyl-4-COT
4-COT
+
+
+
(+)
+
NT
NT
NT
NT
Point mutations in vitro Salmonella/microsome test D N A damage in vivo SCE test Chinese hamster
NT
LHM
+
Point mutations in vivo Mammalian spot test Mouse Chromosomal aberrations in vivo Chromosome analysis Chinese hamster Nucleus anomaly t e s t / micronucleus test Chinese hamster/mouse Dominant lethal test mouse Heritable translocation test mouse
NT
m
NT
NT = not tested.
per plate) in the Ames test with TA1538 and TA98 after metabolic activation. LHM did not show any evidence either of clastogenic potential in vivo, or of tumorigenicity in male and female mice and rats (unpublished results) (Table 1). Ethyl methanesulfonate (EMS) and N-ethyl-Nnitrosourea (ENU) served as positive controls (see the reviews by Russell et al., 1981; Braun et al., 1982). Because positive results in the Ames test indicate the induction of point mutations in vitro, it was of interest to know whether these compounds might induce point mutations in vivo. Therefore, in the studies presented here they were tested in the mammalian (mouse) spot test (Russell and Major, 1957; Fahrig, 1975), an in vivo assay capable of detecting genetic effects of several kinds, including point mutations, minute deficiencies, deletions of various amounts of chromosomal material and somatic recombination. The mammalian spot test is proposed as a relatively rapid
mammalian in vivo test for the identification of potential mutagens and carcinogens (Russell et al., 1981). The method involves exposing the melanoblasts of embryos in utero, the target cell population, to the compound to be tested. Mutation induction is monitored post-natally by checking the fur of the young mice for color spots that result from expression of a recessive gene involved in coat-color determination. Embryos heterozygous for several coat-color genes on a homozygous a / a (non-agouti) background are obtained by crosses of C57BL × T-stock (for comprehensive reviews, see Fahrig, 1978; Russell et al., 1981; for recommendations concerning the practical application in routine testing, see Braun et al., 1982). Materials and methods
Inbred female C 5 7 B L / 6 J Han-SPF mice (purchased from Zentralinstitut fiir Versuchstierzucht, Hannover, F.R.G.) were acclimatized for at
221 least 14 days. Rotation-bred T-stock males were bred by us, derived from animals supplied by Dr. Fahrig, Zentrallaboratorium for Mutagenitatspri~fung, Freiburg, F.R.G. The mice were housed in Macrolon type-II cages with wood-chip bedding (C57BL/6J: 5 F/cage; T-stock: 1 M/cage). Animal-room temperature was controlled at approx. 21-23°C, the relative humidity at approx. 50-65%, air changes at 10-12 times/h, electric lights from 6 a.m. to 6 p.m. All animals had free access to food (Altromin R plus Frolic) and tap water, both provided ad libitum. Embryos of the genotype a/a; b~ + ; cChp/ ++ ; d s e / + + ; s / + (black coat, dark eyes) were produced by mating 9-14-week-old virgin females of the C57BL strain (a/a; otherwise wild type) to fertile males of the T-stock ( a / a = non-agouti; b / b = brown; cchp/cchp chinchilla and pinkeyed dilution; d se/d se = dilute and short ear; s / s = piebald spotting). The mating ratio was 1 M + 2 F in the chlordimeform group and 1 M ÷ 5 F in the LHM test. Pregnant and lactating females were kept individually in Macrolon type-II cages with wood-chip bedding. The treatment followed the setup of Lang (1978). In the study of CDF and its metabolites, there where 108-114 randomly selected ear-marked pregnant females per group. Each of these received the test chemical by gavage on 3 successive mornings, namely, days 8, 9, and 10 of embryonic development, designating the day of observation of the vaginal plug as day 1 (the morning of this day is day 0.25 by Russell's timing (Russell and Major, 1957)). Doses were as follows: 160 mg C D F / k g body weight as aqueous solution, 100 mg formyl-4-COT/kg body weight as aqueous microcrystalline suspension, or 100 mg 4 - C O T / k g body weight as aqueous solution. In preliminary studies, 160 mg C D F / k g body weight was found to be the highest no-effect dose and 100 m g / k g formyi-4COT or 4-COT was a dose between the non-toxic (80 mg/kg) dose level, and a level (160 mg/kg) at which toxic effects were noted. Accordingly, control females were given the vehicle (0.9 g NaCI; 0.085 g Myrj 53 ad 100 ml bidistilled water) at 10 ml/kg. EMS (100 mg/kg, dissolved in physiological saline) served as positive control and was injected intraperitoneally on days 8, 9, and 10. The =
test compounds were provided by Schering (Berlin/Bergkamen, F.R.G.), while EMS (lot No. 7306) was obtained from Ferak (Berlin, F.R.G.). 3 doses of LHM, namely 0.1, 1.0, and 10.0 mg/kg, were given as aqueous solutions by gavage in the manner described above. There were 84-94 randomly selected pregnant females per dose group. It was assumed from dose-finding studies that 10 m g / k g would be a dose level at which toxic effects might be noted. In rats, 0.3 mg/kg, given on each of days 6-15 p.c. had led to a total embryo-lethal effect, presumably induced by inhibition of the prolactin secretion followed by corpora lutea insufficiency (unpublished results). Control females were given the vehicle (physiological saline) at 10 ml/kg. ENU (10 or 20 mg/kg, dissolved in physiological saline) served as positive control and was injected on the same days intraperitoneally. LHM was provided by Schering (Berlin/Bergkamen, F.R.G.); ENU (lot No.: control C) was obtained from Serva (Heidelberg, F.R.G.) and was dissolved daily immediately before application; the pH was 4.2-4.9. Coded offspring were checked for the presence of coat-color spots by at least 2 examiners each at the ages of approx. 2, 3, and 4 weeks. Examination of spots was performed by direct visual inspection. Coat-color spots can be produced either by expression of the recessive gene at the marked loci (Spots of Genetic Relevance, SGR; mostly referred to Recessive Spots, RS) or by a decrease in melanocyte precursor cells (White Mid Ventral Spots, WMVS, indicating cytotoxicity) (Russell and Major, 1957; Eahrig, 1978). SGR are randomly located and generally of greyish or brownish color. WMVS (containing no pigment) are presumed to represent pigment cell death or specific division delay of melanoblasts (Russell and Major, 1957; Fahrig, 1978). According to the recommendations of Braun et al. (1982) no attempt to classify the spots by microscopy (Fahrig, 1978; Hart et al., 1982) has been made in the present experiment. Also morphological malformations were not monitored. For statistical evaluation, the one-tailed Fisher's exact test was performed to compare relative frequencies of variables. Means of variables were compared by the Dunnett test.
222
Results and discussion
The highest LHM and ENU dose levels tested reduced significantly ( P <0.01) the body weight gain of the pregnant females after 2 days of treatment at days 8 and 9 of gestation. This examination was not performed with the C D F group. The test design, the outcome of matings as well as the frequency of SGR and WMVS in the various test and control groups are summarized in Tables 2 and 3. Table 2 clearly shows that 4-COT and EMS have a definite activity. Compared to the concurrent negative control, both compounds induced a significant increase ( P < 0.05) in offspring with SGR; EMS, moreover, also increased the frequency of offspring with WMVS. In contrast to the other groups the number of females with litters surviving to observation decreased clearly after formyl-4-COT treatment compared to the vehicle control, whereas the mean litter size did not differ significantly in all groups. Table 3 shows that LHM did not increase the frequency of offspring with SGR or WMVS compared to the vehicle control. ENU significantly
( P < 0.05) increased the frequency of offspring with SGR and WMVS in a dose-dependent manner. Miiller et al. (1980) obtained, after a single i.p. treatment with 10 or 20 m g / k g ENU at different days of embryonic development, a frequency of offspring with SGR of 4% (day 10) or 7% (day 10) to 9% (day 9), respectively. In the present study these doses of ENU induced, when given on each of days 8, 9, and 10 of gestation (for total exposures of 30 and 60 mg/kg), 10 and 29% of offspring with SGR. This is well in line with expectations based on additivity of separate doses. 3 times 20 m g / k g ENU in the present study seemed more effective than a single i.p. injection with 75 mg/kg, yielding 19.2 (Russell; cited in Braun et al., 1982) or 11.7% (Neuh~iuser-Klaus; cited in Braun et al., 1982) offspring with SGR on different stages of embryonic development, but Russell and Montgomery (1982) showed that there is evidence for a flattening in the dose curve between 50 and 75 mg/kg. For 50 m g / k g they observed an SGR frequency of 20.3% (day 10). Thus, multiple treatment with ENU, a compound characterized by its rapid decomposition under
TABLE 2 T H E EFFECT OF C H L O R D I M E F O R M . HCI, N-FORMYL-4-CHLORO-o-TOLUIDINE, 4-CHLORO-o-TOLUIDINE. HCI A N D ETHYL M E T H A N E S U L F O N A T E (EMS) IN THE MAMMALIAN SPOT TEST Treatment
Dosage
Females
(mg/kg) (route and number of doses)
treated
Offspring with litters surviving to observation
survived to observation N
X
S.D.
with WMVS
with spots of genetic relevance
N
N
%
%
Vehicle control
0 (p.o.; 3 a)
102
56
336
6.0
1.8
1
0.3
3
0.9
Chlordimeform. HCI
160 (p.o.; 3 ~)
108
54
331
6.1
2.1
0
0
3
0.9
N-Formyl-4-chloroo-toluidine
100 (p.o.; 3 a)
114
31
172
5.6
1.9
4
2.3
1
0.6
4-Chloroo-toluidine- HCI
100 (p.o.; 3 * )
108
56
346
6.2
2.0
0
0
11 *
3.2
EMS
100 (i.p.; 3 ~)
107
45
283
6.3
1.7
12 *
4.2
15 *
5.3
Treated on days 8, 9, and 10 of embryonic development. * P < 0.05 Fisher's exact test (one-tailed).
223 TABLE 3 THE EFFECT OF L1SURIDE HYDROGEN MALEATE (LHM) AND N - E T H Y L - N - N I T R O S O U R E A (ENU) IN THE MAMMALIAN SPOT TEST Dosage
Females
(mg/kg) (route and number of doses)
treated
Vehicle control
0 (p.o.; 3 ")
94
LHM
0.1 (p.o.; 3 a) 1.0 (p.o.; 3 ~) 10 (p.o.; 3 a)
Treatment
LHM LHM ENU ENU
10 (i.p.; 3 ~) 20 (i.p.; 3 a )
Offspring with litters surviving to observation
survived to observation
with WMVS N
with spots of genetic relevance
N
.,~
S.D.
•
N
%
39
275
7.1
2.0
0
0
2
0.7
94
49
287
5.9
2.2
0
0
1
0.4
93
51
326
6.4
2.3
0
0
2
0.6
91
45
310
6.9
1.7
0
0
2
0.6
84
55
365
6.6
1.9
9*
2.5
37 *
10.1
84
44
215
4.9
1.9
25 *
11.6
62 *
28.8
Treated on days 8, 9, and 10 of embryonic development. * P < 0.05 Fisher's exact test (one-tailed).
physiological conditions, seems to result in an a d d i t i v i t y o f spot frequencies o f s e p a r a t e doses. T h e results of the vehicle controls (0.7 a n d 0.9% S G R ) a n d the positive control E M S (5.3% S G R ) are c o m p a r a b l e with earlier findings (Lang, 1978), in which n o n e out of 275 vehicle c o n t r o l a n i m a l s showed an S G R , a n d the frequency of S G R in the E M S group was 5.5%. T h e C 5 7 B L / 6 J mice in the earlier s t u d y were from a n o t h e r source. ( F o r further E M S results see Russell et al. (1981).) T h e positive effect of 4 - C O T and the negative effects with C D F and f o r m y l - 4 - C O T in the m a m m a l i a n spot test c o n f i r m the A m e s test findings; however, all 3 c o m p o u n d s were carcinogenic in the m o u s e (see T a b l e 1). A t first sight, this seems to be s o m e w h a t c o n t r a d i c t o r y . If the carcinogenic effect is based on D N A d a m a g e resulting in s o m a t i c m u t a t i o n s , as d e m o n s t r a t e d in the m a m m a l i a n spot test, the following a s s u m p t i o n m a y explain the discrepancy. T h e significant increase in offspring with S G R after 4 - C O T t r e a t m e n t means that the reactive i n t e r m e d i a t e reached the target cells b e y o n d the p l a c e n t a l barrier. Since there are indications that
the reactive m e t a b o l i t e of C D F in mice is formed via 4 - C O T ( u n p u b l i s h e d results), it m a y be supp o s e d that, when C D F and f o r m y l - 4 - C O T were a d m i n i s t e r e d to the animals in the m a m m a l i a n spot test, the m e t a b o l i c turnover of both comp o u n d s in the p r e g n a n t mouse did not lead to an effective level of the finally reactive i n t e r m e d i a t e at the e m b r y o n i c target cells. T h e r e are indications that C D F m a y induce m i c r o s o m a l m o n o o x y g e n a s e s in mice a n d rats (unp u b l i s h e d results), i.e. it might be possible to reach higher c o n c e n t r a t i o n s of the reactive m e t a b o l i t e also in the m a m m a l i a n spot test by multiple ( m o r e than 3 times) t r e a t m e n t with high doses of C D F . However, this a p p r o a c h might be limited by emb r y o t o x i c i t y occurring at higher dose levels. R e g a r d i n g the carcinogenic potential of C D F a n d its principal metabolites one has to consider, besides the organospecificity, that the mice in the long-term studies (see T a b l e 1) were treated continuously with high doses (lifetime feeding). The negative effect of L H M in the m a m m a l i a n s p o t test d e m o n s t r a t e s that the weakly positive A m e s test findings at very high c o n c e n t r a t i o n s
224 w e r e n o t i n d i c a t i v e o f a m u t a g e n i c e f f e c t in vivo. T h i s c o n c l u s i o n is s u p p o r t e d b y t h e n e g a t i v e res u l t s in c a r c i n o g e n i c i t y s t u d i e s w i t h t h i s c o m p o u n d in m i c e a n d rats. Summarizing the interpretation of our data. a p o s i t i v e s p o t test r e s u l t i n d i c a t e s a n in v i v o g e n o t o x i c p o t e n t i a l a n d c l a s s i f i e s t h e c o m p o u n d as a p o t e n t i a l c a r c i n o g e n . A c c o r d i n g t o R u s s e l l et al. ( 1 9 8 1 ) a n d B r a u n et al. (1982), so far, all a g e n t s f o u n d p o s i t i v e in t h e m a m m a l i a n s p o t test h a v e e i t h e r b e e n c a r c i n o g e n i c o r n o t yet b e e n t e s t e d for carcinogenicity. On the other hand, compounds c a r c i n o g e n i c in t h e m o u s e m a y b e n e g a t i v e in t h e m o u s e s p o t test. C o n c e r n i n g t h e A m e s test, t h e r e s u l t s p r e s e n t e d in t h i s p a p e r s u p p o r t t h e g e n e r a l l y a c c e p t e d v i e w that positive bacterial test results do not necess a r i l y c o r r e l a t e w i t h p o s i t i v e r e s u l t s e i t h e r in in vivo mammalian mutagenicity or carcinogenicity studies. Acknowledgement I t h a n k D r . R. F a h r i g for s u p p l y i n g t h e T - s t o c k .
References Braun, R., L.B. Russell and J. Sch6neich (1982) Workshop on the practical application of the mammalian spot test in routine mutagenicity testing of drugs and other chemicals, Mutation Res., 97, 155-161. Fahrig, R. (1975) A mammalian spot test: Induction of genetic alterations in pigment cells of mouse embryos with X-rays and chemical mutagens, Mol. Gen. Genet., 138, 309-314. Fahrig, R. (1978) The mammalian spot test: A sensitive in vivo method for the detection of genetic alterations in somatic
cells of mice, in: A. Hollaender and F.J. de Serres (Eds.), Chemical Mutagens, Plenum, New York, pp. 151-176. FAO (1979) FAO Plant Production and Protection Paper, 15 Suppl., Pesticide Residues in Food: 1978 Evaluations. Food and Agriculture Organization of the United Nations, Rome, pp. 51-78. FAO (1980) FAO Plant Production and Protection Paper, 20 Suppl., Pesticide Residues in Food: 1979 Evaluations, Food and Agriculture Organization of the United Nations, Rome, pp. 129-135. FAO (1981) FAO Plant Production and Protection Paper, 26 Suppl., Pesticide Residues in Food: 1980 Evaluations, Food and Agriculture Organization of the United Nations, Rome, pp. 89-96. Hart, J.W., N.J. Jensen and S. Larsen (1982) Fluorescence microscopy as an aid in differentiation of coat-colour mosaics in the mammalian spot test, Mutation Res., 105, 357-361. Lang, R. (1978) Mammalian spot test with moxnidazole, a 5-nitroimidazole, Experientia, 34, 500--501. Lang, R., and I.-D. Adler (1982) Studies on the mutagenic potential of the pesticide chlordimeform and its principal metabolites in the mouse heritable translocation assay, Mutation Res.. 92. 243-248. MOiler, E.W., J. Braun and H.G. Miltenburger (1980) On the correlation between chemically induced somatic mutations (fur spot test) and cytogenetic damage in mice, Tenth Annual Meeting of EEMS on Environmental Mutagenesis, Athens, 14-19 September 1980. Russell, L.B. (1979) In vivo somatic mutation systems in the mouse, Genetics, 92, 153-163. Russell, L.B., and M.H. Major (1957) Radiation-induced presumed somatic mutations in the house mouse, Genetics, 42, 161-175. Russell, L.B., and C.S. Montgomery (1982) Supermutagenicity of ethylnitrosourea in the mouse spot test, Mutation Res., 92, 193-204. Russell, LB., P.B. Selby, E. yon Halle, W. Sheridan and L. Valcovic (1981) Use of the mouse spot test in chemical mutagenesis; Interpretation of past data and recommendations for future work, Mutation Res., 86, 355-379.
219
Elsevier MTR 00846
The mammalian spot test and its use for testing of mutagenic and carcinogenic potential: experience with the pesticide chlordimeform, its principal metabolites and the drug lisuride hydrogen maleate R. Lang Department o/Experimental Toxicololo', Sehering, Berlin/Bergkamen (F.R. G.)
(Received 12 April 1983) (Revision received22 July 1983) (Accepted 17 October 1983)
Summa~ The mammalian spot test has recently been demonstrated to be a promising method for the detection of somatic mutations induced by chemicals and is at present under validation as an in vivo screening test for carcinogenic potential. The pesticide chlordimeform, its principal, metabolites N-formyl-4-chloro-o-toluidine and 4-chloro-otoluidine, and the drug lisuride hydrogen maleate, as well as the known mutagens ethyl methanesulfonate (EMS) and N-ethyl-N-nitrosourea (ENU) were tested in the mammalian spot test. Female C57BL/6J mice were mated to T-stock males and treated by gavage with maximal tolerated doses of the test compounds, or by intraperitoneal injection with EMS and ENU at days 8, 9 and 10 of pregnancy. Mutation induction was monitored post-natally by checking the fur of the offspring for color spots that result from expression of a recessive gene involved in coat-color determination. Of the environmental chemicals tested, only 4-chloro-o-toluidine was mutagenic in the spot test. This positive result was in agreement with in vitro experiments and its carcinogenic potential in the mouse. However, the mouse carcinogens chlordimeform and N-formyl-4-chloro-o-toluidine were negative, as was the non-carcinogen lisuride hydrogen maleate. N-Formyl-4-chloro-o-toluidine and lisuride hydrogen maleate have been reported to be weakly positive in the Ames test. The positive controls EMS and ENU showed a clear mutagenic response.
The insecticide and acaracide chlordimeformHCi [N,N'-dimethyl-N'-(2-methyl-4-chlorophenyl)-formamidine-HCI; CDF] and its 2 principal metabolites, N-formyl-4-chloro-o-toluidine (formyl-4-COT) and 4-chloro-o-toluidine. HCI (4COT), were carcinogenic in the mouse but not in rats (FAO, 1979, 1980, 1981). Additionally, the metabolites showed some mutagenic potential in short-term bioassays (FAO, 1979, 1980, 1981); 4-COT was found to be clearly mutagenic after metabolic activation in Salmonella typhimurium strains TA98 and TA100, but the formyl metabo0165-1218/84/$03.00 © 1984 ElsevierScience Publishers B.V.
lite showed a very weak response with TA100. C D F was negative in the Ames test (FAO, 1979, 1980, 1981; unpublished results). None of the 3 compounds showed a clastogenic potential (FAO, 1979, 1980, 1981), and none induced translocation heterozygosity in the mouse heritable translocation test (Lang and Adler, 1982) (Table 1). Lisuride hydrogen maleate [3-(10,10a-didehydro-7-methyl-9a-ergolinyl)-l,l-diethylurea, hydrogen maleate; LHM], an ergot alkaloid with dopamine agonist activity, showed weak mutagenic effects at very high concentrations (mg range
220 TABLE 1 PRINCIPAL RESULTS OF T U M O R I G E N I C I T Y A N D MUTAGENICITY STUDIES WITH CDF. FORMYL-4-COT, 4-COT, A N D LHM
Tumorigenicity Mouse Rat
CDF
Formyl-4-COT
4-COT
+
+
+
(+)
+
NT
NT
NT
NT
Point mutations in vitro Salmonella/microsome test D N A damage in vivo SCE test Chinese hamster
NT
LHM
+
Point mutations in vivo Mammalian spot test Mouse Chromosomal aberrations in vivo Chromosome analysis Chinese hamster Nucleus anomaly t e s t / micronucleus test Chinese hamster/mouse Dominant lethal test mouse Heritable translocation test mouse
NT
m
NT
NT = not tested.
per plate) in the Ames test with TA1538 and TA98 after metabolic activation. LHM did not show any evidence either of clastogenic potential in vivo, or of tumorigenicity in male and female mice and rats (unpublished results) (Table 1). Ethyl methanesulfonate (EMS) and N-ethyl-Nnitrosourea (ENU) served as positive controls (see the reviews by Russell et al., 1981; Braun et al., 1982). Because positive results in the Ames test indicate the induction of point mutations in vitro, it was of interest to know whether these compounds might induce point mutations in vivo. Therefore, in the studies presented here they were tested in the mammalian (mouse) spot test (Russell and Major, 1957; Fahrig, 1975), an in vivo assay capable of detecting genetic effects of several kinds, including point mutations, minute deficiencies, deletions of various amounts of chromosomal material and somatic recombination. The mammalian spot test is proposed as a relatively rapid
mammalian in vivo test for the identification of potential mutagens and carcinogens (Russell et al., 1981). The method involves exposing the melanoblasts of embryos in utero, the target cell population, to the compound to be tested. Mutation induction is monitored post-natally by checking the fur of the young mice for color spots that result from expression of a recessive gene involved in coat-color determination. Embryos heterozygous for several coat-color genes on a homozygous a / a (non-agouti) background are obtained by crosses of C57BL × T-stock (for comprehensive reviews, see Fahrig, 1978; Russell et al., 1981; for recommendations concerning the practical application in routine testing, see Braun et al., 1982). Materials and methods
Inbred female C 5 7 B L / 6 J Han-SPF mice (purchased from Zentralinstitut fiir Versuchstierzucht, Hannover, F.R.G.) were acclimatized for at
221 least 14 days. Rotation-bred T-stock males were bred by us, derived from animals supplied by Dr. Fahrig, Zentrallaboratorium for Mutagenitatspri~fung, Freiburg, F.R.G. The mice were housed in Macrolon type-II cages with wood-chip bedding (C57BL/6J: 5 F/cage; T-stock: 1 M/cage). Animal-room temperature was controlled at approx. 21-23°C, the relative humidity at approx. 50-65%, air changes at 10-12 times/h, electric lights from 6 a.m. to 6 p.m. All animals had free access to food (Altromin R plus Frolic) and tap water, both provided ad libitum. Embryos of the genotype a/a; b~ + ; cChp/ ++ ; d s e / + + ; s / + (black coat, dark eyes) were produced by mating 9-14-week-old virgin females of the C57BL strain (a/a; otherwise wild type) to fertile males of the T-stock ( a / a = non-agouti; b / b = brown; cchp/cchp chinchilla and pinkeyed dilution; d se/d se = dilute and short ear; s / s = piebald spotting). The mating ratio was 1 M + 2 F in the chlordimeform group and 1 M ÷ 5 F in the LHM test. Pregnant and lactating females were kept individually in Macrolon type-II cages with wood-chip bedding. The treatment followed the setup of Lang (1978). In the study of CDF and its metabolites, there where 108-114 randomly selected ear-marked pregnant females per group. Each of these received the test chemical by gavage on 3 successive mornings, namely, days 8, 9, and 10 of embryonic development, designating the day of observation of the vaginal plug as day 1 (the morning of this day is day 0.25 by Russell's timing (Russell and Major, 1957)). Doses were as follows: 160 mg C D F / k g body weight as aqueous solution, 100 mg formyl-4-COT/kg body weight as aqueous microcrystalline suspension, or 100 mg 4 - C O T / k g body weight as aqueous solution. In preliminary studies, 160 mg C D F / k g body weight was found to be the highest no-effect dose and 100 m g / k g formyi-4COT or 4-COT was a dose between the non-toxic (80 mg/kg) dose level, and a level (160 mg/kg) at which toxic effects were noted. Accordingly, control females were given the vehicle (0.9 g NaCI; 0.085 g Myrj 53 ad 100 ml bidistilled water) at 10 ml/kg. EMS (100 mg/kg, dissolved in physiological saline) served as positive control and was injected intraperitoneally on days 8, 9, and 10. The =
test compounds were provided by Schering (Berlin/Bergkamen, F.R.G.), while EMS (lot No. 7306) was obtained from Ferak (Berlin, F.R.G.). 3 doses of LHM, namely 0.1, 1.0, and 10.0 mg/kg, were given as aqueous solutions by gavage in the manner described above. There were 84-94 randomly selected pregnant females per dose group. It was assumed from dose-finding studies that 10 m g / k g would be a dose level at which toxic effects might be noted. In rats, 0.3 mg/kg, given on each of days 6-15 p.c. had led to a total embryo-lethal effect, presumably induced by inhibition of the prolactin secretion followed by corpora lutea insufficiency (unpublished results). Control females were given the vehicle (physiological saline) at 10 ml/kg. ENU (10 or 20 mg/kg, dissolved in physiological saline) served as positive control and was injected on the same days intraperitoneally. LHM was provided by Schering (Berlin/Bergkamen, F.R.G.); ENU (lot No.: control C) was obtained from Serva (Heidelberg, F.R.G.) and was dissolved daily immediately before application; the pH was 4.2-4.9. Coded offspring were checked for the presence of coat-color spots by at least 2 examiners each at the ages of approx. 2, 3, and 4 weeks. Examination of spots was performed by direct visual inspection. Coat-color spots can be produced either by expression of the recessive gene at the marked loci (Spots of Genetic Relevance, SGR; mostly referred to Recessive Spots, RS) or by a decrease in melanocyte precursor cells (White Mid Ventral Spots, WMVS, indicating cytotoxicity) (Russell and Major, 1957; Eahrig, 1978). SGR are randomly located and generally of greyish or brownish color. WMVS (containing no pigment) are presumed to represent pigment cell death or specific division delay of melanoblasts (Russell and Major, 1957; Fahrig, 1978). According to the recommendations of Braun et al. (1982) no attempt to classify the spots by microscopy (Fahrig, 1978; Hart et al., 1982) has been made in the present experiment. Also morphological malformations were not monitored. For statistical evaluation, the one-tailed Fisher's exact test was performed to compare relative frequencies of variables. Means of variables were compared by the Dunnett test.
222
Results and discussion
The highest LHM and ENU dose levels tested reduced significantly ( P <0.01) the body weight gain of the pregnant females after 2 days of treatment at days 8 and 9 of gestation. This examination was not performed with the C D F group. The test design, the outcome of matings as well as the frequency of SGR and WMVS in the various test and control groups are summarized in Tables 2 and 3. Table 2 clearly shows that 4-COT and EMS have a definite activity. Compared to the concurrent negative control, both compounds induced a significant increase ( P < 0.05) in offspring with SGR; EMS, moreover, also increased the frequency of offspring with WMVS. In contrast to the other groups the number of females with litters surviving to observation decreased clearly after formyl-4-COT treatment compared to the vehicle control, whereas the mean litter size did not differ significantly in all groups. Table 3 shows that LHM did not increase the frequency of offspring with SGR or WMVS compared to the vehicle control. ENU significantly
( P < 0.05) increased the frequency of offspring with SGR and WMVS in a dose-dependent manner. Miiller et al. (1980) obtained, after a single i.p. treatment with 10 or 20 m g / k g ENU at different days of embryonic development, a frequency of offspring with SGR of 4% (day 10) or 7% (day 10) to 9% (day 9), respectively. In the present study these doses of ENU induced, when given on each of days 8, 9, and 10 of gestation (for total exposures of 30 and 60 mg/kg), 10 and 29% of offspring with SGR. This is well in line with expectations based on additivity of separate doses. 3 times 20 m g / k g ENU in the present study seemed more effective than a single i.p. injection with 75 mg/kg, yielding 19.2 (Russell; cited in Braun et al., 1982) or 11.7% (Neuh~iuser-Klaus; cited in Braun et al., 1982) offspring with SGR on different stages of embryonic development, but Russell and Montgomery (1982) showed that there is evidence for a flattening in the dose curve between 50 and 75 mg/kg. For 50 m g / k g they observed an SGR frequency of 20.3% (day 10). Thus, multiple treatment with ENU, a compound characterized by its rapid decomposition under
TABLE 2 T H E EFFECT OF C H L O R D I M E F O R M . HCI, N-FORMYL-4-CHLORO-o-TOLUIDINE, 4-CHLORO-o-TOLUIDINE. HCI A N D ETHYL M E T H A N E S U L F O N A T E (EMS) IN THE MAMMALIAN SPOT TEST Treatment
Dosage
Females
(mg/kg) (route and number of doses)
treated
Offspring with litters surviving to observation
survived to observation N
X
S.D.
with WMVS
with spots of genetic relevance
N
N
%
%
Vehicle control
0 (p.o.; 3 a)
102
56
336
6.0
1.8
1
0.3
3
0.9
Chlordimeform. HCI
160 (p.o.; 3 ~)
108
54
331
6.1
2.1
0
0
3
0.9
N-Formyl-4-chloroo-toluidine
100 (p.o.; 3 a)
114
31
172
5.6
1.9
4
2.3
1
0.6
4-Chloroo-toluidine- HCI
100 (p.o.; 3 * )
108
56
346
6.2
2.0
0
0
11 *
3.2
EMS
100 (i.p.; 3 ~)
107
45
283
6.3
1.7
12 *
4.2
15 *
5.3
Treated on days 8, 9, and 10 of embryonic development. * P < 0.05 Fisher's exact test (one-tailed).
223 TABLE 3 THE EFFECT OF L1SURIDE HYDROGEN MALEATE (LHM) AND N - E T H Y L - N - N I T R O S O U R E A (ENU) IN THE MAMMALIAN SPOT TEST Dosage
Females
(mg/kg) (route and number of doses)
treated
Vehicle control
0 (p.o.; 3 ")
94
LHM
0.1 (p.o.; 3 a) 1.0 (p.o.; 3 ~) 10 (p.o.; 3 a)
Treatment
LHM LHM ENU ENU
10 (i.p.; 3 ~) 20 (i.p.; 3 a )
Offspring with litters surviving to observation
survived to observation
with WMVS N
with spots of genetic relevance
N
.,~
S.D.
•
N
%
39
275
7.1
2.0
0
0
2
0.7
94
49
287
5.9
2.2
0
0
1
0.4
93
51
326
6.4
2.3
0
0
2
0.6
91
45
310
6.9
1.7
0
0
2
0.6
84
55
365
6.6
1.9
9*
2.5
37 *
10.1
84
44
215
4.9
1.9
25 *
11.6
62 *
28.8
Treated on days 8, 9, and 10 of embryonic development. * P < 0.05 Fisher's exact test (one-tailed).
physiological conditions, seems to result in an a d d i t i v i t y o f spot frequencies o f s e p a r a t e doses. T h e results of the vehicle controls (0.7 a n d 0.9% S G R ) a n d the positive control E M S (5.3% S G R ) are c o m p a r a b l e with earlier findings (Lang, 1978), in which n o n e out of 275 vehicle c o n t r o l a n i m a l s showed an S G R , a n d the frequency of S G R in the E M S group was 5.5%. T h e C 5 7 B L / 6 J mice in the earlier s t u d y were from a n o t h e r source. ( F o r further E M S results see Russell et al. (1981).) T h e positive effect of 4 - C O T and the negative effects with C D F and f o r m y l - 4 - C O T in the m a m m a l i a n spot test c o n f i r m the A m e s test findings; however, all 3 c o m p o u n d s were carcinogenic in the m o u s e (see T a b l e 1). A t first sight, this seems to be s o m e w h a t c o n t r a d i c t o r y . If the carcinogenic effect is based on D N A d a m a g e resulting in s o m a t i c m u t a t i o n s , as d e m o n s t r a t e d in the m a m m a l i a n spot test, the following a s s u m p t i o n m a y explain the discrepancy. T h e significant increase in offspring with S G R after 4 - C O T t r e a t m e n t means that the reactive i n t e r m e d i a t e reached the target cells b e y o n d the p l a c e n t a l barrier. Since there are indications that
the reactive m e t a b o l i t e of C D F in mice is formed via 4 - C O T ( u n p u b l i s h e d results), it m a y be supp o s e d that, when C D F and f o r m y l - 4 - C O T were a d m i n i s t e r e d to the animals in the m a m m a l i a n spot test, the m e t a b o l i c turnover of both comp o u n d s in the p r e g n a n t mouse did not lead to an effective level of the finally reactive i n t e r m e d i a t e at the e m b r y o n i c target cells. T h e r e are indications that C D F m a y induce m i c r o s o m a l m o n o o x y g e n a s e s in mice a n d rats (unp u b l i s h e d results), i.e. it might be possible to reach higher c o n c e n t r a t i o n s of the reactive m e t a b o l i t e also in the m a m m a l i a n spot test by multiple ( m o r e than 3 times) t r e a t m e n t with high doses of C D F . However, this a p p r o a c h might be limited by emb r y o t o x i c i t y occurring at higher dose levels. R e g a r d i n g the carcinogenic potential of C D F a n d its principal metabolites one has to consider, besides the organospecificity, that the mice in the long-term studies (see T a b l e 1) were treated continuously with high doses (lifetime feeding). The negative effect of L H M in the m a m m a l i a n s p o t test d e m o n s t r a t e s that the weakly positive A m e s test findings at very high c o n c e n t r a t i o n s
224 w e r e n o t i n d i c a t i v e o f a m u t a g e n i c e f f e c t in vivo. T h i s c o n c l u s i o n is s u p p o r t e d b y t h e n e g a t i v e res u l t s in c a r c i n o g e n i c i t y s t u d i e s w i t h t h i s c o m p o u n d in m i c e a n d rats. Summarizing the interpretation of our data. a p o s i t i v e s p o t test r e s u l t i n d i c a t e s a n in v i v o g e n o t o x i c p o t e n t i a l a n d c l a s s i f i e s t h e c o m p o u n d as a p o t e n t i a l c a r c i n o g e n . A c c o r d i n g t o R u s s e l l et al. ( 1 9 8 1 ) a n d B r a u n et al. (1982), so far, all a g e n t s f o u n d p o s i t i v e in t h e m a m m a l i a n s p o t test h a v e e i t h e r b e e n c a r c i n o g e n i c o r n o t yet b e e n t e s t e d for carcinogenicity. On the other hand, compounds c a r c i n o g e n i c in t h e m o u s e m a y b e n e g a t i v e in t h e m o u s e s p o t test. C o n c e r n i n g t h e A m e s test, t h e r e s u l t s p r e s e n t e d in t h i s p a p e r s u p p o r t t h e g e n e r a l l y a c c e p t e d v i e w that positive bacterial test results do not necess a r i l y c o r r e l a t e w i t h p o s i t i v e r e s u l t s e i t h e r in in vivo mammalian mutagenicity or carcinogenicity studies. Acknowledgement I t h a n k D r . R. F a h r i g for s u p p l y i n g t h e T - s t o c k .
References Braun, R., L.B. Russell and J. Sch6neich (1982) Workshop on the practical application of the mammalian spot test in routine mutagenicity testing of drugs and other chemicals, Mutation Res., 97, 155-161. Fahrig, R. (1975) A mammalian spot test: Induction of genetic alterations in pigment cells of mouse embryos with X-rays and chemical mutagens, Mol. Gen. Genet., 138, 309-314. Fahrig, R. (1978) The mammalian spot test: A sensitive in vivo method for the detection of genetic alterations in somatic
cells of mice, in: A. Hollaender and F.J. de Serres (Eds.), Chemical Mutagens, Plenum, New York, pp. 151-176. FAO (1979) FAO Plant Production and Protection Paper, 15 Suppl., Pesticide Residues in Food: 1978 Evaluations. Food and Agriculture Organization of the United Nations, Rome, pp. 51-78. FAO (1980) FAO Plant Production and Protection Paper, 20 Suppl., Pesticide Residues in Food: 1979 Evaluations, Food and Agriculture Organization of the United Nations, Rome, pp. 129-135. FAO (1981) FAO Plant Production and Protection Paper, 26 Suppl., Pesticide Residues in Food: 1980 Evaluations, Food and Agriculture Organization of the United Nations, Rome, pp. 89-96. Hart, J.W., N.J. Jensen and S. Larsen (1982) Fluorescence microscopy as an aid in differentiation of coat-colour mosaics in the mammalian spot test, Mutation Res., 105, 357-361. Lang, R. (1978) Mammalian spot test with moxnidazole, a 5-nitroimidazole, Experientia, 34, 500--501. Lang, R., and I.-D. Adler (1982) Studies on the mutagenic potential of the pesticide chlordimeform and its principal metabolites in the mouse heritable translocation assay, Mutation Res.. 92. 243-248. MOiler, E.W., J. Braun and H.G. Miltenburger (1980) On the correlation between chemically induced somatic mutations (fur spot test) and cytogenetic damage in mice, Tenth Annual Meeting of EEMS on Environmental Mutagenesis, Athens, 14-19 September 1980. Russell, L.B. (1979) In vivo somatic mutation systems in the mouse, Genetics, 92, 153-163. Russell, L.B., and M.H. Major (1957) Radiation-induced presumed somatic mutations in the house mouse, Genetics, 42, 161-175. Russell, L.B., and C.S. Montgomery (1982) Supermutagenicity of ethylnitrosourea in the mouse spot test, Mutation Res., 92, 193-204. Russell, LB., P.B. Selby, E. yon Halle, W. Sheridan and L. Valcovic (1981) Use of the mouse spot test in chemical mutagenesis; Interpretation of past data and recommendations for future work, Mutation Res., 86, 355-379.