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The application of mutagenicity tests to the prediction of carcinogenic activity of chemicals and drugs
Pharmacological Research Communications, Vol. 16, No. 5, 1984
421
TilE APPLICATION OF MUTAGENICITY TESTS TO THE PREDICTION OF CARCINOGENIC ACTIVITY OF CHEMICALS AND DRUGS P. Dolara and G. Caderni Institute of Pharmacology and Toxicology, University of Florence, Italy
Receivedm final ~rm 30c~ber 1983 MUTACENESIS AS A WAY OF PREDICTING CARCINOGENESIS Numerous tests have been developed that can measure the mutagenic activity of chemicals and drugs.
Since many carcinogens are also mutagens an
extensive effort has been carried out to develop new mutagenesis tests that can be reliable predictors of carcinogenicity. After a decade of effort, however, the main question:
is there a causal
relationship between mutagenicity and carcinogenicity? remains unanswered (ICPEMC, ]982).
It has been empirically demonstrated that a high percent-
age of substaltces that give a positive mutagenic response are also animal carcinogens, but a causal link between the two phenomena has not yet been established. However, there is general agreement about the existence of two groups of chemical carcinogens:
a) genotoxic carcinogens, that produce a change
in the genetic information of cells, and b) carcinogenic enhancer and tumor promoters that act somewhere else in the cascade of events that separate the initiation of a tumor from its clinical onset.
Since mutagenesis is
aimed at detecting the finst type of activity, it is inherently incapable of detecting tumor promoters or enhancers.
Notwithstanding this limitation,
because of their rapidity and low cost, mutagenesJs tests have attained a large popularity for studying the toxicity of chemicals and drugs. CURRENT METHODOLOGIES FOR MIITAGENICITY TESTING More than iO0 different mutagenicity tests are currently in use, but we will outline only the prir=eipal ones in the table which follows.
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© 1984 The Italian Pharmacological Society
Pharmacological Resea,,ch Communications, Vol. 16, No. 5, 1984
422
Tab_le ! .
Outl/ne
of l'rimary M u r a l s
SYSTEM
Salmonella typhimurium Escherichia coli
.
.
.
gene mutation.
Forward and Rever~e H . t a t i o n Assays
DNA damage
gene mutation
Mammalian Cells In Vitro Chinese hamster ovary liCPR'r, TK and Na/K cells (CllO) ATPase Assays V79 Chinese hamster lung cells LSI78Y mouse lymphoma cells A l k a ] i n e E l u t i c m of Various human and rodent cell I ines DNA DNA Re pai r S i s t e r Chromat id Ext:hange (SCE) Mouse c e l t l i n e BAI,BI 3T3 T r a n s f o r m a t ion Assay and C311/10 'l'J
.
gene mutation
.
Schizosaccharomyces pombe Saccharomyces cerevisiae 3.
Ames T e s t WP2 Assay Pol A Test
Yeasts
2. i
TYPE OF TEST
Bacterial Cells
I.
_
COMMON NAME
gene mutation
DNA damage DNA damage DNA damage
neoplastlc transformation
Manaaals In Vivo Mice
S p e c i f i c Locus T e s t Dominant Lethal Test Micrommleus In Vivo C y t o g e n e t i c s Spermutest
5.
Host Mediated Assay
6.
Insects Drosophila melanogaster
gune m~tation chromo soma I damage chromosomal damage chromosomal damage morphoiogie damage
(See numbers 1 and 2 and gene m u t a t i o n in 3) Sex-I inked Recessive Lethal Assay
gene mutation
Pharmacological Research Communications, Vol. 16, No. 5, I984
423
BRIEF OUTLINE OF THE INDIVIDUAL METHODS Since the available mutagenicity tests are at very different stages of validation, some being fully developed, others in experimental phase~ their performance has been currently described with the following terminology: sensitivit[: gens tested;
number of mutagenic
carcinogens~number of total carcino-
accurac%: number of correct results (positive and negative)/number of total chemicals tested; ~ : number of non-mutagenic non-carcinogens/total number of non-carcinogens tested; r@liability or predictive yalue: of positive results obtained.
number of mutagenic carcinogens/number
Table 2 shows the performance of the most commonly used mutagenicity tests. For some of the less frequently used, not enough data are available. +
Table 2.
Performance of some mutagenicity tests
Test systems
Sensitivity
Specificity
Predictive Value
Bacterial cells Salmonella typhimurium E. coli WP2
0.74 0.67
0.66 0.73
0.85 0.87
Yeast cells Saccharomyces cerevisiae (mitotic recombination)
0.55
0.69
0.86
Mammalian cells in vitro Gene mutation In vitro transformation (BHK21CLI3)
0.94 O.83
O. 75 O. 63
O. 92 O. 78
Man~nals in vivo Micronucleus assay (mice) Spermatest (mice)
0.55 0.42
0.69 0.81
0.85 0.81
Insects Drosophyia SLRC
0.57
0.89
0.86
++
+
mean values obtained from Purchase (1982) ++IARC (1980), pp. 114-123.
++
++
424
Pharmacological Research Communications, VoL 16, No. 5, 1984
A very brlef description of the most important tests in use follows in Sections 1-6. ].
Methods using bacteria~ cells:
The most popular test was developed by Ames and coworkers (1975), using 5 auxotr6phic stralns of Salmonella typhhnuri,!n b The cells are incubated with the putative mutagens, with and without metabolic activation (an induce4 rat liver $9 preparation), and after 2 days the colonies reverting to prototrophy are scored. A similar protocol was developed by others with E. coli (Herman and Dworkin, 1971). The reliability of the Ames test has been recently reviewed by several authors (Purchase, 1982; Mohn, 1981; Grafe et al. 1981; IARC, 1980, pp. 85-106). The method has been used with over 3,000 chemicals in a large number of laboratories. It has a good sensitivity and predictive value, although it does not perform well with some chemicals such as hydrazines, nitrosamines and metal carcinogens. The quantitative variability among different laboratories, however, is noteworthy (Grafe et al., 1981). Tests using E. coli are widely in use, have a similar performance and are capable of detecting some metal carcinogens (Vennit and Levy, L
1974). 2. .
Methods using yeast cells: .
2
-
•
- -
,
.
.
.
.
.
Mutation assays are based upon a discolor~ition of yeast colonies that are defective in a gene regulating adenine biosynthesis and therefore accumulate a red pigment. Gene mutations may block previous steps of adenine biosynthesis, and the resulting colonies wi]l lose their pigments. Reverse mutation assays measure the growth of prototrophs on deficient media. Mitotic recombination and gene conversion can be measured with methods using yeast cells (Zimmerman et al.,. 1975). The performance of yeast assays is similar to bacterial assays, although the sensitivity is lower and the validation has been obtained on a more restricted experimental sampling (IARC, 1980, pp. 135-153). 3. Methods using man~alian ce]Is in vitro: A. Gene mutationVarious strains of cells can be used that become resistant to a certain chemical (like 8-azaguanine, 6-thioguanfne, 5-Br-deoxy"-'~',,,,~,na,~.._~.=~,~ .... uhen a specific mutation in their genome occurs (.Ne,.-:-"rd~ Flanders, 1981). They have a high predictive value and sensitivity but ale longer and more expensive than bacterial and yeast tests (IARC, 1980, pp. iO7-133). B. Alkaline elution of DNA is a relatively s~mple technique in which DNA damage is detected using tile selective permeability of millJpore membranes for small molecular weight DNA, whlch ~s produced by the action of nucleases on damaged DNA or as a result of the susceptibillty of damaged DNA to alkaline cleavage. The method has not been extensively validated but seems promising on tlle limited number of compounds so far tested (Kohn et al., 1974; Swenberg et al., 1976; I'arodf et of., ]982).
Pharmacological Research Communications, Vol. 16, No. 5, 1984
425
C. DNA repair measures DNA synthesis induced by genetic damage followed by DNA repair. It is determined by measuring the incorporation of labeled thymidine into DNA after the exposure to a chemical mutagen, blocking DNA replication with hydroxyurea. The test has a good predictive value but a low sensitivity (Williams et al., 1982) and is not adviced as a primary screening test (Larsen et al., 1982). D. S i s t e r c h r o m a t i d e x c h a n g e (SCE) m e a s u r e s t h e number o f e x c h a n g e s o f s u b - s e c t i o n s o f c h r o m a t i d s a f t e r t h e e x p o s u r e to a c h e m i c a l m u t a g e n . The cells are grown for two generations in the presence of 5-Br-deoxyuridine to label individual chromatlds and later stained with a fluorescent dye to evidentiate homologous segments that have been exchanged between sister chromatlds (Latt, 1973,; Kato 1974). The molecular mechanism by which SCEs occur is not known but their frequency is increased by the exposure to mutagens. SCE assays have been used with hundreds of chemicals but they have not been validated as extensively as bacterial mutagenesis methods (IARC, 1980, pp. 227-247). E. T~ansformation assays dlffer from the prevlous]~ discussed tests because they measure neoplastic transformation in Xit[o that is very close to the process of carcinogenesis in viva. In these methods cellular lines are treated with a putative carcinogen and after several passages in vitro the number of transformed loci are scored (DiPaolo et al;, 1969; DiPaolo et al., 1972; Kakunaga, 1973). These tests have a good sensitivity and predictive value but are technically difficult, slow and costly. 4. , Methods us in..g mammals in v i v o : A. Mouse specific locus test is an assay to detect heritable pointmutations in mammals. Treated wild-type animals are mated with homozygote strains for some recessive allels. If a mutation in any of the loci represented in the recessive strain occurs in tile wild-type mice, this will result in mutant offspring (Ehling, 1978). Even though the method requires large animal facilities and a long time to be performed it is the only established assay for detecting gene mutations in whole man~nals (Russel and Matter, 198£); ICPEMC, 1983:1). g. Dominant lethal test in mice detects any genetic damage in the treated animal that causes the c]e:~th of the offspring (Bateman, [977). The test is relatively quick and simple to perform, however, it is not very sensitive (1ARC, 1980, pp. 248-251). C. Micronucleus assay detects cht'omosomal damage caused by dastogens or spindle poisons. Acentric chromosomes are excluded from anaphase groups and in thedaughter nuclei form separated "micronuclei" that are particularly easy to evidentiate (Schmidt, 1976; ICPEMCa,1983). This test ks not very sensitive but very often included in test batteries (Trzos, 1978; Purchase, 1982).because of its good predictive value. D. In viva cytogenetics: many transformed cells have chromosomal aberrations, so that the detection of this damage can be used as # predictive carcinogenicity test (Mitelman and Levan, 1978). chromosomal
4 26
Pharmacological Research Comm~micatJons, Vol. !6, No, 5, !984
aberrations are scored in bone marrow cells. The method has a low sensitivir.y (Matter, 1980). E. Spermatest detects the inct'e~ist.,of mc,rl)hological spermal abnorma/ities after treatment with a putative carcinogen. Many substances have been tested with this tQchnique (Heddle and Bruce, 1977; Topham, 1980a; Topham, 19805), The spermatest has shown a relatively good specificity and predictive value together with a low sensitivity. 5.
Host-mediated assay:
In host-mediated assays, cells like bacteria,yeasts or mammal [an ceils are injected into the animal (it~ the venous system or in the peritoneum). After an adequate incubation time tile cells are recovered from the liver or the peritoneum and tested for mutagenicity as described i'n sections ], 2 and 3 above (gent mutations in marmna]ian cells). The test can be useful for studying the pharmacodynamics t~f carcinogens in different organs (Fahrig, 1977; Simmon et el., 1979; Sirianni et el., ]979; Legator et el., 1982). 6.
Insects:
The most conmlonly used m u t a g e n i c i t y t e s t w i t h D r o s o p h i l a me]anogaster i s t h e s e x - l i n k ~ d r e c e s s i v e l e t h a l ;3ssay. For t h i s a s s a y f e m a l e s t h a t h a v e a p a r t i c i J l a r X chromosome (Base X chromosome) a r e mated w i t h t r e a t e d w i l d type males. I f a l e t h a l X m u t a t i o n o c c u r s Jn m a l e s , in t h e F_ g e n e r a t i o n this will result in the absence of an entire ~:lass of males ~W~rg]er, 1977).
This a@say is well validated, has good specificity and predictive value, but very few laboratories know how ~o do it (IARC, 1980, pp. 157-184; Purchase, 1982; ICPEMC, 1983a). A FEW COMMENTS ABOUT THE PERFORMANCE OF DIFFERENT TEST SYSTEMS In the previous section and in Table 2 we have briefly reviewed the performance of individual tests.
It'appears clearly that the predictive values of
most currently used systems are very close to 0.8-0.9, whereas sensitivity and
specificity are more variable.
However, in evaluating these results, it should
be remembered that different predictivities are obtained when changing the prevalence of carcinogens in a group of chemicals to be tested (ICPEMC 1982, 1983b). It has been shown (Purchase, 1982) that using an assay with a sensitivity and specificity of 0.9 when testing 500 carcinogens and 500 non-carcinogens, 50 false positive (non-carcinogenic mutagens) would be found, giving the expected reliability of .9.However~ by testing a group of chemicals with 5% carcinogens (950 non-carcinogens and 50 carcinogens) 45 carcinogens (90%) would be positive in the test but 95 non-carcinogens (10%) would turn out
Pharmacological Research Communications, Vol. 16, No. 5, 1984 positive as well.
427
The total count of positives would be, therefore, 140,
and the reliability (mutagenic carcinogens/total mutagenic compounds) would therefore be only .32 (~5/140).
In the real world the r~tio of carcinogens/
non-carcinogens is presumably very low, and the reliability of individual tests in predicting carcinogenicity of randomly selected chemicals would be, therefore, extremely poor. To improve the relatively poor performance of single mutagenicity tests, the idea of batteries of tests has been developed. assumption is that by using systems differing
The underlying theoretical
in genetic end points and in
levels of cellular complexity, the limitations of each individual test would be overcome. This premise is generally accepted by the scientific community and is the basis for the existing regulations in this field. however, devoid of problems.
It is not,
In the following section we will discuss this
issue. CONBINATION OF INDIVIDUAL TESTS IN A BATTERY:
In a large international,
AN EXPERIMENTAL EVALUATION
interlaboratory study (De Serres and Ashby,
1981) a group of 62 chemicals, of which 25 were carcinogens, were tested with various
methodologies
ill d i f f e r ~ ' n t
tl,e c o m p l e t i o n o f t h e s t u d y . were c h o s e n b e c a u s e
E l e v e n of t h e c a r c i n o g e n s
they normally are i.lac:~e
~leth~dolc~g[es u s e d ,~ere b a c t e r i a l re.ration
assays,
]aboratc~L'ies and d e c o d e d o n l y a f t e r
yeast
assays,
repaic
(problem carcinogens)
in b a c t e r i a l
assays.
and ~hage i n d ~ l c t i o a ,
in v i t r c J mm,malJan a s s a y s ,
The
baecerlal
and in v i v o a s s a y s .
The results of the different tests are shown in Table3, where the numDe~ not in parentheses represents the values obtained considering the results of the problem carcinogens that and amounts n o t i n p a r e n t h e s e s
lower the overall performance of the tests, include
the problem carcinogens.
The performance of different systems in Table 3 is considerably worse than that reported in Table 2, probably because of the abundance of problem carcinogens tested and because the researchers did not know the structure of the substances being tested.
Considering all chemicals tested, the
accuracy can be ranked in the following way:
in vitro mammalian>bacterial
Pharmaco/ogica/ Research Communications, Vo/. 16, No. 5, 1984
428 Table3.
MutagenicityTest?
Bacterial Repair
Performance . . . . . . . . . . . . .
Bacterial Mutatit~n
Yeast Assays
In Vitro Manmmlian
~.58) 0.51
~.63) 0.67
In Vivo Assays
Sensitivity
~.65 ) 0.57 +
~.80 0.64
Specificity
0.74 ) 0.58
~.91) 0.67
~.70) 0.62
~.82) 0.61
@.90) 0.80
Accuracy
~.68) 0.59
~.73) 0.61
~.61) 0.56
~.68) 0.65
~.48) 0.52
)
(0.37) 0,36
÷
Values including problem carcinogens. (),Values not including problem carcinogens. (Data obtained from De Serres and Ashby, 1981)
mutation> bacterial repalr>yeast>-in vivo.
Excluding the problem carcino-
gens the first two would be reversed. In this study the number of results obtained with bacterial and yeast assays largely outnumbered those obtained with more complicated tests.
To
evaluate the perform;race of ba'~teries of tests, we selected from the available data only the first eight results on Salmonella . . . . . . . . ty p himurium, eight on yeasts, cell
eight
on in, vitr_~o marmtm] i a ,
transformation),
in P r q s o p h ! l a , ities
obtained,
eight
T h i s same s e t
putting
together
were tab,lated,
a t random f o u r d i f f e r e n t
lethal
sperm a b n o r m a l -
for each chemical,
B a t t e r i e s of t e s t s results
each group of mutagenesls assays as shown inTable 4.
and
recessive
in mice,
o f a s s a y s was c o n s i d e c e d
and negative v a l u e s
gene mutation
in v i v o ( s e x - l i n k e d
SCE in mice bo,~e marrow, m i c r o n u c l e u s
in mice).
and p o s i t i v e
and f i n a l l y
ass~ays (DNA r e p a i r ,
obtained
were from
If fewer than three
tests were available for each set, we considered them ineligible as a battery.
In the case of methionlne (Table 4) we considered for example 6
batteries.out of 8 possible.
Pharmaco/ogicalResearch Communications, Vol. 16, No. 5, 1984
429
Table 4.
Results of a battery of tests on methionine
Battery
Re s ul t s of i nd ~v idua Salmonella Yeast In vitro ,mamIn vivo typhimurium assay m a l i a n assay assays
No. i
-
-
+
n.a.
3
-
+
-
-
4
-
+I-
n.a.
-
-
-
-
n . a .
n . a .
-
-
-
n . a .
n . a .
5 6
+
+ +
n.a. -
-
1
tests
Battery evaluation Case i+
Case 2+
+ m
+ +
+
n.a. = not available Note that the final battery evaluation for methionine as shown in Table 4 differs between Case l+and Case 2+ the table).
(see the sixth and seventh columns of
Case l+evaluation is based upon the presence of at least one
positive result in the battery.
Case 2%,on the other hand, is based upon
the presence of at least 2 positives in the battery.
It is obvious that
Case l+evaluation is .the more conservative interpretation of the results of the battery, according to which even methionine genic.
would be considered muta-
The chances of misclassifying methionine are reduced by using Case 2+
evaluation of the batteries. We have analyzed the data of the interlaboratory study according to these criteria and the results are shown in Table 5.
It
is evident that carcino-
genic chemicals are well classified (81.2% of correct results) using Case i+ criterion.
Using the same criterion, however, only 34% of non-carcinogens
are properly classified.
Using Case 2+criterion the non-carcinogens are
better classified but alot of carcinogens are passed over (41.6%). It seems to us that whichever criteria are used to analyze the battery test results, a consistent number of misclassifications are going to occur. Taking one positive result as a measure of mutagenicity will produce a number of false positives among the non-carcinogenic chemicals.
For example, as
shown in Table 5, ascorbic acid and l-naphthylamine--which are not carcinogenic--are both classified as mutagens using Case l+criterion (0% correct
Pharmacological Research Communications, Vol. 16, No. 5, 1984
430
Table
3 No. positire batterles
Total no. batteries
Case
Percent of correct results Case
i+
2+
I+
2+
7 3 2 8 8 3 4 8 7
2 O O 8 6 5 4 8 6
8 7 8 8 8 8 5 8 8
87 42 25 100 i00 I00 80
2~ O O iOO 75 62 80 IOO 75
8
5
8
3
8
1 7 2 2
8 8 7 6
I00 62 37 I00 86 83 IOO
5 3 8 6 5
IOO
iOO 87 tOO
40 50 lOO
40 iOO 1OO 80
40 I00 100
Ca r c i a o g e n i c Ch en*i c91 4-dimethylamlnoazobenzene
Chloroform Diethylstilbestrol 4-nitroqulnoline-N-ox[de 2-naphthylamine Benzidine 2"acetylaminofluorene Benzo(a)pyrene Hydrazine sulphate Hexamethylphosphoramide Ethylenethlourea Safrole Epichlorhydrin DL-ethionine N-nltrosomorpholine ~-propr~olactone Urethane DimethyIcarbamoyl chloride Cyclophosphamide 3-aminotriazole 4,4Lmethylenebis(2-chloroani Me t hy [azoxymethano lacetate 9, IO-dimethylan~hracene o-To]uidine Auramine
line)
5
5
5
5 7
2 4
5 8
3 2
3 2
5
5
3 4
3 3
3 3
3 l
IOO
87
3
5 5 3
5 4 5 average
=
62 37 12 87 28 33
75
60 75
60
20
81,2 58 .4
N o n - c a r c i n o g g n ~ c Ch(~mi c a I_s_s.................. 3-me t h y l - 4 - n i t roqu i no I i n e - N - o x i d e
Dini trosopentamethyl erie tet rami ne y-butyrolactone 3,3',5,5L~etramethyIbeuzidine Pyrene Anthracene Azoxyhenzene 4-acetylmninofluorene 1-naphthylamine Diphenylnitrosamlne Dimethylformamide l,l,l-trichloroethane 4-d ime thy lain inee zobenz e n e-4-su Iphon i~ic id Ascorbic acid Isopropyl N-(3-ch]oropheny])carbamate Hethionine Sucrose
8 4
8 O
8 6
0 33
O IOO
3 I
6 8
50 87
].00 IOO
6
O O O
8
25
1OO
2 4 4 8 3
O 3 3 6 2
5 5 4 8 6
60 20 O O 50
leo 4O 25 25 66
3 I .4
1 l 3
8 4 8
62
87
3 4 4
2 3 l
3 4 6
2
3
3
average =
75
75
50
62
O O 33
33 25 83
33
1OO
34
65.9
Pharmacological Research Communicatlbns, VoL 16, No. 5, 1984 results).
Taking
two positive
431
results as an indication of mutagenigity,
on the other hand, reduces the number of errorsfor non-carcinogenic chemicals but reduces considerably the predictivity of the test for carcinogenic chemi-cats. The criteria for analyzing the results of a battery of tests have never been thoroughly discussed in the literature.
If the goal is to reduce
the number of potentially mutagenlc and carcinogenic chemicals present in the environment, then we would propose the use of Case l~criterion in assessing all c~emicals.
However, one'needs to be aware of the extreme imprecislon
of this approach.
MUTAGENESIS TESTING AND DRUGS:
TWO TYPICAL CASES
Metronidazole is carcinogenic in mice after oral administration (Rustia and Shublck, 1972), and induces mammary fibroadenomas in rats (Cohen et al., 1973).
It was shown to be mutagenic in Salmonella (McCann et al., 19757, to
produce mutagenic urine in humans (Legator et al., 1975) and to induce chromosomal aberrations in long-term treatments in humans (Mitelman et el., 19767.
llowever, it is very weakly mutagenic on nitroreductase-deficient
strain TAIOOFr I of Salmonella, not mutagenic in V79 mammalian celts (Dayan et al., 1982); it is inactive on DNA repair of primary rat hepatocytes, and does not induce geuotoxic
efgects
on human l y m p h o c y t e s in v i t r o
(Lambert,
19797. Similarly, isoniazide, which is carcinogenic in mice (Morl et el., 1960) is a weak mutagen on Sa]monella (Wade eta].,
]98]), is mutagenic in yeasts
(Zetterberg and Bostr3m, ]9817 and in host mediated assays (Jansen et al., 1980), inactive in DNA repair tests (Wade, ]98]), does not damage human lymphocyte chromosomes in vivo (Jansen, 1980) and is inactive in dominant lethal tests in mice (Rohrborn et at., 19727. Human carcinogenicity data are not yet adequate for both drugs (Goldman~ et al., 19777.
If the toxicity of these two drugs had to be evaluated on
the basis of the results of a battery of tests, both drugs would be classified as mutagens and likely carclnogens.
However, there is closer agreement
Pharmacologica/ Research Communications, Vo/. 16, No. 5, 1984
432
between animal carcinogeniclty data and bacterial mutagenesis results, whereas more complicated mammalian systems, in principle closer to the animal model, perform very poorly.
It is, therefore, very difficult to reach
any meaningful conclusion about the risks connected with the use of these Zwo drugs, if one had to consider mutagenicJty data alone. REGULATORY APPROACHES TO MUTAGENiCITY TESTING IN THE PHARMACEUTICAL FIELD At the beginning of the 197Os an important OMS report (~10) appeared proposing the inclusion of mutageniclty testing in the routine evaluation of drug toxicity (OMS, 1971).
The report had certainly a broad influence
and in that decade more than 50 countries prescribed the use of mutagenicity data in support of new drugs' applications.
These regulations in different
countries are analyzed in detail in ~ recent publication of the International Federation of Pharmaceutical Manufacturing Associations (IFPM, 1980). The Organization for Economic Cooperation (OECD), which is comprised of 24 industrialized countries, has proposed a protocol recommending a minimum pre-marketing data-set on drugs, including gene mutation on prokaryotes and chromosome aberrations in vitro and in vivo (for a discussion, see Lo Prieno, 1981).
Similar recommendations are contained in the sixth amendment of a
directive of the Council of European Co~nunities (EEC, 1979), stating that new chemicals had to be tested for mutageniclty by the manufacturer prior to markethlg. For medicinal compounds in particular, a technical commission of the European communities (EEC, 1980) submitted a proposal to make mutagenicity studie~ obligatory for all new drugs. A con~ittee on Proprietary Medicinal Products (CMPC) has issued guidelines (discussed by Zb~nden, 1980) on dew drugs, based upon the following: a)
bacterial
b)
in v i t r o
c e l l ckwomosome damage a n a l y s i s
c)
in v i t r o
gene m u t a t i o n in ma~naltan c e ~ I s o r i n v i v a z e s t s w i t h
r
,
t e s t s with e s t a b l i s h e d
strains
Drosophila melanogaster d)
in viva chromosome damage analysis i, rodents.
PharmacologicalResearchCommun~a~on~Vo~ 1~ No. ~ 1984
433
Although t h e r e i s a c o n c o r d a n c e on t h e g e n e r a l p r i n c i p l e s s i t y of m u t a g e n i c i t y ' d a t a ,
there is considerable
variation
and t h e n e c e s among d i f f e r e n t
countries on the requirements for the assessment of drug safety. in the EEC, a 1977 I t a l i a n
law r e q u i r e s data on p r o k a r y o t e s ,
For example,
eukaryotes
and DNA repair (DM, 1977), whereas the Federal Republic of Germany requires tests to detect both gene and chromosomal mutations (ICPEMC, 19B3b) like dominant lethal test and bone marrow test. A h o m o g e n i z a t i o n o f the jz~terna~Jo~al reg~]atlons wou)d be welcome in
this field and has been advocated by the pharmaceutical industry (see, for
example, Mazu~ and Berthe,]983).
Due to national variations and d i f f e r e n c e s
of opinions in the scientific co1~lunity, con~on international guidelines on drug m u t a g e n i c i t y have not been i n t r o d u c e d .
CONCLUSIONS
From the previous discussion it is evident that, not w~thstanding the considerable amount of work dedicated to the development of genetic toxicology tests, numerous problems remain unsolved.
Mutagenesis tests, in fact,
are plagued by imprecision and by the intrinsic incapability of detecting some classes of carcinogens.
On the other hand, given the difficulty of
extrapolating from animal carcinogenicity studies to human carcinogenicity, it would be unwise to expect a nonproblematic link between mutagenicity and carcinogenicity. When testing new drugs or chemical compounds, if the goal is to find substances that have a minimum of risk of inducing mutations and cancerp then a prescreening with several mutagenesis techniques seems to be justified, and this has been the current opinion of the scientific community.
It
should be clear, however, that the introduction of mutagenicity studies might keep a certain number of potentially useful compounds out of the market,
it is also true that the presence of mutagenic activity ~er se
should discourage the manufacturing of certain chemicals because of possible negative effects on the genetic structure of living organisms, independent from their possible carcinogenic activity.
Given the choicebetween chemi-
cals and drugs that have the same use but are differentiated by their
Pharmacological Research Communications, Vol. 16, No. 5, 1984
434
genetic effects, it is wise to develop non-mutagenic substances. well known that all methodologies have limitations.
It is also
Mutagenesi8 testing
seems to have more imperfections than most of the current methods in use in toxicology.
We believe, however, that the intelligent application of
mutagenielty tests can give useful data concerning the reactivity, metabolism and toxicity of a compound.
It may also be useful for tracing chemicals
and their metabolites in body fluids or in the environment.
It would also be
useful to define more precisely the guidelines for interpreting mutagenicity tests in regulatory approaches.
In this regard it seems appropriate to dis-
cuss again if the expectation of improving the reliability of mutagenicity experiments with complex systems and organisms has been fulfilled.
If no one
questions the usefulness of new, refined methods in genetic research, when dealing with industrial application more consideration should be given to methods =hat produce acceptable results in a shorter time and at a lower cost.
ACKNOWLEDGEMENTS:
We thank Mary Forrest for revision of the manuscript.
REFERENCES Ames B.N., McCann J. aud Yan~asaki E. (1975) Mutat. Res. 31, 347-364. Bateman A.J. (1977) In: Kielbey B.J. Legator H. Nichols W. and Ramel (3. eds. Handbook of M u t a g e n i c i t y T e s t P r o c e d u r e s , Amsterdam E l s e v i e r / N o r t h Holland Biomedical P r e s s , 325-334. Cohen S.M., E r t w e r k E . , Esch A.M., C r o v e t t i A..I. and Bryan G.T. (1973) d. N a t l . Cancer I n s t . 51, 4 0 3 - 4 1 7 . Dayan J . , C r a j e r M.C. and Deguingand S. (1982) Murat. Res. 102, 1-12. D e c r e t o M i n l s t e r i a l e , 25 Agosto ]977, G.U. 238. De S e r r e s F . J . and Ashby J . (1981) In: E v a l u a t i o n of Short-Term T e s t s f o r C a r c i n o g e n i c i t y - - P r o g r e s s in M u t a t i o n R e s e a r c h , Vol. ]; E l s e v i e r / N o r t h HollarJd, New York. DiPaolo J . A . , Donovan P . , Nelson R.I.. (1969) J . N a t l . Cancer It2st. 42, 867-876. DiPaolt) J.A., Takano, K. and Popescu N.C. (|972) Cancer Res. 32, 26862695. EEC Council Directive 79/83]/EEC Sept. 1979. EEC P r o p o s a l f o r a Council D i r e c t i v e Amending D i r e c t i v e 65/65 EEC, 75/318 EEC and 75/319 EEC. O . J . no. C 355, 3 1 . 1 2 . ] 9 8 0 , p. 1.
Pharmacological Research Communications, VoL 16, No. 5, 1984 E h l i n g U.H. (19781 In:, l l o l l a e n d e r A., De S e r r e s F . J . ( e d s ) , Chemical H u t a gens, Vo]. 5, Plenum P r e s s , New York, London, p. 233. F a h r i g R. (1977) Handbook ~ l u t a g e n i c i t y T e s t P r o c e e d i n g s , p. 135-147. Goldman P . , I n g e l f i n g e r J . A . and Priedman P.A. (19771 I n : O r i g i n of tluman C a n c e r , H i a t t H . H . , Watson J . D . and Winsten J . A . ( e d s ) Cold S p r i n g Harhor L a b o r a t o r y , p. 465-474. Orafe A., M a t t e r n I . E . and Green M. (19811 Murat. Res. 85, 391-410. lteddle J . A . and Bruce W.R. (19771 In: O r i g i n s of ltuman C a n c e r , H i a t t i t . H . , Watson J . D . a n d Winsten J . A . ( e d s ) Cold S p r i n g liorbor L a b o r a t o r y , p. 1549. Herman R.K. and Dworkin N.B. (19711 J . B a c t e r i o l . 106, 5 4 3 - 5 5 0 . t t o w a r d - F l a n d e r s P. (19811 Murat. r e s . 86, 307-327. IARC Monographs on t h e E v a l u a t i o n of the C a r c i n o g e n i c R i s k of Chemicals to ttumans, Suppl. 2 ( 1 9 8 0 ) , IARC, I.yon. ICPEMC R e p o r t (19821 b t u t a t , r e s . 99, 73-91. ICPEMC R e p o r t (1983a) Murat. Res. 114, 117-177. llCPEMC Report (1983b) Murat. Res. 114, 179-216. IFPM (1980) Legal and P r a c t i c a l R e q u i r e m e n t s f o r t h e R e g i s t r a t i o n of Drugs (Medic£nal P r o d u c t s ) f o r lfuman Huse, Z u r i c h . J a n s e n J . D . , C]emmesen J . and Sundaram K. (1980) Murat. Res. 76, 8 5 - 1 1 2 . Kakunaga T. (19731 . I n t . Jo Cancer 42, 463-473. Kate H. (19741 N a t u r e 25, 7 0 - 7 2 . Kohn K.W., Friedman C.A., Ewig R.A.G. and I q b a l Z.M. (1974) B i o c h e m i s t r y 13, 4134-4139. Lambert B., L i n d b l a d A. and Ringb~rg U. (19791 ~lutat. Res. 67, 2 8 1 - 2 8 7 . Larsen K.H., Brash D., C l e a v e r J . E . , Ilart R.W., Maher V.bl., P a i n t e r R . B . , Sega G.A. (19821 Murat. Res. 98, 287-218. L a t t S.A. (19731 Prot'. N a t l . Ac.ad. S c i . USA 70, 3395-3399. L e g a t o r M.S., Conner T.H. ,nd S t o e c k e l H . . ( 1 9 7 5 1 S c i e n c e 188, 1118-1119. L e g a t o r M.S., Buedlng E . , Batz~nger R.-, Conner T.II., E i s e n s t a d t E . , Farrow M.G., F i c s o r G., Ilsie A., Seed J . and S t a f f o r d R.S. (19821 Murat. Res. 98, 319-374. L o p r i e n o N. (19811 I~iol. [ t | t . 3, 2-14. M u t t e r B.E. and Tstlchimoto T. (1980) Arch. 'l't~xi~'ot. 46, 8 9 - 9 8 . Mazu~ G. and B e r t h e J . (19831 In: Quo V a d i s , Rancucci R. and W i l l i a m s G.M. ( e d s ) , E l s e v i e r , Amsterdam, p. 8 4 - 9 1 . McCann J . , Choi E . , Yamasaki E . , and Amt:s B.N. (19751 P r e c . N a t l . Acad. S c i . USA 72, 5135-5139. Mitelman F . , l t a r t l e y - A s h B. at~d U r s i n g B. (19761 Lancet i~, 802. Mitelman F. and l,evan G. (19781 l l e r i d i t a s 89, 2 0 7 - 2 3 2 . Mohn G . R . ( 1 9 8 l ) Murat. Res. 87, 191-210. Mori K., Yasuno A. and blatsumoto K. (1960) Gann 51, 8 3 - 8 9 . OblS (1971) R a p p o r t s q'echniqtles 482. P a r o d i S . , T a n n i n g h e r bl., Boero P. a~d S a n t i L. ( ] 9 8 2 ) Murat. Res. 93, 1-24. P u r c h a s e I . F . t l . (19821 Murat. Res. 99, 5 3 - 7 1 . r ~ h r b o r n O., Proppittg P. and lhJselmaier t4.(1972) bltJtat. R e s . 16, 189-194. Russel L.B. and M a t t e r B.E. (1980) M,ltat. Res. 75, 279-302. R ~ s t i a M. and Shubik P. (19721 J . N a t l . Cancer I n s t . 48, 7 2 1 - 7 2 9 .
435
436
Pharmacological Research Communications, Vo/. 16, No. 5, 1984
Sehmidt W. (1976) In: Chemical Mutagens" Principles and Methods for Their Detection, Hollaender A. (ed.) Vol. 4, New York, Pl~num Press, p. 31-53. Simmon V.F., Rosenkranz li.S., Zeiger E. and Poirier L.A. (1979) J. Natl. Cancer Inst. 62, 911-918. S~rianni S.R., Furukawa M. and Huang C.C. (1979)Mut~t. Res. 64, 259-267. Swenherg J.A. and Petzold G.L. and [larbach P.R. (1976) Biochem. Biophys. Res. Comm. 72, 732-738. Topham J.C. (198Oa) Murat. Res. 69, 149-155. Topham J.C. (!98Ob)Murat. Res. 70, IO9-114. Tt'zos R.J., Petzold G.I,.~ Brunden M.N. and Swenberg J.A. (1978) Murat. Kes. 58, 79-86. Vennit S. and Levy L.S. (1974) Nature 250, 493-495. Wade D.R., Lohman P.H.M., Mattern I.E. and Berends F. (1981) Murat. Res. 89, 9-20. Williams G.M., Laspia M.F. and Dunkel U.C. (1982) Murat. Res. 97, 359-370. W{]rgler F.E., Sohels F.ll. and Vogel E. (1977) In: Handbook of Mutagen~clcy Test Procedures, Ki|hey (ed.), Elsevier/North Holland, Amsterdam, p. 335373. Zblnden C. (1980) Arch. Toxicol. 46, I-7. Zetterberg G. and Bostr~m G. (198]) Mr,tat. Res., 91, 215-2|9. Zinmlerman F.K., Kern R. and Rasenberger H. (1975) Murat. Res. 28, 381-388.
421
TilE APPLICATION OF MUTAGENICITY TESTS TO THE PREDICTION OF CARCINOGENIC ACTIVITY OF CHEMICALS AND DRUGS P. Dolara and G. Caderni Institute of Pharmacology and Toxicology, University of Florence, Italy
Receivedm final ~rm 30c~ber 1983 MUTACENESIS AS A WAY OF PREDICTING CARCINOGENESIS Numerous tests have been developed that can measure the mutagenic activity of chemicals and drugs.
Since many carcinogens are also mutagens an
extensive effort has been carried out to develop new mutagenesis tests that can be reliable predictors of carcinogenicity. After a decade of effort, however, the main question:
is there a causal
relationship between mutagenicity and carcinogenicity? remains unanswered (ICPEMC, ]982).
It has been empirically demonstrated that a high percent-
age of substaltces that give a positive mutagenic response are also animal carcinogens, but a causal link between the two phenomena has not yet been established. However, there is general agreement about the existence of two groups of chemical carcinogens:
a) genotoxic carcinogens, that produce a change
in the genetic information of cells, and b) carcinogenic enhancer and tumor promoters that act somewhere else in the cascade of events that separate the initiation of a tumor from its clinical onset.
Since mutagenesis is
aimed at detecting the finst type of activity, it is inherently incapable of detecting tumor promoters or enhancers.
Notwithstanding this limitation,
because of their rapidity and low cost, mutagenesJs tests have attained a large popularity for studying the toxicity of chemicals and drugs. CURRENT METHODOLOGIES FOR MIITAGENICITY TESTING More than iO0 different mutagenicity tests are currently in use, but we will outline only the prir=eipal ones in the table which follows.
0031 --6989/84/050421-16/$03.00/0
© 1984 The Italian Pharmacological Society
Pharmacological Resea,,ch Communications, Vol. 16, No. 5, 1984
422
Tab_le ! .
Outl/ne
of l'rimary M u r a l s
SYSTEM
Salmonella typhimurium Escherichia coli
.
.
.
gene mutation.
Forward and Rever~e H . t a t i o n Assays
DNA damage
gene mutation
Mammalian Cells In Vitro Chinese hamster ovary liCPR'r, TK and Na/K cells (CllO) ATPase Assays V79 Chinese hamster lung cells LSI78Y mouse lymphoma cells A l k a ] i n e E l u t i c m of Various human and rodent cell I ines DNA DNA Re pai r S i s t e r Chromat id Ext:hange (SCE) Mouse c e l t l i n e BAI,BI 3T3 T r a n s f o r m a t ion Assay and C311/10 'l'J
.
gene mutation
.
Schizosaccharomyces pombe Saccharomyces cerevisiae 3.
Ames T e s t WP2 Assay Pol A Test
Yeasts
2. i
TYPE OF TEST
Bacterial Cells
I.
_
COMMON NAME
gene mutation
DNA damage DNA damage DNA damage
neoplastlc transformation
Manaaals In Vivo Mice
S p e c i f i c Locus T e s t Dominant Lethal Test Micrommleus In Vivo C y t o g e n e t i c s Spermutest
5.
Host Mediated Assay
6.
Insects Drosophila melanogaster
gune m~tation chromo soma I damage chromosomal damage chromosomal damage morphoiogie damage
(See numbers 1 and 2 and gene m u t a t i o n in 3) Sex-I inked Recessive Lethal Assay
gene mutation
Pharmacological Research Communications, Vol. 16, No. 5, I984
423
BRIEF OUTLINE OF THE INDIVIDUAL METHODS Since the available mutagenicity tests are at very different stages of validation, some being fully developed, others in experimental phase~ their performance has been currently described with the following terminology: sensitivit[: gens tested;
number of mutagenic
carcinogens~number of total carcino-
accurac%: number of correct results (positive and negative)/number of total chemicals tested; ~ : number of non-mutagenic non-carcinogens/total number of non-carcinogens tested; r@liability or predictive yalue: of positive results obtained.
number of mutagenic carcinogens/number
Table 2 shows the performance of the most commonly used mutagenicity tests. For some of the less frequently used, not enough data are available. +
Table 2.
Performance of some mutagenicity tests
Test systems
Sensitivity
Specificity
Predictive Value
Bacterial cells Salmonella typhimurium E. coli WP2
0.74 0.67
0.66 0.73
0.85 0.87
Yeast cells Saccharomyces cerevisiae (mitotic recombination)
0.55
0.69
0.86
Mammalian cells in vitro Gene mutation In vitro transformation (BHK21CLI3)
0.94 O.83
O. 75 O. 63
O. 92 O. 78
Man~nals in vivo Micronucleus assay (mice) Spermatest (mice)
0.55 0.42
0.69 0.81
0.85 0.81
Insects Drosophyia SLRC
0.57
0.89
0.86
++
+
mean values obtained from Purchase (1982) ++IARC (1980), pp. 114-123.
++
++
424
Pharmacological Research Communications, VoL 16, No. 5, 1984
A very brlef description of the most important tests in use follows in Sections 1-6. ].
Methods using bacteria~ cells:
The most popular test was developed by Ames and coworkers (1975), using 5 auxotr6phic stralns of Salmonella typhhnuri,!n b The cells are incubated with the putative mutagens, with and without metabolic activation (an induce4 rat liver $9 preparation), and after 2 days the colonies reverting to prototrophy are scored. A similar protocol was developed by others with E. coli (Herman and Dworkin, 1971). The reliability of the Ames test has been recently reviewed by several authors (Purchase, 1982; Mohn, 1981; Grafe et al. 1981; IARC, 1980, pp. 85-106). The method has been used with over 3,000 chemicals in a large number of laboratories. It has a good sensitivity and predictive value, although it does not perform well with some chemicals such as hydrazines, nitrosamines and metal carcinogens. The quantitative variability among different laboratories, however, is noteworthy (Grafe et al., 1981). Tests using E. coli are widely in use, have a similar performance and are capable of detecting some metal carcinogens (Vennit and Levy, L
1974). 2. .
Methods using yeast cells: .
2
-
•
- -
,
.
.
.
.
.
Mutation assays are based upon a discolor~ition of yeast colonies that are defective in a gene regulating adenine biosynthesis and therefore accumulate a red pigment. Gene mutations may block previous steps of adenine biosynthesis, and the resulting colonies wi]l lose their pigments. Reverse mutation assays measure the growth of prototrophs on deficient media. Mitotic recombination and gene conversion can be measured with methods using yeast cells (Zimmerman et al.,. 1975). The performance of yeast assays is similar to bacterial assays, although the sensitivity is lower and the validation has been obtained on a more restricted experimental sampling (IARC, 1980, pp. 135-153). 3. Methods using man~alian ce]Is in vitro: A. Gene mutationVarious strains of cells can be used that become resistant to a certain chemical (like 8-azaguanine, 6-thioguanfne, 5-Br-deoxy"-'~',,,,~,na,~.._~.=~,~ .... uhen a specific mutation in their genome occurs (.Ne,.-:-"rd~ Flanders, 1981). They have a high predictive value and sensitivity but ale longer and more expensive than bacterial and yeast tests (IARC, 1980, pp. iO7-133). B. Alkaline elution of DNA is a relatively s~mple technique in which DNA damage is detected using tile selective permeability of millJpore membranes for small molecular weight DNA, whlch ~s produced by the action of nucleases on damaged DNA or as a result of the susceptibillty of damaged DNA to alkaline cleavage. The method has not been extensively validated but seems promising on tlle limited number of compounds so far tested (Kohn et al., 1974; Swenberg et al., 1976; I'arodf et of., ]982).
Pharmacological Research Communications, Vol. 16, No. 5, 1984
425
C. DNA repair measures DNA synthesis induced by genetic damage followed by DNA repair. It is determined by measuring the incorporation of labeled thymidine into DNA after the exposure to a chemical mutagen, blocking DNA replication with hydroxyurea. The test has a good predictive value but a low sensitivity (Williams et al., 1982) and is not adviced as a primary screening test (Larsen et al., 1982). D. S i s t e r c h r o m a t i d e x c h a n g e (SCE) m e a s u r e s t h e number o f e x c h a n g e s o f s u b - s e c t i o n s o f c h r o m a t i d s a f t e r t h e e x p o s u r e to a c h e m i c a l m u t a g e n . The cells are grown for two generations in the presence of 5-Br-deoxyuridine to label individual chromatlds and later stained with a fluorescent dye to evidentiate homologous segments that have been exchanged between sister chromatlds (Latt, 1973,; Kato 1974). The molecular mechanism by which SCEs occur is not known but their frequency is increased by the exposure to mutagens. SCE assays have been used with hundreds of chemicals but they have not been validated as extensively as bacterial mutagenesis methods (IARC, 1980, pp. 227-247). E. T~ansformation assays dlffer from the prevlous]~ discussed tests because they measure neoplastic transformation in Xit[o that is very close to the process of carcinogenesis in viva. In these methods cellular lines are treated with a putative carcinogen and after several passages in vitro the number of transformed loci are scored (DiPaolo et al;, 1969; DiPaolo et al., 1972; Kakunaga, 1973). These tests have a good sensitivity and predictive value but are technically difficult, slow and costly. 4. , Methods us in..g mammals in v i v o : A. Mouse specific locus test is an assay to detect heritable pointmutations in mammals. Treated wild-type animals are mated with homozygote strains for some recessive allels. If a mutation in any of the loci represented in the recessive strain occurs in tile wild-type mice, this will result in mutant offspring (Ehling, 1978). Even though the method requires large animal facilities and a long time to be performed it is the only established assay for detecting gene mutations in whole man~nals (Russel and Matter, 198£); ICPEMC, 1983:1). g. Dominant lethal test in mice detects any genetic damage in the treated animal that causes the c]e:~th of the offspring (Bateman, [977). The test is relatively quick and simple to perform, however, it is not very sensitive (1ARC, 1980, pp. 248-251). C. Micronucleus assay detects cht'omosomal damage caused by dastogens or spindle poisons. Acentric chromosomes are excluded from anaphase groups and in thedaughter nuclei form separated "micronuclei" that are particularly easy to evidentiate (Schmidt, 1976; ICPEMCa,1983). This test ks not very sensitive but very often included in test batteries (Trzos, 1978; Purchase, 1982).because of its good predictive value. D. In viva cytogenetics: many transformed cells have chromosomal aberrations, so that the detection of this damage can be used as # predictive carcinogenicity test (Mitelman and Levan, 1978). chromosomal
4 26
Pharmacological Research Comm~micatJons, Vol. !6, No, 5, !984
aberrations are scored in bone marrow cells. The method has a low sensitivir.y (Matter, 1980). E. Spermatest detects the inct'e~ist.,of mc,rl)hological spermal abnorma/ities after treatment with a putative carcinogen. Many substances have been tested with this tQchnique (Heddle and Bruce, 1977; Topham, 1980a; Topham, 19805), The spermatest has shown a relatively good specificity and predictive value together with a low sensitivity. 5.
Host-mediated assay:
In host-mediated assays, cells like bacteria,yeasts or mammal [an ceils are injected into the animal (it~ the venous system or in the peritoneum). After an adequate incubation time tile cells are recovered from the liver or the peritoneum and tested for mutagenicity as described i'n sections ], 2 and 3 above (gent mutations in marmna]ian cells). The test can be useful for studying the pharmacodynamics t~f carcinogens in different organs (Fahrig, 1977; Simmon et el., 1979; Sirianni et el., ]979; Legator et el., 1982). 6.
Insects:
The most conmlonly used m u t a g e n i c i t y t e s t w i t h D r o s o p h i l a me]anogaster i s t h e s e x - l i n k ~ d r e c e s s i v e l e t h a l ;3ssay. For t h i s a s s a y f e m a l e s t h a t h a v e a p a r t i c i J l a r X chromosome (Base X chromosome) a r e mated w i t h t r e a t e d w i l d type males. I f a l e t h a l X m u t a t i o n o c c u r s Jn m a l e s , in t h e F_ g e n e r a t i o n this will result in the absence of an entire ~:lass of males ~W~rg]er, 1977).
This a@say is well validated, has good specificity and predictive value, but very few laboratories know how ~o do it (IARC, 1980, pp. 157-184; Purchase, 1982; ICPEMC, 1983a). A FEW COMMENTS ABOUT THE PERFORMANCE OF DIFFERENT TEST SYSTEMS In the previous section and in Table 2 we have briefly reviewed the performance of individual tests.
It'appears clearly that the predictive values of
most currently used systems are very close to 0.8-0.9, whereas sensitivity and
specificity are more variable.
However, in evaluating these results, it should
be remembered that different predictivities are obtained when changing the prevalence of carcinogens in a group of chemicals to be tested (ICPEMC 1982, 1983b). It has been shown (Purchase, 1982) that using an assay with a sensitivity and specificity of 0.9 when testing 500 carcinogens and 500 non-carcinogens, 50 false positive (non-carcinogenic mutagens) would be found, giving the expected reliability of .9.However~ by testing a group of chemicals with 5% carcinogens (950 non-carcinogens and 50 carcinogens) 45 carcinogens (90%) would be positive in the test but 95 non-carcinogens (10%) would turn out
Pharmacological Research Communications, Vol. 16, No. 5, 1984 positive as well.
427
The total count of positives would be, therefore, 140,
and the reliability (mutagenic carcinogens/total mutagenic compounds) would therefore be only .32 (~5/140).
In the real world the r~tio of carcinogens/
non-carcinogens is presumably very low, and the reliability of individual tests in predicting carcinogenicity of randomly selected chemicals would be, therefore, extremely poor. To improve the relatively poor performance of single mutagenicity tests, the idea of batteries of tests has been developed. assumption is that by using systems differing
The underlying theoretical
in genetic end points and in
levels of cellular complexity, the limitations of each individual test would be overcome. This premise is generally accepted by the scientific community and is the basis for the existing regulations in this field. however, devoid of problems.
It is not,
In the following section we will discuss this
issue. CONBINATION OF INDIVIDUAL TESTS IN A BATTERY:
In a large international,
AN EXPERIMENTAL EVALUATION
interlaboratory study (De Serres and Ashby,
1981) a group of 62 chemicals, of which 25 were carcinogens, were tested with various
methodologies
ill d i f f e r ~ ' n t
tl,e c o m p l e t i o n o f t h e s t u d y . were c h o s e n b e c a u s e
E l e v e n of t h e c a r c i n o g e n s
they normally are i.lac:~e
~leth~dolc~g[es u s e d ,~ere b a c t e r i a l re.ration
assays,
]aboratc~L'ies and d e c o d e d o n l y a f t e r
yeast
assays,
repaic
(problem carcinogens)
in b a c t e r i a l
assays.
and ~hage i n d ~ l c t i o a ,
in v i t r c J mm,malJan a s s a y s ,
The
baecerlal
and in v i v o a s s a y s .
The results of the different tests are shown in Table3, where the numDe~ not in parentheses represents the values obtained considering the results of the problem carcinogens that and amounts n o t i n p a r e n t h e s e s
lower the overall performance of the tests, include
the problem carcinogens.
The performance of different systems in Table 3 is considerably worse than that reported in Table 2, probably because of the abundance of problem carcinogens tested and because the researchers did not know the structure of the substances being tested.
Considering all chemicals tested, the
accuracy can be ranked in the following way:
in vitro mammalian>bacterial
Pharmaco/ogica/ Research Communications, Vo/. 16, No. 5, 1984
428 Table3.
MutagenicityTest?
Bacterial Repair
Performance . . . . . . . . . . . . .
Bacterial Mutatit~n
Yeast Assays
In Vitro Manmmlian
~.58) 0.51
~.63) 0.67
In Vivo Assays
Sensitivity
~.65 ) 0.57 +
~.80 0.64
Specificity
0.74 ) 0.58
~.91) 0.67
~.70) 0.62
~.82) 0.61
@.90) 0.80
Accuracy
~.68) 0.59
~.73) 0.61
~.61) 0.56
~.68) 0.65
~.48) 0.52
)
(0.37) 0,36
÷
Values including problem carcinogens. (),Values not including problem carcinogens. (Data obtained from De Serres and Ashby, 1981)
mutation> bacterial repalr>yeast>-in vivo.
Excluding the problem carcino-
gens the first two would be reversed. In this study the number of results obtained with bacterial and yeast assays largely outnumbered those obtained with more complicated tests.
To
evaluate the perform;race of ba'~teries of tests, we selected from the available data only the first eight results on Salmonella . . . . . . . . ty p himurium, eight on yeasts, cell
eight
on in, vitr_~o marmtm] i a ,
transformation),
in P r q s o p h ! l a , ities
obtained,
eight
T h i s same s e t
putting
together
were tab,lated,
a t random f o u r d i f f e r e n t
lethal
sperm a b n o r m a l -
for each chemical,
B a t t e r i e s of t e s t s results
each group of mutagenesls assays as shown inTable 4.
and
recessive
in mice,
o f a s s a y s was c o n s i d e c e d
and negative v a l u e s
gene mutation
in v i v o ( s e x - l i n k e d
SCE in mice bo,~e marrow, m i c r o n u c l e u s
in mice).
and p o s i t i v e
and f i n a l l y
ass~ays (DNA r e p a i r ,
obtained
were from
If fewer than three
tests were available for each set, we considered them ineligible as a battery.
In the case of methionlne (Table 4) we considered for example 6
batteries.out of 8 possible.
Pharmaco/ogicalResearch Communications, Vol. 16, No. 5, 1984
429
Table 4.
Results of a battery of tests on methionine
Battery
Re s ul t s of i nd ~v idua Salmonella Yeast In vitro ,mamIn vivo typhimurium assay m a l i a n assay assays
No. i
-
-
+
n.a.
3
-
+
-
-
4
-
+I-
n.a.
-
-
-
-
n . a .
n . a .
-
-
-
n . a .
n . a .
5 6
+
+ +
n.a. -
-
1
tests
Battery evaluation Case i+
Case 2+
+ m
+ +
+
n.a. = not available Note that the final battery evaluation for methionine as shown in Table 4 differs between Case l+and Case 2+ the table).
(see the sixth and seventh columns of
Case l+evaluation is based upon the presence of at least one
positive result in the battery.
Case 2%,on the other hand, is based upon
the presence of at least 2 positives in the battery.
It is obvious that
Case l+evaluation is .the more conservative interpretation of the results of the battery, according to which even methionine genic.
would be considered muta-
The chances of misclassifying methionine are reduced by using Case 2+
evaluation of the batteries. We have analyzed the data of the interlaboratory study according to these criteria and the results are shown in Table 5.
It
is evident that carcino-
genic chemicals are well classified (81.2% of correct results) using Case i+ criterion.
Using the same criterion, however, only 34% of non-carcinogens
are properly classified.
Using Case 2+criterion the non-carcinogens are
better classified but alot of carcinogens are passed over (41.6%). It seems to us that whichever criteria are used to analyze the battery test results, a consistent number of misclassifications are going to occur. Taking one positive result as a measure of mutagenicity will produce a number of false positives among the non-carcinogenic chemicals.
For example, as
shown in Table 5, ascorbic acid and l-naphthylamine--which are not carcinogenic--are both classified as mutagens using Case l+criterion (0% correct
Pharmacological Research Communications, Vol. 16, No. 5, 1984
430
Table
3 No. positire batterles
Total no. batteries
Case
Percent of correct results Case
i+
2+
I+
2+
7 3 2 8 8 3 4 8 7
2 O O 8 6 5 4 8 6
8 7 8 8 8 8 5 8 8
87 42 25 100 i00 I00 80
2~ O O iOO 75 62 80 IOO 75
8
5
8
3
8
1 7 2 2
8 8 7 6
I00 62 37 I00 86 83 IOO
5 3 8 6 5
IOO
iOO 87 tOO
40 50 lOO
40 iOO 1OO 80
40 I00 100
Ca r c i a o g e n i c Ch en*i c91 4-dimethylamlnoazobenzene
Chloroform Diethylstilbestrol 4-nitroqulnoline-N-ox[de 2-naphthylamine Benzidine 2"acetylaminofluorene Benzo(a)pyrene Hydrazine sulphate Hexamethylphosphoramide Ethylenethlourea Safrole Epichlorhydrin DL-ethionine N-nltrosomorpholine ~-propr~olactone Urethane DimethyIcarbamoyl chloride Cyclophosphamide 3-aminotriazole 4,4Lmethylenebis(2-chloroani Me t hy [azoxymethano lacetate 9, IO-dimethylan~hracene o-To]uidine Auramine
line)
5
5
5
5 7
2 4
5 8
3 2
3 2
5
5
3 4
3 3
3 3
3 l
IOO
87
3
5 5 3
5 4 5 average
=
62 37 12 87 28 33
75
60 75
60
20
81,2 58 .4
N o n - c a r c i n o g g n ~ c Ch(~mi c a I_s_s.................. 3-me t h y l - 4 - n i t roqu i no I i n e - N - o x i d e
Dini trosopentamethyl erie tet rami ne y-butyrolactone 3,3',5,5L~etramethyIbeuzidine Pyrene Anthracene Azoxyhenzene 4-acetylmninofluorene 1-naphthylamine Diphenylnitrosamlne Dimethylformamide l,l,l-trichloroethane 4-d ime thy lain inee zobenz e n e-4-su Iphon i~ic id Ascorbic acid Isopropyl N-(3-ch]oropheny])carbamate Hethionine Sucrose
8 4
8 O
8 6
0 33
O IOO
3 I
6 8
50 87
].00 IOO
6
O O O
8
25
1OO
2 4 4 8 3
O 3 3 6 2
5 5 4 8 6
60 20 O O 50
leo 4O 25 25 66
3 I .4
1 l 3
8 4 8
62
87
3 4 4
2 3 l
3 4 6
2
3
3
average =
75
75
50
62
O O 33
33 25 83
33
1OO
34
65.9
Pharmacological Research Communicatlbns, VoL 16, No. 5, 1984 results).
Taking
two positive
431
results as an indication of mutagenigity,
on the other hand, reduces the number of errorsfor non-carcinogenic chemicals but reduces considerably the predictivity of the test for carcinogenic chemi-cats. The criteria for analyzing the results of a battery of tests have never been thoroughly discussed in the literature.
If the goal is to reduce
the number of potentially mutagenlc and carcinogenic chemicals present in the environment, then we would propose the use of Case l~criterion in assessing all c~emicals.
However, one'needs to be aware of the extreme imprecislon
of this approach.
MUTAGENESIS TESTING AND DRUGS:
TWO TYPICAL CASES
Metronidazole is carcinogenic in mice after oral administration (Rustia and Shublck, 1972), and induces mammary fibroadenomas in rats (Cohen et al., 1973).
It was shown to be mutagenic in Salmonella (McCann et al., 19757, to
produce mutagenic urine in humans (Legator et al., 1975) and to induce chromosomal aberrations in long-term treatments in humans (Mitelman et el., 19767.
llowever, it is very weakly mutagenic on nitroreductase-deficient
strain TAIOOFr I of Salmonella, not mutagenic in V79 mammalian celts (Dayan et al., 1982); it is inactive on DNA repair of primary rat hepatocytes, and does not induce geuotoxic
efgects
on human l y m p h o c y t e s in v i t r o
(Lambert,
19797. Similarly, isoniazide, which is carcinogenic in mice (Morl et el., 1960) is a weak mutagen on Sa]monella (Wade eta].,
]98]), is mutagenic in yeasts
(Zetterberg and Bostr3m, ]9817 and in host mediated assays (Jansen et al., 1980), inactive in DNA repair tests (Wade, ]98]), does not damage human lymphocyte chromosomes in vivo (Jansen, 1980) and is inactive in dominant lethal tests in mice (Rohrborn et at., 19727. Human carcinogenicity data are not yet adequate for both drugs (Goldman~ et al., 19777.
If the toxicity of these two drugs had to be evaluated on
the basis of the results of a battery of tests, both drugs would be classified as mutagens and likely carclnogens.
However, there is closer agreement
Pharmacologica/ Research Communications, Vo/. 16, No. 5, 1984
432
between animal carcinogeniclty data and bacterial mutagenesis results, whereas more complicated mammalian systems, in principle closer to the animal model, perform very poorly.
It is, therefore, very difficult to reach
any meaningful conclusion about the risks connected with the use of these Zwo drugs, if one had to consider mutagenicJty data alone. REGULATORY APPROACHES TO MUTAGENiCITY TESTING IN THE PHARMACEUTICAL FIELD At the beginning of the 197Os an important OMS report (~10) appeared proposing the inclusion of mutageniclty testing in the routine evaluation of drug toxicity (OMS, 1971).
The report had certainly a broad influence
and in that decade more than 50 countries prescribed the use of mutagenicity data in support of new drugs' applications.
These regulations in different
countries are analyzed in detail in ~ recent publication of the International Federation of Pharmaceutical Manufacturing Associations (IFPM, 1980). The Organization for Economic Cooperation (OECD), which is comprised of 24 industrialized countries, has proposed a protocol recommending a minimum pre-marketing data-set on drugs, including gene mutation on prokaryotes and chromosome aberrations in vitro and in vivo (for a discussion, see Lo Prieno, 1981).
Similar recommendations are contained in the sixth amendment of a
directive of the Council of European Co~nunities (EEC, 1979), stating that new chemicals had to be tested for mutageniclty by the manufacturer prior to markethlg. For medicinal compounds in particular, a technical commission of the European communities (EEC, 1980) submitted a proposal to make mutagenicity studie~ obligatory for all new drugs. A con~ittee on Proprietary Medicinal Products (CMPC) has issued guidelines (discussed by Zb~nden, 1980) on dew drugs, based upon the following: a)
bacterial
b)
in v i t r o
c e l l ckwomosome damage a n a l y s i s
c)
in v i t r o
gene m u t a t i o n in ma~naltan c e ~ I s o r i n v i v a z e s t s w i t h
r
,
t e s t s with e s t a b l i s h e d
strains
Drosophila melanogaster d)
in viva chromosome damage analysis i, rodents.
PharmacologicalResearchCommun~a~on~Vo~ 1~ No. ~ 1984
433
Although t h e r e i s a c o n c o r d a n c e on t h e g e n e r a l p r i n c i p l e s s i t y of m u t a g e n i c i t y ' d a t a ,
there is considerable
variation
and t h e n e c e s among d i f f e r e n t
countries on the requirements for the assessment of drug safety. in the EEC, a 1977 I t a l i a n
law r e q u i r e s data on p r o k a r y o t e s ,
For example,
eukaryotes
and DNA repair (DM, 1977), whereas the Federal Republic of Germany requires tests to detect both gene and chromosomal mutations (ICPEMC, 19B3b) like dominant lethal test and bone marrow test. A h o m o g e n i z a t i o n o f the jz~terna~Jo~al reg~]atlons wou)d be welcome in
this field and has been advocated by the pharmaceutical industry (see, for
example, Mazu~ and Berthe,]983).
Due to national variations and d i f f e r e n c e s
of opinions in the scientific co1~lunity, con~on international guidelines on drug m u t a g e n i c i t y have not been i n t r o d u c e d .
CONCLUSIONS
From the previous discussion it is evident that, not w~thstanding the considerable amount of work dedicated to the development of genetic toxicology tests, numerous problems remain unsolved.
Mutagenesis tests, in fact,
are plagued by imprecision and by the intrinsic incapability of detecting some classes of carcinogens.
On the other hand, given the difficulty of
extrapolating from animal carcinogenicity studies to human carcinogenicity, it would be unwise to expect a nonproblematic link between mutagenicity and carcinogenicity. When testing new drugs or chemical compounds, if the goal is to find substances that have a minimum of risk of inducing mutations and cancerp then a prescreening with several mutagenesis techniques seems to be justified, and this has been the current opinion of the scientific community.
It
should be clear, however, that the introduction of mutagenicity studies might keep a certain number of potentially useful compounds out of the market,
it is also true that the presence of mutagenic activity ~er se
should discourage the manufacturing of certain chemicals because of possible negative effects on the genetic structure of living organisms, independent from their possible carcinogenic activity.
Given the choicebetween chemi-
cals and drugs that have the same use but are differentiated by their
Pharmacological Research Communications, Vol. 16, No. 5, 1984
434
genetic effects, it is wise to develop non-mutagenic substances. well known that all methodologies have limitations.
It is also
Mutagenesi8 testing
seems to have more imperfections than most of the current methods in use in toxicology.
We believe, however, that the intelligent application of
mutagenielty tests can give useful data concerning the reactivity, metabolism and toxicity of a compound.
It may also be useful for tracing chemicals
and their metabolites in body fluids or in the environment.
It would also be
useful to define more precisely the guidelines for interpreting mutagenicity tests in regulatory approaches.
In this regard it seems appropriate to dis-
cuss again if the expectation of improving the reliability of mutagenicity experiments with complex systems and organisms has been fulfilled.
If no one
questions the usefulness of new, refined methods in genetic research, when dealing with industrial application more consideration should be given to methods =hat produce acceptable results in a shorter time and at a lower cost.
ACKNOWLEDGEMENTS:
We thank Mary Forrest for revision of the manuscript.
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