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Experience with mutagenicity testing of new drugs: viewpoint of a regulatory agency
Mutation Research, 182 (1987) 187-192 Elsevier
187
MTR 08652
Experience with mutagenicity testing of new drugs" viewpoint of a regulatory agency Stephan Madle, Anke Korte
*
a n d R o l f Bass
Institute for Drugs, Federal Health Office, Seestrasse 10, D-IO00 Berlin 65 (Federal Republic of Germany) (Received 5 November 1986) (Revision received 16 March 1987) (Accepted 23 March 1987)
Keywords: Drugs, new; Mutagenicity testing; Regulations; Registration.
Summary Quality and quantity of mutagenicity testing were analyzed for drugs with new active compounds which were submitted for registration in the Federal Republic of Germany from mid 1982 to mid 1986. A large variety of deficiencies was found, applying to selection and number of mutagenicity tests as well as to test performances. Only 65 out of the 144 drugs submitted for registration were tested sufficiently in the initial phase of registration. From 1982 to 1986 this situation has not been changed markedly. Inadequate test performance still remains the main reason for insufficient testing, leading in some cases to artificially positive results. For in vivo tests the selection of test species was mainly motivated by technical reasons and not by characteristics of the test compound. Most of the insufficiencies were eliminated during the second phase of registration. In some cases insufficient mutagenicity testing led to consequences concerning risk-benefit assessment of the drug and its regulation.
In the Federal Republic of Germany detailed mutagenicity testing has been required for the registration of drugs for several years. In this communication we describe quantity and quality of mutagenicity testing of new drugs submitted for approval to the Institute for Drugs of the Federal Health Office. Evaluation of mutagenicity testing was restricted to the aspect of possible genetic
* Present address: City of Hamburg, Health Department, Tesdorpfstrasse 8, D-2000 Hamburg (Federal Republic of
Germany). Correspondence: Dr. S. Madle, Institute for Drugs, Federal Health Office, Seestrasse 10, D-1000 Berlin 65 (Federal Republic of Germany).
hazard for human germ cells (in this publication the aspect carcinogenicity was disregarded). Complete mutagenicity testing is not required for all drugs. Type of active compound, indication for medical use, resorption, etc. are relevant criteria for determining whether a complete testing is necessary or not. E.g., complete routine testing is not adequate for cytostatics and hormones. The 144 drugs which were selected for the present analysis all contained new active compounds and were evaluated as needing complete testing for mutagenic effects. Mutagenicity testing of drugs in the Federal Republic of Germany is not regulated in detail with regard to the choice of test systems and protocols. This is in agreement with European
0165-1161/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
188 Rules and Regulations and corresponds to our philosophy that mutagenicity testing of drugs should be adapted to physico-chemical properties of the active compound(s) and to pharmacokinetics and metabolism. Therefore, evaluation of mutagenicity testing of drugs cannot be standardized. According to our approach test strategies may differ from compound to compound, but 3 minimum criteria should be fulfilled: (a) testing for the induction of gene mutations; (b) testing for the induction of chromosome mutations; (c) adequate inclusion of metabolization, in vitro: activation systems ($9 mix, hepatocytes); in vivo: relevant test species. These criteria are in agreement with the description of 4 categories of test systems in the Notes for Guidance for the Testing of Medicinal Products for their Mutagenic Potential (EEC, Notice No. 84/C 293/04, Annex II): test for gene mutations in bacteria; test for chromosome mutations in mammalian cells in vitro; test for gene mutations in eukaryotic cells in vitro; in vivo-test (for chromosome mutation). Performing one test from each of these categories should result in an appropriate test battery in most cases. We like to emphasize that from our point of view there is no necessity for a strict adherence to these categories. The very broad spectrum of genotoxicity tests available allows the selection of suitable test systems for each compound. Furthermore, most of the systems enable adaptation of the test conditions, if necessary. In addition to the evaluation of the combination of test systems we routinely analyze the quality of test performance. Here the standard for evaluation is given by scientific literature (standard protocols, size of test samples, number of experiments, preparation times, type of metabolization, handling of positive controls, criteria for analysis, criteria for equivocal/unequivocal resuits). Evaluation of test performance is routinely adapted to the present state of the art. Tests performed insufficiently with respect to the mentioned criteria often lead to equivocal results, which may indicate a mutagenic potential. Since the main goal of our work is to ensure safety of drugs, the term "suspicion" is used in a broad sense: clear positive results as well as unclarified indications for genotoxic effects lead to the judgement "suspicious".
In the Federal Republic of Germany registration of drugs is divided into two phases. In phase I extensive documents required for the evaluation of quality, safety and efficacy are submitted. In most cases these documents are not adequate for registration. The insufficiencies of the documentation are listed and sent to the applicant for comments. In phase II of the registration process, the applicant submits supplementary information, e.g. from further investigations. Following this the Federal Health Office decides about registration.
Evaluation of phase I mutagenicity testing (Table 1) The 144 drugs selected were submitted for application between mid 1982 and mid 1986. In phase I of registration, a sufficient exclusion of a mutagenic potential was reached for 45% only. 13% of the drugs were under suspicion of mutagenicity. An additional 42% were tested insufficiently. In these cases the minimum criteria were not met due to the lack of tests or unacceptable performances. ("Suspicious" drugs with insufficient testing were considered as "suspicious" only.) Frequencies of "suspicion" and "insufficient testing" varied considerably from year to year without a clear tendency.
Evaluation of phase H mutagenicity testing (Table 2) Up to now 49 drugs have reached phase II of registration. From Table 1 79 drugs had to be expected in phase II; the difference being due to 1 refusal of an application (submission 1983) and 8 withdrawals of applications after phase I (submissions 1983, 6, and 1984, 2); for 21 drugs phase II documents have not been submitted up to now (submissions 1985, 10, and 1986, 11). For most of the 49 drugs dealt with in phase II, suspicion was eliminated or mutagenicity testing was completed without further problems. For 11 drugs, however, mutagenic effects could not be excluded sufficiently after phase II. Three of these had been insufficiently tested in phase I and a suspicion arose from the investigations submitted in phase II.
Evaluation of mutagenicity testing after phase I1 11 drugs were critical with respect to mutagenicity after phase II of registration. 4 of them were still insufficiently tested (without being "suspi-
189 TABLE 1 DRUG-MUTAGENICITY TESTING: EVALUATION IN PHASE I Year of submission of the application
Number of drugs assessed
1982 1983 1984 1985 1986 1982-86
Inadequate testing
Adequate testing; no mutagenic potential
Unclarified suspicion
Insufficient testing a
22 54 24 23 21
12 (54%) 18 (33%) 14 (58%) 11 (48%) 10 (48%)
5 (235[) 7 (135[) 3 (135[) 1 (4%) 2 (95[)
5 (235[) 29 (54%) 7 (29%) 11 (48%) 9 (435[)
144
65 (45%)
18 (135[)
61 (425[)
" Compounds with suspicion are not included.
cious"). They belonged to chemical classes which probably do not induce mutagenic effects. Other toxicological problems were not apparent and great benefits of the drugs were to be expected. Therefore, they were registered and given the opportunity to complete the documentation after registration. In all 4 cases the approval under conditions was accepted, the necessary tests were performed and no further problems arose. For the remaining 7 "suspicious" compounds the type of suspicion and our approach to solve the problems shall be described as follows: Compound A, a nitroimidazole. There were positive results from tests for gene mutations in bacteria and yeast, tests for chromosome mutations were negative. The application was withdrawn after phase II due to several toxicological and clinical problems.
Compound B, an antibiotic. A small battery of mutagenicity tests was submitted in phase I. An in vitro chromosome aberration test had been performed according to an inadequate design and exhibited clearly increased aberration frequencies. Clarification of this result was asked for. Only data from yeast tests were submitted in phase II which were unable to clarify the problem. The expected benefit of the drug led to approval under the condition that further tests be carried out. Then a well-performed clearly negative chromosome aberration test (same system as in phase I) was submitted. Compound C, a nucleoside analogue. This drug clearly induced chromosome aberrations in vitro and in vivo. The specific chemical properties of the compound led us to assume a threshold for the underlying mechanism of action. Experimental re-
TABLE 2 DRUG-MUTAGENICITY TESTING: EVALUATION IN PHASE II (INADEQUATE TESTING IN PHASE I) Year of submission of the application
Number of drugs assessed
Phase I: suspicion
Phase II: elimination of suspicion; sufficient testing
1982 1983 1984 1985 1986
10 29 8 2
5 6
2 5
1
1
1982-86
49
12
8
Phase I: insufficient testing
Phase II: sufficient testing; no suspicion
Phase II: mutagenicity cannot he excluded sufficiently
5 23 7 2
2 19 7 2
6 5
37
30
11
190
suits supported this assumption. To ensure that the threshold concentration was not exceeded in man, we asked for supplementary pharmacokinetic investigations (including determinations of concentrations in germ tissue). For several reasons, among them mutagenicity, the compound was then registered only for such uses and daily doses leading to concentrations of the drug well below the experimentally determined threshold.
Compound D, an antirheumatic drug. Whereas in phase I absolutely insufficient mutagenicity testing was presented, in phase II data from extensive testing were submitted. Some of the tests were repeatedly positive. The resulting negative riskbenefit assessment led to the rejection of the application. The applicant did not accept the rejection and performed further investigations which demonstrated that the positive effects observed were due to in vitro artefacts irrelevant for man. The drug accordingly was approved for marketing. Additionally, in this case it became obvious that in phase I of registration information was withheld by the applicant. In phase II more than 10 tests were submitted which had been performed long ago. If this information had been submitted initially, the problem could have been solved within phase II of registration. Compound E, an antipsychotic drug. In phase I for analysis of gene mutations a mammalian cell test was submitted repeatedly showing positive results. We asked for clarification. As in phase II only a non-reproduced Ames test (with very strong toxic effects of the test compound) was submitted, the suspicion was not invalidated to a sufficient extent. The application was rejected for several toxocological and clinical reasons. Compound F, a narcotic analgesic. A completely insufficient testing was presented in phase I. In phase II a positive Ames test was submitted as the only gene mutation test (the other tests were acceptable and negative). Upon approval under conditions extensive data from gene mutation tests with Salmonella and mammalian cells and SCE tests - - all of which were negative - - invalidated the suspicion.
Compound G, a narcotic analgesic. Phase I was supplied without mutagenicity testing. In phase II several tests were presented, most of them with adequate performances. Three tests showed positive, though not alarming, responses. Approval was granted with the condition that this matter should be further clarified. The results of further studies confirmed suspicion; the drug now carried the stigma of mutagenic potential. Consideration on how to prevent genetic effects in man (e.g., restricting indications for medical use) were done. According to § 28.3 of the (German) National Drug Law (Arzneimittelgesetz) approval of drugs can be granted with the condition of further investigations when essential therapeutic benefit is expected and toxicological risks appear to be relatively low. As described above, for 7 of the drugs regulated after phase II of registration conditional approval was granted. 6 of these compounds were considered as being suspicious due to invalid testing only, further investigations led to a sufficient exclusion of genetic effects in man. For 1 drug risk-benefit assessment led to approval under conditions, although there were very clear indications for a mutagenic potential. Further investigations performed after registration confirmed mutagenicity of the compound. Since there was no reason why extrapolation to man was not justified, measures were taken to prevent genetic effects in man.
Use of single test systems and evaluation of test pedormances For the 114 drugs which have completed the registration process, 515 tests were conducted. On average this means that 4.5 tests were conducted per compound. In toto, 613 tests have been evaluated up to now (Table 3). 59% of all tests were conducted in vitro, 41% in vivo. Bacterial tests were performed most frequently (151 tests, repair test excluded). For 11 compounds two different types of bacterial gene mutation tests were performed. Micronucleus tests in vivo were quite frequent, too (99 tests). The rather infrequent use of SCE tests (22 tests in vitro, 15 tests in vivo) and the relatively high number of 30 dominant lethal assays (fertility studies not included) was surprising. From our
191 TABLE 3 DRUG-MUTAGENICITY TESTING: EVALUATION OF TEST SYSTEMSEMPLOYED Test system
Tests sub- Inademitred (n) quate performances (%)
Gene mutations in bacteria Micronucleus test in vivo (bone marrow) Gene mutations in mammalian cells in vitro Chromosome mutations in vivo (bone marrow) Chromosomeaberrations in mammalian cells in vitro Yeast assays Dominant lethal assay SCE in mammalian cells in vitro UDS in mammalian cells in vitro SCE in vivo (bone marrow) Other systems a All systems In vitro tests In vivo tests a
Tests leading to a suspicion (n(%))
151
32
4 (2.6%)
99
32
1 (1.0%)
72
22
9 (12.5%)
66
33
2 (3.0%)
57 39 30
58 51 37
4 (7.0%) 1 (2.6%) 0
22
45
2 (9.1%)
18
44
2 (11.1%)
15 44
27 61
0 3 (6.8%)
613 39 360 (59%) 38 253 (41%) 40
28 (4.6%) 22 (6.1%) 5 (2.0%)
Host-mediated assay, 17; chromosomal aberration test in spermatogonia, 7; Drosophila test, 6; chromosomalanalysis with human lymphocytes in vivo, 4 (chromosome aberrations, 3; SCE, 1); sperm morphology test, 3; mammalian spot test, 2; chromosomalaberration test with metaphase-II occytes, 1; UDS in testes in vivo, 1; DNA binding in testes, 1; micronucleustest in vitro, 1.
point of view the latter is not suitable for routine testing due to insensitivity and great expense. 39% of all tests were performed inadequately, thus rendering the results unacceptable. Quality of test performance did not differ clearly between tests conducted in vitro and in vivo (38 vs. 40%). This high percentage of inadequate test perfdrmances was quite astonishing. Therefore, we would like to point out that the time-lag between perfor-
mance of a test and submission of the protocol was regarded. Test protocols were evaluated according to the state of the art of that time, when the tests were performed (and not according to the now present state of the art). At the time of submission most mutagenicity tests date back 3-5 years, older test protocols up to 10 years are seldom submitted. In general this time-lag does not lead to great problems, since the protocols of the frequently employed test systems have not changed markedly in the last years. Further to formal mistakes (e.g., incomplete protocols) the following reasons contributed to the statement of inadequate performance: lack of clarification of equivocal results, too small test samples (e.g., in vitro, results not confirmed by a second experiment; in vivo, too small numbers of animals and target cells) and employment of low concentrations/doses only without given reasons. Chromosome aberration tests in vitro were rejected most frequently (58%), although this system has been used in mutation research for many years and its experimental peculiarities are quite well understood. For this system additional criticism emanates from omitting metabolization systems (quite frequently for tests submitted up to 1983) and from unclear criteria used for chromosomal analysis. 4.6% of the tests yielded results which gave suspicion for a mutagenic activity of the compound. In vitro tests (6.1%) differed clearly from in vivo tests (2.0%). From the high frequency of positive results a higher sensitivity with respect to the detection of genetic effects might be deduced for in vitro tests. But for evaluation of the sensitivity of test systems only well-performed tests should be considered. Evaluation of the "positive" gene mutations tests in vitro (the system with the highest frequency of positive results, 12.5%) showed that most of the "positives" were obtained from inadequately performed tests. Reinvestigations by wellperformed assays of the same system eliminated the suspicion. Therefore it seemed to us that the higher "sensitivity" of gene mutation tests with mammalian cell cultures is correlated to their great complexity and is rather due to insufficient performance by increasing the variability of genetic effects in different cultures of one experiment. Great variations are - - by chance - - expected to lead to
192 TABLE 4 TEST SPECIES EMPLOYED FOR IN VIVO ASSAYS Test system
Tests submitted
Rat
Mouse
Chinese hamster
(n) Micronucleus test in vivo (bone marrow) Chromosome mutations in vivo (bone marrow) SCE in vivo (bone marrow) Dominant lethal assay
102
6
85
11
66
17
16
33
15
-
2
13
30
3
27
-
"positive" results. Therefore, from our point of view results of in vitro tests should be confirmed by a second experiment for exclusion of artificial findings. One of the main problems for in vivo mutagenicity tests consists in the choice of a relevant test species. For the drugs analyzed here the selection of test species seemed to be motivated mainly by practical reasons (Table 4), although the use of
mice for chromosomal analyses seems to be neither practical nor appropriate for routine testing. Species were seldom selected according to properties of the test compound (metabolism/pharmacokinetics) in order to mimic the human situation. Due to a high technical suitability 46 of the 81 chromosomal analyses in vivo were performed with Chinese hamsters. On the other hand, Chinese hamsters were never used for other toxicological or pharmacological studies. These investigations were routinely performed with mice or rats. Resuits from mutagenicity testing obtained with mice or rats are therefore expected to yield more meaningful results. None of the Chinese hamster tests were accompanied by analyses of resorption, distribution, metabohsm and elimination of the test compound in this species, leading to "black box-results". Therefore, our conclusion is that (negative) results from in vivo tests of drugs are scarcely relevant when the species used are not adequately investigated. Furthermore, if for some reason an adequate test species cannot be employed, extended in vitro testing (including different metabolization systems, e.g., $9 mix and hepatocytes) may lead to a more meaningful test battery than the use of a species at random.
187
MTR 08652
Experience with mutagenicity testing of new drugs" viewpoint of a regulatory agency Stephan Madle, Anke Korte
*
a n d R o l f Bass
Institute for Drugs, Federal Health Office, Seestrasse 10, D-IO00 Berlin 65 (Federal Republic of Germany) (Received 5 November 1986) (Revision received 16 March 1987) (Accepted 23 March 1987)
Keywords: Drugs, new; Mutagenicity testing; Regulations; Registration.
Summary Quality and quantity of mutagenicity testing were analyzed for drugs with new active compounds which were submitted for registration in the Federal Republic of Germany from mid 1982 to mid 1986. A large variety of deficiencies was found, applying to selection and number of mutagenicity tests as well as to test performances. Only 65 out of the 144 drugs submitted for registration were tested sufficiently in the initial phase of registration. From 1982 to 1986 this situation has not been changed markedly. Inadequate test performance still remains the main reason for insufficient testing, leading in some cases to artificially positive results. For in vivo tests the selection of test species was mainly motivated by technical reasons and not by characteristics of the test compound. Most of the insufficiencies were eliminated during the second phase of registration. In some cases insufficient mutagenicity testing led to consequences concerning risk-benefit assessment of the drug and its regulation.
In the Federal Republic of Germany detailed mutagenicity testing has been required for the registration of drugs for several years. In this communication we describe quantity and quality of mutagenicity testing of new drugs submitted for approval to the Institute for Drugs of the Federal Health Office. Evaluation of mutagenicity testing was restricted to the aspect of possible genetic
* Present address: City of Hamburg, Health Department, Tesdorpfstrasse 8, D-2000 Hamburg (Federal Republic of
Germany). Correspondence: Dr. S. Madle, Institute for Drugs, Federal Health Office, Seestrasse 10, D-1000 Berlin 65 (Federal Republic of Germany).
hazard for human germ cells (in this publication the aspect carcinogenicity was disregarded). Complete mutagenicity testing is not required for all drugs. Type of active compound, indication for medical use, resorption, etc. are relevant criteria for determining whether a complete testing is necessary or not. E.g., complete routine testing is not adequate for cytostatics and hormones. The 144 drugs which were selected for the present analysis all contained new active compounds and were evaluated as needing complete testing for mutagenic effects. Mutagenicity testing of drugs in the Federal Republic of Germany is not regulated in detail with regard to the choice of test systems and protocols. This is in agreement with European
0165-1161/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
188 Rules and Regulations and corresponds to our philosophy that mutagenicity testing of drugs should be adapted to physico-chemical properties of the active compound(s) and to pharmacokinetics and metabolism. Therefore, evaluation of mutagenicity testing of drugs cannot be standardized. According to our approach test strategies may differ from compound to compound, but 3 minimum criteria should be fulfilled: (a) testing for the induction of gene mutations; (b) testing for the induction of chromosome mutations; (c) adequate inclusion of metabolization, in vitro: activation systems ($9 mix, hepatocytes); in vivo: relevant test species. These criteria are in agreement with the description of 4 categories of test systems in the Notes for Guidance for the Testing of Medicinal Products for their Mutagenic Potential (EEC, Notice No. 84/C 293/04, Annex II): test for gene mutations in bacteria; test for chromosome mutations in mammalian cells in vitro; test for gene mutations in eukaryotic cells in vitro; in vivo-test (for chromosome mutation). Performing one test from each of these categories should result in an appropriate test battery in most cases. We like to emphasize that from our point of view there is no necessity for a strict adherence to these categories. The very broad spectrum of genotoxicity tests available allows the selection of suitable test systems for each compound. Furthermore, most of the systems enable adaptation of the test conditions, if necessary. In addition to the evaluation of the combination of test systems we routinely analyze the quality of test performance. Here the standard for evaluation is given by scientific literature (standard protocols, size of test samples, number of experiments, preparation times, type of metabolization, handling of positive controls, criteria for analysis, criteria for equivocal/unequivocal resuits). Evaluation of test performance is routinely adapted to the present state of the art. Tests performed insufficiently with respect to the mentioned criteria often lead to equivocal results, which may indicate a mutagenic potential. Since the main goal of our work is to ensure safety of drugs, the term "suspicion" is used in a broad sense: clear positive results as well as unclarified indications for genotoxic effects lead to the judgement "suspicious".
In the Federal Republic of Germany registration of drugs is divided into two phases. In phase I extensive documents required for the evaluation of quality, safety and efficacy are submitted. In most cases these documents are not adequate for registration. The insufficiencies of the documentation are listed and sent to the applicant for comments. In phase II of the registration process, the applicant submits supplementary information, e.g. from further investigations. Following this the Federal Health Office decides about registration.
Evaluation of phase I mutagenicity testing (Table 1) The 144 drugs selected were submitted for application between mid 1982 and mid 1986. In phase I of registration, a sufficient exclusion of a mutagenic potential was reached for 45% only. 13% of the drugs were under suspicion of mutagenicity. An additional 42% were tested insufficiently. In these cases the minimum criteria were not met due to the lack of tests or unacceptable performances. ("Suspicious" drugs with insufficient testing were considered as "suspicious" only.) Frequencies of "suspicion" and "insufficient testing" varied considerably from year to year without a clear tendency.
Evaluation of phase H mutagenicity testing (Table 2) Up to now 49 drugs have reached phase II of registration. From Table 1 79 drugs had to be expected in phase II; the difference being due to 1 refusal of an application (submission 1983) and 8 withdrawals of applications after phase I (submissions 1983, 6, and 1984, 2); for 21 drugs phase II documents have not been submitted up to now (submissions 1985, 10, and 1986, 11). For most of the 49 drugs dealt with in phase II, suspicion was eliminated or mutagenicity testing was completed without further problems. For 11 drugs, however, mutagenic effects could not be excluded sufficiently after phase II. Three of these had been insufficiently tested in phase I and a suspicion arose from the investigations submitted in phase II.
Evaluation of mutagenicity testing after phase I1 11 drugs were critical with respect to mutagenicity after phase II of registration. 4 of them were still insufficiently tested (without being "suspi-
189 TABLE 1 DRUG-MUTAGENICITY TESTING: EVALUATION IN PHASE I Year of submission of the application
Number of drugs assessed
1982 1983 1984 1985 1986 1982-86
Inadequate testing
Adequate testing; no mutagenic potential
Unclarified suspicion
Insufficient testing a
22 54 24 23 21
12 (54%) 18 (33%) 14 (58%) 11 (48%) 10 (48%)
5 (235[) 7 (135[) 3 (135[) 1 (4%) 2 (95[)
5 (235[) 29 (54%) 7 (29%) 11 (48%) 9 (435[)
144
65 (45%)
18 (135[)
61 (425[)
" Compounds with suspicion are not included.
cious"). They belonged to chemical classes which probably do not induce mutagenic effects. Other toxicological problems were not apparent and great benefits of the drugs were to be expected. Therefore, they were registered and given the opportunity to complete the documentation after registration. In all 4 cases the approval under conditions was accepted, the necessary tests were performed and no further problems arose. For the remaining 7 "suspicious" compounds the type of suspicion and our approach to solve the problems shall be described as follows: Compound A, a nitroimidazole. There were positive results from tests for gene mutations in bacteria and yeast, tests for chromosome mutations were negative. The application was withdrawn after phase II due to several toxicological and clinical problems.
Compound B, an antibiotic. A small battery of mutagenicity tests was submitted in phase I. An in vitro chromosome aberration test had been performed according to an inadequate design and exhibited clearly increased aberration frequencies. Clarification of this result was asked for. Only data from yeast tests were submitted in phase II which were unable to clarify the problem. The expected benefit of the drug led to approval under the condition that further tests be carried out. Then a well-performed clearly negative chromosome aberration test (same system as in phase I) was submitted. Compound C, a nucleoside analogue. This drug clearly induced chromosome aberrations in vitro and in vivo. The specific chemical properties of the compound led us to assume a threshold for the underlying mechanism of action. Experimental re-
TABLE 2 DRUG-MUTAGENICITY TESTING: EVALUATION IN PHASE II (INADEQUATE TESTING IN PHASE I) Year of submission of the application
Number of drugs assessed
Phase I: suspicion
Phase II: elimination of suspicion; sufficient testing
1982 1983 1984 1985 1986
10 29 8 2
5 6
2 5
1
1
1982-86
49
12
8
Phase I: insufficient testing
Phase II: sufficient testing; no suspicion
Phase II: mutagenicity cannot he excluded sufficiently
5 23 7 2
2 19 7 2
6 5
37
30
11
190
suits supported this assumption. To ensure that the threshold concentration was not exceeded in man, we asked for supplementary pharmacokinetic investigations (including determinations of concentrations in germ tissue). For several reasons, among them mutagenicity, the compound was then registered only for such uses and daily doses leading to concentrations of the drug well below the experimentally determined threshold.
Compound D, an antirheumatic drug. Whereas in phase I absolutely insufficient mutagenicity testing was presented, in phase II data from extensive testing were submitted. Some of the tests were repeatedly positive. The resulting negative riskbenefit assessment led to the rejection of the application. The applicant did not accept the rejection and performed further investigations which demonstrated that the positive effects observed were due to in vitro artefacts irrelevant for man. The drug accordingly was approved for marketing. Additionally, in this case it became obvious that in phase I of registration information was withheld by the applicant. In phase II more than 10 tests were submitted which had been performed long ago. If this information had been submitted initially, the problem could have been solved within phase II of registration. Compound E, an antipsychotic drug. In phase I for analysis of gene mutations a mammalian cell test was submitted repeatedly showing positive results. We asked for clarification. As in phase II only a non-reproduced Ames test (with very strong toxic effects of the test compound) was submitted, the suspicion was not invalidated to a sufficient extent. The application was rejected for several toxocological and clinical reasons. Compound F, a narcotic analgesic. A completely insufficient testing was presented in phase I. In phase II a positive Ames test was submitted as the only gene mutation test (the other tests were acceptable and negative). Upon approval under conditions extensive data from gene mutation tests with Salmonella and mammalian cells and SCE tests - - all of which were negative - - invalidated the suspicion.
Compound G, a narcotic analgesic. Phase I was supplied without mutagenicity testing. In phase II several tests were presented, most of them with adequate performances. Three tests showed positive, though not alarming, responses. Approval was granted with the condition that this matter should be further clarified. The results of further studies confirmed suspicion; the drug now carried the stigma of mutagenic potential. Consideration on how to prevent genetic effects in man (e.g., restricting indications for medical use) were done. According to § 28.3 of the (German) National Drug Law (Arzneimittelgesetz) approval of drugs can be granted with the condition of further investigations when essential therapeutic benefit is expected and toxicological risks appear to be relatively low. As described above, for 7 of the drugs regulated after phase II of registration conditional approval was granted. 6 of these compounds were considered as being suspicious due to invalid testing only, further investigations led to a sufficient exclusion of genetic effects in man. For 1 drug risk-benefit assessment led to approval under conditions, although there were very clear indications for a mutagenic potential. Further investigations performed after registration confirmed mutagenicity of the compound. Since there was no reason why extrapolation to man was not justified, measures were taken to prevent genetic effects in man.
Use of single test systems and evaluation of test pedormances For the 114 drugs which have completed the registration process, 515 tests were conducted. On average this means that 4.5 tests were conducted per compound. In toto, 613 tests have been evaluated up to now (Table 3). 59% of all tests were conducted in vitro, 41% in vivo. Bacterial tests were performed most frequently (151 tests, repair test excluded). For 11 compounds two different types of bacterial gene mutation tests were performed. Micronucleus tests in vivo were quite frequent, too (99 tests). The rather infrequent use of SCE tests (22 tests in vitro, 15 tests in vivo) and the relatively high number of 30 dominant lethal assays (fertility studies not included) was surprising. From our
191 TABLE 3 DRUG-MUTAGENICITY TESTING: EVALUATION OF TEST SYSTEMSEMPLOYED Test system
Tests sub- Inademitred (n) quate performances (%)
Gene mutations in bacteria Micronucleus test in vivo (bone marrow) Gene mutations in mammalian cells in vitro Chromosome mutations in vivo (bone marrow) Chromosomeaberrations in mammalian cells in vitro Yeast assays Dominant lethal assay SCE in mammalian cells in vitro UDS in mammalian cells in vitro SCE in vivo (bone marrow) Other systems a All systems In vitro tests In vivo tests a
Tests leading to a suspicion (n(%))
151
32
4 (2.6%)
99
32
1 (1.0%)
72
22
9 (12.5%)
66
33
2 (3.0%)
57 39 30
58 51 37
4 (7.0%) 1 (2.6%) 0
22
45
2 (9.1%)
18
44
2 (11.1%)
15 44
27 61
0 3 (6.8%)
613 39 360 (59%) 38 253 (41%) 40
28 (4.6%) 22 (6.1%) 5 (2.0%)
Host-mediated assay, 17; chromosomal aberration test in spermatogonia, 7; Drosophila test, 6; chromosomalanalysis with human lymphocytes in vivo, 4 (chromosome aberrations, 3; SCE, 1); sperm morphology test, 3; mammalian spot test, 2; chromosomalaberration test with metaphase-II occytes, 1; UDS in testes in vivo, 1; DNA binding in testes, 1; micronucleustest in vitro, 1.
point of view the latter is not suitable for routine testing due to insensitivity and great expense. 39% of all tests were performed inadequately, thus rendering the results unacceptable. Quality of test performance did not differ clearly between tests conducted in vitro and in vivo (38 vs. 40%). This high percentage of inadequate test perfdrmances was quite astonishing. Therefore, we would like to point out that the time-lag between perfor-
mance of a test and submission of the protocol was regarded. Test protocols were evaluated according to the state of the art of that time, when the tests were performed (and not according to the now present state of the art). At the time of submission most mutagenicity tests date back 3-5 years, older test protocols up to 10 years are seldom submitted. In general this time-lag does not lead to great problems, since the protocols of the frequently employed test systems have not changed markedly in the last years. Further to formal mistakes (e.g., incomplete protocols) the following reasons contributed to the statement of inadequate performance: lack of clarification of equivocal results, too small test samples (e.g., in vitro, results not confirmed by a second experiment; in vivo, too small numbers of animals and target cells) and employment of low concentrations/doses only without given reasons. Chromosome aberration tests in vitro were rejected most frequently (58%), although this system has been used in mutation research for many years and its experimental peculiarities are quite well understood. For this system additional criticism emanates from omitting metabolization systems (quite frequently for tests submitted up to 1983) and from unclear criteria used for chromosomal analysis. 4.6% of the tests yielded results which gave suspicion for a mutagenic activity of the compound. In vitro tests (6.1%) differed clearly from in vivo tests (2.0%). From the high frequency of positive results a higher sensitivity with respect to the detection of genetic effects might be deduced for in vitro tests. But for evaluation of the sensitivity of test systems only well-performed tests should be considered. Evaluation of the "positive" gene mutations tests in vitro (the system with the highest frequency of positive results, 12.5%) showed that most of the "positives" were obtained from inadequately performed tests. Reinvestigations by wellperformed assays of the same system eliminated the suspicion. Therefore it seemed to us that the higher "sensitivity" of gene mutation tests with mammalian cell cultures is correlated to their great complexity and is rather due to insufficient performance by increasing the variability of genetic effects in different cultures of one experiment. Great variations are - - by chance - - expected to lead to
192 TABLE 4 TEST SPECIES EMPLOYED FOR IN VIVO ASSAYS Test system
Tests submitted
Rat
Mouse
Chinese hamster
(n) Micronucleus test in vivo (bone marrow) Chromosome mutations in vivo (bone marrow) SCE in vivo (bone marrow) Dominant lethal assay
102
6
85
11
66
17
16
33
15
-
2
13
30
3
27
-
"positive" results. Therefore, from our point of view results of in vitro tests should be confirmed by a second experiment for exclusion of artificial findings. One of the main problems for in vivo mutagenicity tests consists in the choice of a relevant test species. For the drugs analyzed here the selection of test species seemed to be motivated mainly by practical reasons (Table 4), although the use of
mice for chromosomal analyses seems to be neither practical nor appropriate for routine testing. Species were seldom selected according to properties of the test compound (metabolism/pharmacokinetics) in order to mimic the human situation. Due to a high technical suitability 46 of the 81 chromosomal analyses in vivo were performed with Chinese hamsters. On the other hand, Chinese hamsters were never used for other toxicological or pharmacological studies. These investigations were routinely performed with mice or rats. Resuits from mutagenicity testing obtained with mice or rats are therefore expected to yield more meaningful results. None of the Chinese hamster tests were accompanied by analyses of resorption, distribution, metabohsm and elimination of the test compound in this species, leading to "black box-results". Therefore, our conclusion is that (negative) results from in vivo tests of drugs are scarcely relevant when the species used are not adequately investigated. Furthermore, if for some reason an adequate test species cannot be employed, extended in vitro testing (including different metabolization systems, e.g., $9 mix and hepatocytes) may lead to a more meaningful test battery than the use of a species at random.