umu test using test results of 486 chemicals and comparison with the Ames test and carcinogenicity data

Genetic Toxicology

ELSEVIER

Mutation Research 369 (1996) 129-145

Validation of the SOS/umu test using test results of 486 chemicals and comparison with the Ames test and carcinogenicity data Georg Reifferscheid *, Jiirgen Heil A M M U G . UniL'ersit3" o f Mainz, Obere Zahlbacher Str. 63, 55101 Mainz, German3,

Received 14 November 1995; revised 9 January 1996;accepted 9 January 1996

Abstract The present study gives a comprehensive update of all u m u genotoxicity assay results published so far. The available data of 486 chemicals investigated with the u m u test are compared with the Ames test (274 compounds) as well as rodent carcinogenicity data (179 compounds). On the whole, there is good agreement between the u m u test and the Ames test results, with a concordance of about 90%. The u m u test was able to detect 86% of the Ames mutagens, while the Ames test (using at least 5 strains) detected 97% of the u m u positive compounds. The elimination of TA102 from the set of Ames tester strains reduced the percentage of detectable u m u genotoxins from 97 to 86%. The agreement between carcinogenesis and u m u response was 65%, which is comparable to earlier studies concerning rodent carcinogenesis and Salmonella mutagenesis. The present compilation of u m u results provides a database that can be used for the comparison of the SOS-inducing activity of chemicals and their mutagenicity, respectively, carcinogenicity. The results presented here clearly demonstrate that a chemical which induces the expression of the u m u operon can be regarded a rodent carcinogen with a high degree of certainty (93%). K e w ' o r d s : umu

test; Ames test; Genotoxicity;Mutagenicity;Carcinogenicity

1. Introduction Genotoxins and mutagens are considered to be substantially involved in the development of the carcinogenic process. Unquestionably, continuing degenerative changes of the genetic apparatus, caused by whatever mechanism, lead to the formation of a variety of further diseases. Beginning in the early 1970s, many procedures

* Corresponding author. Tel.: +49 (6131) 173314; Fax: +49 (6131) 173364; E-mail: [email protected].

have been published and are now widely in use to detect potential mutagens and carcinogens. Since that time, many tests have been further developed by adapting the increasing knowledge about the molecular mechanisms of mutagenicity, but only a few methods have been proven to be sufficiently sensitive for the detection of genotoxins and mutagens in environmental samples, and again, only a few test systems can be considered as valid. An appropriate method designed for extensive use (i.e., routine testing) should integrate scientific, ecological and economic aspects as far as possible. The

0165-1218/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PH S0165-1218(96)00007-9

130

G. Reifferscheid, J. Heil / Mutation Research 369 (1996) 129 145

basic statistics of the test system, such as the sensitivity, specificity and accuracy, which result from the comparative study of a multitude of chemicals ( > 100), should be known. Ecological aspects include measures, such as non-radioactive handling, the avoidance of waste or the cutting down of the use of animal products, such as $9 or fetal calf serum. Last, but not least, the costs per assay play an important role in the acceptance of a specific test system. Increasing scientific and public interests in environmental genotoxicity reflect the urgent need for standardized and valid procedures for the determination of genotoxicity, respectively, mutagenicity. Thus, the German Institute for Standardization (DIN) has decided to standardize appropriate methods. International standardization by the ISO (International Standardization Organization) is at its beginning. As a result of discussions among experts and extensive prescreenings in order to select test systems which are able to reflect the genotoxic potentials of native waste waters, the DIN standardization procedure started with two bacterial genotoxicity tests, the Salmoneila/microsome assay (Ames test) and, at the recommendation of our laboratory, the umu test system. The umu test, although less well-known as the Ames test, is convincing by its simple performance and the excellent reproducibility of its results.

2. Theoretical background The umu test, first developed and published by Oda et al. (1985), is based on the ability of DNAdamaging agents to induce the expression of the umu operon. In connection with the damage inducible genes (din genes) recA, lexA and umuD, the umuC-gene is essentially involved in bacterial mutagenesis via the so-called SOS-pathway (Little and Mount, 1982; Rajagopalan et al., 1992). LexA protein is the repressor of all cellular din genes. Lesions such as single-stranded DNA, depurinic and depyrimidinic sites or even free deoxynucleotides seem to activate RecA to a LexA processing form that facilitates cleavage of LexA repressor, thus leading to the derepression of all din genes. RecA protein appears to have further roles in the mutagenic process: first, it facilitates cleavage of UmuD protein,

thereby generating a mutagenically active form (UmuD'); and second, it interacts physically with UmuD ('or the functionally homologous MucA' in the plasmid pKM101-containing Ames tester strains TA98 and TAI00), UmuC and DNA-polymerase III in order to correctly position these mutagenesis proteins at lesions in the DNA (Rajagopalan et al., 1992; Frank et al., 1993). Bacteria lacking the umuC gene are nearly non-mutable by UV-light and the most genotoxic chemicals (Bagg et al., 1981, Perry and Walker, 1982). It is sure that MucAB as well as UmuDC proteins act in a recA- and lexA-dependent way as components of a multiprotein complex, which recently was designated as 'mutasome' (Rajagopalan et al., 1992). Due to the participation of umuC in the mutagenic process leading to both point- and frameshiftmutations, only one single bacterial strain is necessary to detect different kinds of mutagens. Nevertheless, additional strains have been developed in order to increase sensitivity for the detection of genotoxins belonging to certain classes, such as, e.g., nitroarenes (Oda et al., 1992, 1993, 1995). All umu strains carry the plasmid pSKl002 which bears an umuD gene and an umuC gene fused with lacZ, the structural gene for [3-galactosidase (Shinagawa et al., 1983). The induction of the mutator gene umuC by DNA-damaging agents is detected by measuring intracellular [3-galactosidase levels (Miller, 1972). Both, the Ames tester strains and the umu strains have been genetically modified. A reduction of the lipopolysaccharide structure of the cell wall (rfa) facilitates an increased permeability, especially for hydrophobic chemicals such as polyaromatic hydrocarbons. A further sensitivity enhancing alteration compared with the wild-type strains is a deficiency in a general pathway for the excision of damaged bases from the DNA: the nucleotide-excision repair. Incision near conformational distortions of the DNA, caused by a variety of bulky adducts, occurs via the formation of a multiprotein complex consisting of 3 proteins, collectively called UvrABC excinuclease. Consequently, the irreversible deletion in the uurB region leads to a loss of repair efficiency. The Ames tester strains TA98 and TAI00 carry the plasmid pKMl01, which bears the so-called 'mucAB genes'. Extensive studies have shown that

131

G. Reifferscheid, J. Heil / Mutation Research 369 (1996) 129-145 Table 1 Genotoxicity of chemical compounds tested in the umu test and comparison with the Ames test and carcinogenicity results

Genotoxicity I Mutagenicity Compound

CAS.Reg.No.

umu test R e f .

Acetaldehyde Acetamide Acetone Acetonitril 2-Acetylaminofluorene (+Sg) Acid Black 26, 50, 108, 110 and 156 (+as) Acid Black t07 iAcid Black 155 Acid Blue 41, 62, 83, 90, 92, 112, 113, 127, 129, 138, 140, 175 and t85 (¢.~9) Acid Brown 2 Acid Brown 13 Acid Brown 19 Acid Brown 294 Acid Green 26, 28, 40 and 41 (~:$9) Acid Orange 61, 59, 67 and 149 (~L-Sg) Acid Red 114 (+¢9) Acid Red 111,119, 129, 138, 143:1, 257, 266, 274, 276, 336 and 365 (±$9) Acid Violet 34, 48 and 97 (*$9) Acid Yellow 38, 48, 72, 110 and 142 (~:$9) Acridine Orange Acrinol (+S9) Acrylonitrile Actlnomycin D Adriamycin (Adr) Aflatoxin B1 Aflatoxin GI AH2e (dental material) Alaun (Potassium aluminium sulfate) Aldrin AI kylaminoethylglycice 9-Amlnoacrldine 2-Aminoanthracene (+ $9) o--Aminoazotoluene 2-Aminobiphenyl 4-AminobJphenyl 6-Amlnochrysene (+ $9) 2 -Am ino-3,4-dim ethyl-3H-im idazol (4,5-P/quinoline; MelQ (+ $9) 2-Am ino-3,6-dimethyl-imidazo 4,5-f)quinoliue;MelQx (+ SO) 3-Amino-l,4-dimethyl-6H-pyrido [3,4-b]indole; Trp-P-I (+ sg) 3-Amlno-l-methyl-SH-pyrido [4,3-b]indole; Trp-P-2 (+ $9) 2-Aminofluorene (+,9>9) 2-Amino-6-.methyldipyrido( 1,2-a:3 ",2"d)imidazole (Glu-P-1;+ sg)

75-07-0 60-35-5 67-64-1 75-05-8 53-96-3

Ames test Ref.

Caminogenicity EPA

91 40;91 40;91 91 40;49;91 26 25 25

-38 - 32;61 - 32;61 ;90 -38 + 32;61 n.d. n,d, n.d.

26 25 26 25 25 26 26 26

n.d.

n.d,

90-45-9 613-13-8 97-56-3 90-41-5 92-67-1 2642-98-0

26 26 26 + 40;58 + 62 o 40 - 45;91 + 40;45 + 40;91 + 68 + 20 91 - 91 62 ÷ 45;91 + 40;59 +" 49;85 - 40 + 68 + 49;68

77094-11-2

+

40;49

+72

÷

40;49

+72

62450-06-0

+

49;58

+ 61;72;73

P

62450-07-1 153-78-6

+ +

49;58 49;91

+ 61;72;73 + 30;32;34

P/LP

67730-11-4

+

40

+72

+ + +

+ + + -

6459-94-5

-

49¢-38-2 1837-57-6 107-13-1 50-76-0 25316-40-9 1162-65-8 1165-39-5 778424-9 309-00-2

P

n.d. n.d. n.d. n.d. n.d. n.d. +54

n.d. n.d, + 32 + 62;86 + 1;61 59;67 +32 +32 +32 + 66;71 n.d. 61 -62 + 32;61 + 32;33;34 +90 +32 +32 + 3;14 m

-

IARC NCI/NTP other summary (m/r) studies response

SP SP

SP

P P/LP

SP

+

+

LP

P P

67 32

+ + ÷

P P/LP

÷

LP

P

P

32

SP

+ t+

P

+

SP

SP

÷

SP SP

P

31

÷ ÷

SP

4-

G. Reifferscheid, J. Heil / Mutation Research 369 (1996) 129-145

132 Table 1 (continued)

Genotoxicity I Mutagenicity Compound

! CAS.Reg.No.

umu test Ref.

2-Amino-dipyrido( 1,2 -a:3',2'-d) imidazole-2-amine (Glu-P-2; + S9) 2-Am ino~3-methyl-3H-im idazo(4,5-rFluinoline; IQ (+ sg) 2-Am ino-l-m ethyl-8-phenylim idazo [4,5-b]pyridine (PhlP) 2 -Am ino<3-m ethyl-gH-py rido (2,3-b)indole; MeA alpha C (+ sg) 2-Am ino-sH-pyrido(2,3-b)indole (+S9) {A alpha C) Aminotriazole ~mmonium heptamolybdate Amphotericin B Amsacrine A n i l i n e " HCI o-A,'lisidine Anthracene (+ as) Aphidicolin Arsenic trioxide Atrazine Atropine sulfate Auramine Avarol (+ SS) Azaserine Azlnphos-Ethyl A z i n ~ y l (+ SS) Beriumacetate (Be 2 + ) " 1 H20 Benzalkoniumdlloride Benz(a)anthracene ( * aS) Benzene Benzidine (+ $9) Benzo(a)pyrene (+ SS) Benzo(e)pyl~le (+ S.9) Benzoic acid Benzoin Benzylchloride bia-Benzimid Bitertanol Bleomycin Bromo acetic acid Bromobenzene 5-Bromo-2"-deoxyuridine 1-Bromo pentane 1-Buty alcoho 2-Butylalcohol Butylated hydroxianisole (BHA) Butylated hydroxytoluene (BHT)

I

Ames

test

Carcinogenicity EPA

IARC

Ref.

NCI/NTP (m/r)

i

other I summary studies i response

67730-10-3

+

68

+72

SP

+

76180-96-6

+

40;49;58

+72

SP

+

105650-23-5

+

49

+84

SP

+

68006-83-7

+

40

+72

SP

+

26148-68-5 61-82-5 12027-87-7 1397-89-3 51264-1 4-3 142-O4-1 90-O4-O 120-12-7 38966-21-1 1327-53-3 1912-24-9 55-48-1 2465-27-2 5,5303-98-5 115-02-8 2642-71-9 86-50-O 103-33-3

+

+72 P

SP SP

+ +

LP

LP

I

I

-

40 40 41 58 91 40;91 49 91 91

-

91

-

91

543-80-6 8001-54-5 56-55-3 71-43-2 92-87-5 50-32-8 192-97-2 65-85-0 119-53-9 100-44-7 23491-453 55179-31-2 11056-06-7 79-08-3 108-86-1 59-14-3 110-53-2 71-36-3 78-92-2 25013-16-5 128-37-0

-

+ + -

40 +,- 91;40 + 58 ÷ 91 91 + 91 + 40

91 63 + 40 40;91 +" 40;49 + 40;91 91 - 40 - 40 + 58 - 91 - 91 + 40;91 + 91 -;+ 40;58 - 40 + 91 - 91 - 91 - 91 - 91

- 32

n.d. -90 +" 15 - 32;64 + ; + " 61;90 - 32;61 n.d. n.d. - 36 - 32;61 + ' , - , - 32;61;90 n.d. + 32 n.d. ±;+ 61 ; 8 8 + 32;61 n.d. -62 + 32 - 1;10 + 32;90 + 32;33;34;61 + 12;32;61;90 - 32;61 - 1 + 32;33;61;88 n.d. n,d, +* 28 n.d. - 32;61 n.d. n.d. - 32;61 n.d. - 90 - 38

LP LP

P

+ + ÷

LP P

SP

P

LP

SP SP SP P

SP SP SP SP LP

N/LP P/LP LP

+ EE(r)/N(m) ~P

÷

÷ + ÷

p P

32!

+ 4.

N

+

P

LP

67

+

I

P/LP SP LP

LP N

+ ÷

G. Reifferscheid, J. Heil / Mutation Research 369 (1996) 129-145

133

Table 1 (continued)

~motoxicity / Motagenicity Compound

urnu test Ref.

CAS.Reg.No.

t-Butylhydroperoxide t -Butylrnethylether di-n-Butylsulfoxide l~-Butyrolactone

75-91-2 1634.04-4 2168-93-6 365.36-46-6

+ • +

91 91 40 40

;Cadmium acetate * 2 H 2 0 Cadmium c h l o r i d e " 6/2 H 2 0

5743-O4-4 7790-78-5 10106.,64-2 10325-94-7

-

91 91 91 41 40

+

91 58

-

91 40 91;40 40

-

Cadmium chloride Cadmium nitrate Caffein Caprolactam Captan epsilon-Caprolactam epsllon-Caprotactone Carbon tetrachlorlde Catech~ Cesium chloride p-Chlo~lide Chloramine T Chloramphenicol 4-Chloroaninne 1 -C hloro-2,4-dinitrobenzene Chloroform Chlo~lnedigluconate l-Chloro-4-~ 8-Chkxo-2-n~uene 6-¢hloro.9-(3-(2-chloroethyllmlrlno)l~Opyl ) amino-2 -methoxyacridine"2HCI 0CR-191) p-Ohiorophenol Chromiurn(VI)oxide Ch~ysene (÷ SS) Ciprofloxacin cLs.PtatJnum Citdnin Clotrimazo( Coba~(n)chkx~e copp~(.)sun~e"

s N2o

m~resoi Cumenehy~opefoxlde Cupferron Cydchexan Danthron Daunomycin Daunorubian 1,2-Diaminob~n,~m~ (+ S9) 2,4-Diamtnotoluene (÷ S) 2,6-Diandnotoluene (+ $ 9 )

58-08-2 105-60-2 133,06-2 105-60-2 502-44-3 56-23-5 126-80-9 7647-17-8

97-00-7 67.,66-3 16472-51-0 100-(X).-5

+ -

41 63 91 4O 91 ;63 58;91 40;91 63

-

91

5367-28-2

-

91

17070-45-0 106-48-9

+ + + + +

34;40;45 63 41 40 87;91 58

+ +

91 91 41;91 91 91 91 91 91

56-77°7 127-65-1 56-75-7 t 06-47-8

1333-82-0 218-01-9 85721o33-1 15663-27-1 518-75-2 23~75-1 7646-79-9 7758,9e-8 108-39-4 80-15-9 135-20-6 110-82-7 11~I~2 23541-50-6 20~30-81~ 95-54-5 95-80-7 823-4O-5

+

+ + +

91 34 40 63 + 40 + 40 +o 49

]

Ames test Ref.

Carcinogenicity EPA

IARC

P

SP SP

NCI/NTP (m/r)

other studies

summary response

+" 2 8 n.d. n.d. + 32;90 n.d, n.d. - 10;38 n.d. - 32;61 -75 + 32;33;61 ;90 -75 - 1;32;61 - 32;61 + 61

+ +

N/L N P/LP

N LP

N

N

N

N

P

SP I

LP

+

n.d. -64 n.d. -32 -64 + 61 -78 -62

LP I

P

SP

SP

+

+ 19 n.d. + 30;32;34;61 +64 + 61 +32 +" 67 + 61 +/n.d. ÷ n.d. n.d. +" + -

61

SP P/U :) LP

+

+

P/LP SP LP

LP

+ +

SP

LP SP

+

61

83 13;90 32;61

+" 83 +34 +32 -63 +24 +32 + 61

SP

+

P

SP

SP/N

+

P

SP

SP

+

134

G. Reifferscheid, J. Heil / Mutation Research 369 (1996) 129-145

Table 1 (continued)

Genotoxicity I Mutagenicity Compound

o-Oianisidine 1,2:3,4-Dibenzoanthracene Oibromoacetonitrile !Dk:hlofluanid t,l-Dichloroacetone 1,2-Dichlorobenzene 1,4..Dtchlorobenzene 2,6 -Ok:hlorobenzonitrile Dichlorotriphenyltrlchloroethane (DOT, J:Sg) Dichromium dipotaszium tetrasulfate Dieldrin 1.2:3.443iepoxybutane Di(2-ethylhexyl)phthalate Oiethylnitrosamine (÷ $9) Diethylst,besterol Diethylsulfate (+ Sg) 2".343i~yt-4-aminoazobenzene 7.12-Oimethylbenzanthracene (÷ $9) 2.4.Obnethyl-t ,3-dloxane Dimethylf~rmamide 1,2-Dimethylhydrazine

DimethyJsulfate [Nmethylsu~oxk:te Di-n..but3d sutfoxide m-Dinib'olc~tr~ ,4,4'..D~#robipheny! 3,7-Din~rofluora~ 3.9 -Dinibofluoranthene 2.7 -DinRn)-9-fluarenone 1.5-Dinit~naphthalene 2.4.Din#Tophenol 1,3-Dinitropyrene t ,S-Ok,imDpyTene t ,8.Oinitropyrene 2,4.Dinitl'otoluene 1,4-Oioxane 1,3-Dioxane 1,3-Dioxc4ane Diphenylsulfoxide Direc.t Black 17 13tm~ Black 112, 117 and 132 (:1:S9) 134re(:t Blue 90, t08, 168, 200 - 203, 237 and 248 (~S0) Direct Brown t96, 2 t 0 and 223 (:11:$8) Direct Green S$ and 80 (:I~SS) D~rect Orange 3S (~SS) D~rect Red 2 (:kSg)

CAS.Reg.No.

umutest R~.

119-90-4 215-58-7 3252-43-5 1085-96-9 513-88-2 95-50-1 106-46-7 1194-65-6 50-29-3

4O 69 91 58 91 40;91 91 91 4O;45 41 40;91 + 91 - 40 + 40;45;91 - 40;91 + 40 - 40 + 40;91 + 4O - 40 -,÷ 40;58 + 40 ÷ 40;45 - 42 + 40;42;58 - 42 + 47;48 + 47 + 47;48 + 47;48 + 91 + 91 - 40 + 40;,47 + 4~,47 + 40;47 ÷ 47;48 91 + 40 - 40 - 40 26 26

60-57-1 296-18-0 117-81-7 55-18-5 56-53-1 64-67-5 97-56-3 57-97-6 76~S-20-1 68-12-2 54O-73-8 62-75-9 77-78-1 75-18-3 67-68-5 2188-93-6 99-65-0 1528-74-1 105735-7%5 22506-53-2 31551-45-8 605-71-0 51-28-5 75321-20-9 42397-64-8 423G7-85-9 121-14-2 123-91-1 505-22-6 646-O6-0 945-51-7 2945-96-2

+ ÷ + + +

26 26 26 26 26

I

Ames~ R~.

+ 61 + 61 +38 + 61 + 61 - 19 - 75 n.d. - 10;59;90 n.d. - 32;61 ÷32 -1 +32 - 1.61 + 32;33;61 ÷;- 32;61 + 32;33;34;61 n.d. - 24 -;+;- 32:52;61 + 32;61 + 59;61 n.d. - 32;61;9O n.d. + 61 +77 +39 +39 +89 +77 - 24;9O +60 +60 + 6;60 +55 -75 + 61 n°d. n.d. + 61 rLd. n.d. n.d. n.d. n.d. + ~ 54

Carcinogenicity EPA

IARC

NCI/NTP (m/r)

P LP

SP LP IE

LP

I I

N LP

SP

LP N

LP SP SP SP SP SP SP

SPIN

P

P P P P P P

P

I.

P

LP SP SP SP

SP LP SP LP LP

LP LP

+ +

P

59

+

P

32

P

32

+ + + + + + + +

5~

÷ + + +

P

LP LP

÷ +

LP SP SP

+ + + + +

LP P

other summary studies response

SP

G. Reifferscheid, J. Heil /Mutation Research 369 (1996) 129-145

135

Table 1 (continued)

Carcinogenicity

Genotoxicity I Mutagenlcity umutest

cAS.Reg.No.

Compound

Ref.

Direct Red 31, 79, 83:1, 89, 224 - 226 and 243 (~$s) Direct Violet 6g (:1~9) Direct Yellow 12, 39, 87 and 130 (:I:Sg) Disperse Blue 60, 87, 145, and 158 (:1:$9) Disperse Brown t (~:Sg) Disperse Orange t3 and 73 (:1~9) Disperse Red 73 Disperse Red 143, 162, 153 and t64 (:1:$9) Disperse Red 145 Disperse Red 157 Disperse Violet 36 and 52 C1:$9) Disperse Yellow 7, 42, t04 and 164 (2S9) Disuffiram (Teb'aethylthiuramdisulfide) I~;thianone Doxorubicinhydroch Iorlde Dulcine (4-Emoxyphlmylurea)

-

26

26 26 26 26 26 26 26 26 26 26

-

+ -

+ + -

97-77-8 3347-22-6 25316-40-9 150-69-6

26

-

+ + -

91 58 45 40

I

Ames test Ref.

n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. + 61 n.d. + 61 n.d.

519-23-3 74011-58-8 106-89-8 106-88-7 96-09-3 1239-45-8 67-21-0 64-17-5 108-93-4 62-500 4245-77-6 75~73-9

+ + + + + +

91 87 40 40 91 40 91 40 + 40;50;91 + 40;91 + 40 + 91

+ 61 +* 87 +82 +32 + 61 + 32;61 - 32;90 - 32;61;90 +;+ 90;61 + 32;33 +32 +90

:gran :uraz.olldone Furfural Furylfuramide (AF-2)

206-44-0 86-73-7 51-21-8 31251-03-3 133-07-3 50-OO-O 110-00-9 67-45-8 96-01-1 3688-53-7

- 40 - 40 + 40;45 - 91 + 91 + 45;91 - 91 + 51 - 40 + 40;45;47

+ ~ (+ sg) - 32;61 + 61 n.d. + 32;61 +° 28 + 61 +44 ÷;+;-;- 24;61;36;75 ÷ 32;33;61

Geosmin Giutaraldehyde Glutaric acid Glyoxal

19700-21-1 111-30-8 110-94-1 4405-13-4

EIIIpticine =noxacin Epichlorhydrtn 1,2-Epoxybutan Epoxystyrene Ethidium bromide (+ $9) d,l-Ethionin SUtylalcohol Ethyte¢~ dlbromide

Ethyt m ~ h a n e ~ a t e

(EMS)

N.EthyI-N'-nitro-N-nitro,~guanidine N.EthyI-N-nitro~uma Fluoranthene Fluonme 5-Fluorouracil Fluotrlmazole Fo~t e ~

+ +

40 47;62 63 91

-4O +* 83 -64 +" 80

EPA IARC

NCI/NTP (m/r)

summary other studies response

N

N

N

61

P

32

P

32

P P

18 16

P

32

P

P IN/L P P P P

I

SP LP SP

LP N(m)/SP(r)

I SP SP

SP LP

SP

lip

P

SP

LP SP

P

SP

SP/LP

G. Reifferscheid, .1. Heil / Mutation Research 369 (1996) 129-145

136 Table 1 (continued)

Genotoxicity I Mutagenicity Compound

Hanllane

Hexabenzobenzene Hexachlorobenzene Hexachlorocyclohexane Hexachloroethane Hexamathylphophorictriam ida Hydrazine sulfate (+ $9) Hydrogen peroxide 8-Hydroxyquinollne Hydroquinone

CAS.Reg.No.

486-84-O

Hyclmxy urea Hydroxylamine

191-07-1 118-74-1 58-89-9 67o72-1 680-31-9 10034-93-2 7722-84-1 148-24-3 123-31-9 127-07-1 78O3-49-8

Indium chloride Iron(Ill)sulfate

1002582-8 10025-22-5

5-Jode-2"-deoxyuridine

umu test Ref.

+ -

46;91 40 91 91 40 40

+ + + -

40 40;91 91 40 91 40

-

41 41

54-42-2

40

Lead acetate Lead nitrate Litium chloride

301-04-2 10099-74-8 7447-41-8

-

Magnesium acetate Magnesium chloride Magnesium perchlorate Magnesium sulfate Manganese acetate Manganese(il)chloride Manganese nitrate

142-72-3 7786-30-3 10034-81-8 7487-88-9 638-38-O 7773-01-5 16377-66-9 69-65-8 106-78-1 637-89-8 91-80-5 3544-23-8 72-43-5 484-2O-8 298-81-7 74-83-9 74-95-3 56-49-5 1120-.97-4 1331-09-5 74-95-3 75-09-2

÷ +;+°

D-l~mni~l Melamine Methapydlene (+ $9) 3-Methoxy-4-aminoazobenzene (+$9) Methoxychlor ¢ M O P (÷ UV) g-MOP (+ I.N) Methylbromide Methylenebromide !3 - M e t h y l d ~ a m h n m e (+ SS) ¢-Methyl-1,3.dioxane 2-Methyl-l,3-dioxolane Methylene bromide Methylenechloride 2-Methylisobomeol Methyl methanesulfonata (MMS) N-MethyI-N-nitro-N-nitrosoguanidine N4Aet~l-N-nit rosourea Methyll~lsulfoxida Methylroeanillnechlorlde

66-27-3 70-25-7 684-93-5 1193-82-4 548-62-9

Ames test

- 57 + 19 -32

+ -

40 40;42 63

1 SP LP

other summary studies response

P/L P P N

LP SP SP LP I I

I

LP N SP/N

+ ± + ÷

LP N SE N(myEE(r)

P N

21 61

+ +

I

n.d. - 32;61

n.d.

91 40 40 40;58 40,58

P P/L

NCI/NTP (m/r)

n.d.

41 41;91 41 41 41 41 41 91 91 40 91 49;85

+ + +

IARC

¢ 61 + 61 n.d, n.d. - 61 -1 + 32 +*;+ 28;61 + 55;61

n.d.

91 91 91 50 91 34;40;45 40 40

EPA

Ref.

41 41 41

+ + + + + + -

Carcinogenicity

P

n.d. n.d.

I I

n.d, n.d. n.d. n.d. + 61 n,d. -75

N

- 75

-40 n.d. + 61 -38 -d56 +* 28 + 37 + 61 + 32;34;61 n.d. n.d. + 61 + 23;61 -40 + 30;32;61 + 32;33 +32 n.d. ±;-;+ 61;62;76

LP N

N I

N(m)/SP(r)

I SP

N

LP

÷

P N(m)

P

;P P P

+ + P

SP

SP

SP SP SP

LP LP LP

32

+

+ P P P

32 32 32

+ + +

G. Reifferscheid, J. Heil / Mutation Research 369 (1996) 129-145

137

]7able 1 (continued)

Genotoxicity I Mutagenicity Compound

CAS.Reg.No.

Methylsulfide Metronldazol Miconazole Mitomycin C Monochloroacetic acid

75-18-3 443-48-1 22916-47-8 50-07-7 79-11-8

NaUdixic acid (sodium-salt) Nalidixic acid (free acid) Naphthalene 1-Naphthylamlne Nickel(ll)chloride * 6 1420 Nicotine Nifuroxazide Nitric oxide i5-Nitroacenephthene i o-Nitroani=ol 2-Nitrobiphenyl $-Nltrochrysene 3-Nitrofluoranthene 2-Nitrofiuorene 5-Nitro-2-furaldehyde Nitrofurantoin Nitrofurazone 5-Nit to-2 4urylacrylacid Nitrogen dioxide 1-Nltronaphthalene 2-Nitronaphthalene 2-Nitrophenol 4-Nltrophenol 4-Nitro-o-phenylenediam ine 2-Nitro-~o-phenylenediam ine 1 -Nitropyrene 4-Nitroquinoline-N-oxide N-Nitrosobutylurea N-Nitrosocimetidine 4-Nit ro=m-N,N-dimethylaniline 4-Nitrolodipherlylamine 3-Nitrotoluene N-Nitrosodiphenylamine N-Nitrosodiethanolamine N-Nitroso-N4)utyI-N-propylamine Norhannane Nystatin

5374-O5-8 389-O8-2 91-20-3 134-32-7 7791 *2O-0 54-11-5 965-52-8 10102-43-9 602-87-9 91-23-6 86-O0-0 7496-02-8 892-21-7 607-57-8 698-63-5 67-20-9 59-87-0 710-25-8 10102-44-0 86-57-7 581-89-5 88-75-5 100-02-7 99-56-9 5307-14-2 5522-43-O 56-57-5 869-01-2 73785-40-7 138-89-6 156-10-5 99-08-1 86-3O-6 1116-54-7 25413-64-3 244-63-3 1400-61-9

l-Octanol Ofloxacin

111-87-5 82419-36-1

Paraquat Pararo=anilineJHCl Pentachloronitrobenzene

4685-1~7 569-81~ ~-88-8

umu test Ref.

+ +

+ + -

+* -

+ -

+ + + + + + + + + ÷ + +

+ + ÷ + + + + + -

+ + + + +

+

Ames test Ref.

40 91 91 40;58 40

n.d.

91 58 40 4,g 41 40 91 27 40;47 91 4O 68 47;48 40;47 51 51 47;51 51 27;40 40;47;48;91 40;47 91 40 34;45;91 91 40;47 40;58 58 40;45 58 91 91 91 91 91 46 91

n.d. +* 87 - 32;61 + 32;61 - 10 - 32;61 + 61 +22 + 32;61 +1- 61 -35 + 14

91 87

+

40;45

-

91

-

91

+8

n.d. +* 28;30 - 32;61

Carcinogenicity EPA

NCI/NTP (m/r)

P

÷ SP

N/L

I LP

P

SP

N/SP N

P

32

+

EE(m)/SP(r) SE(m)

+ +

P

+

÷

SP I

÷

SP LP P/LP LP

N

I I

N

I

SP/LP SP

I

43

+

LP

43

+

÷ ÷

LP SP P P

P

I

SP

LP P

LP SP

SPr/Nm LP SP

n.d.

+* 87 +70 - 10;Sl +1- 61

other summary studies response

P

+6

+ 32 + 61 + 61;44 + 44;88 + 61 +22 +77 +32 13.4. - 32;61 + 30;32;34 +89 + 6;60 +32 +29 +53 + 61 + 61 n.d. + 61 +7 + 61 n.d. n.d.

IARC

P/LP I N/L LP

SP/LP N

P

32

+ +

P

32

+ + +

G. Reifferscheid, J. Heft/Mutation Research 369 (1996) 129-145

138 Table 1 (continued)

Carclnog*nlclty

Genotoxlclty I Mutageni¢ity Compound

Pentachlorophen~ Pe~ylene Pllulnic acid p 69) Phermnthrlne Phenobarbital Phenol pPh*nyl~xllamln* Ph~y*hydnmne Phthlllic mnhydrk~ Polychlorlnmsd blphenyts (KC-6~) Potassium chloride Po~sadum chromate Potassium ¢yankle Pota~dum dk:hromate (Cr207 2-) Po/asslum permangmnm Proca~ln4-HCl PTo~lnokl Prol~mol-2 Propeme sulto¢~ s-Prop~acto~ PTopyten* oxide (÷ ss) Pyrogaik~ Pyrocamchoi Quercetln (+ St)

R e , , , ~ mad~ S (=St) Rtmctlve ~ 16, 16, 21, 4.% 100, 10t, 147 and 148 (:ldSl) R*w:tlve lk'own 21 (:kSS) Reactive Orange 6, 7, 15, r~l, 67 and 72 (:~SS) ReacU~ Rm:l 22 - 24, 43, 4 8 , 1 0 L 100, 111 - 114 (:1:$8) iRm~tlve Red 110 Rm~b~l Vlokd 4 and 22 (~69) teac:Ib'~mYMIow 13 Reactive Yellow 76 Reacthm Yellow 14, 76, 77, 79, 115 and 11(; (:1~,9) Res*q~n talorcln Saccharin

s a , ~ y ~ Selene dioxide Sodium Sodium chkxlde sodium nilxl~ (1402 -)

CAS.Re.No.

87-86-5 198-55-0 88-89-1 85-01-8 5O4)6-6 108-95-2 106-503 100-83-0 85-44-9 37317-41-2 7447-40-7 11073-34-0 151-50-8 7778-50-9 7722-64-7 369-70-1 7%23-8 67-63-0 1120-71-4 57-57-8 75-56-9 129-00-0 87-66-1 120-80-9 117-39-5

umu tm;t Ref.

-

91

-

91 91 40

+

40

-

+

-

+ + -

-

41

+

41 91 40,'91 41

-

+ -

40

-

91

-

÷

40;45

-

26

-

26

-

26

-

26

n.d. ll.d. n.d.

-

26 26 26 26 26 26

n.d. n.d. n.d. n.d. ll.d, n.d.

-

91

-

40

+ -

+

-

+

÷

81-07-2 94-59-7 90-02-8 7446-08-4 26629-22-8 7647-14-5 7632-00-0

- 61 + 61 + 17 - 32,61 1 -64 + 55 +* 52 n,d. - 59 + 61 + 61 n.d. + 59;81;90 +11 - 32 n.d. n.d. + 32;61 + 32;61 + 5;61 - ; - ; - ; + ~ 12;32;61,90 + 4,'64 - 64

91 40 40;40 50 91 62,'63 63

-

+ +

+

5O-55-5 108-46-3

63 91 91 91 40;45

Ames test Ref.

-

-

+ •

÷

91 40;45 40 41 40;45;51 91 40,'58

the - in an evolutionary sense - former 'chromosomal' mucAB genes share a large homology to the plasmid-derived genes umuDC, both in structure and function (Walker, 1984). The two operons con-

EPA

IARC

St:)

NCI/NTP (m/r)

other I summary studies :response

SP

I

I

SP N I

N SP/LP

SP

I

P

SP

P P P

SP SP SP

SP

P

59

+

P

59

+

LP SP

+

I

LP LP SP(m)/LP(r)

+

P

32

I

I

+ 24;74,'90

LP

LP(r)

+

I

ll.d.

75 - 32,61 -

+,- 2;10 - 1,~1 ll.d. n.d. + 30;32;88 - 10 + 32,61 ;90

+ +

P

P

LP

LP SP

I

N

SP SP

LP

+

+ +

N

32

P

321

+

fer enhanced capacities for mutagenesis and survival on their host cells (Perry and Walker, 1982). In both the sensitive Ames tester strains as well as in the umu test, DNA-damaging, replication-inhibit-

G. Reifferscheid, J. Heil / Mutation Research 369 (1996) 129-145

ing agents induce the so-called 'SOS' response. Most genotoxic and mutagenic events measured by these strains derive from lesions which are processed via the error-prone pathway.

139

typhimurium TA1535/pSK1002 and NM2009 as indicated) was carried out by our laboratory or those cited, using either the tube-method described by Oda et al. (1985), with modifications by Whong et al. (1986), or the microplate version developed by Reifferscheid et al. (1991). All compounds investigated in the umu test were assayed without and, if necessary, with metabolic activation by $9 preparation from Aroclor 1254-induced male rats. Genotoxicity data of 381 of the chemicals have been published, detailed data for the remaining 105 (Salmonella

3. Data evaluation

All compounds investigated are listed alphabetically in Table 1. The Ames test data were taken from the international literature as indicated. The umu test Table 1 (continued)

Genotoxicity I Mutagenicity Compound

Sodium w o l f r a m a t e Sterigmatocystin

Streptonigrin Streptozotocin Styrene o x i d e Succinic a n h y d r i d e Tetrahydrofuran Thioacetamide Thiourea o-To,dine o-Toluidine Toluene

Trichloroacetorm 1,2,4-Trichlorobenzene t,1,1 -Trichloroethane Trichloroethylene Trichioronit rom ethane 2,4,6-Trichlorphenol 2,4,7-Trinitro-9-fluomn Tris(2,3-dibromopropyl)phosphate Urethane

CAS.Reg.No.

13472-45-2 10048-13-2 3930-19-6 1_A.~ ' u~_ ?.-66-4 96-09-3 108-30-5 I O9-99-9 62-55-5 62-56-6 119-93-7 95-53-4 108-88-3 918-09-3 120-82-1 71-55-6 79-O1-6 76-O6-2 88-06-2 34263-36-O 126-72-7 51-79-6

Vitrebond

Xylene

1330-20-7

Zearalenon Zinc a c e t a t e Zirconium chloride o x i d e Zirconium n i t r a t e

17924-92-4 557-34-6 13520-92-8 1374689-9

umu test

I I

Ref.

I

+ + + +

-

41 68 91 40;91 40 40 91 40;45;9O 40;91

+e

49

-

40

-

40 91 91

-

40

-

+

40 91 91 34

+

68

-

40

+

20;91

+

+ -

40 -

91

-

41

-

41

-

41

Ames test

Carcinogenicity EPA

IARC

P

SP

Ref.

n,d. +,+ + + + -

61;79 26 32;61 32;61;90 32;61 ;88

SP P/LP SP P/LP LP P

n.d. - 12;32;61;90 P - 32;61 P + 24;61 P - ; + ; + ~ 32;61;90 P -9 + 61 n.d. - 19 -40 P/LP + 37 - 61 P +34 + 61

SP SP SP I I

NCI/NTP other summary (m/r) studies response

+ LP SP

P

32

+ +

N

LP LP LP LP N

+

P

32

+ + + +

I

I

LP

+

SP

SP I N(m) LP SP

SP

SP

+

SP

LP

+

-9

I

N

+ 61

LP

SP(m)/N(r)

- 32;61

P

n.d.

n.d. n.d. n.d.

+

140

G. Re~fferscheid, J. Heil / Mutation Research 369 (1996) 129-145

g e n o t o x i n s a n d n o n - g e n o t o x i n s o n l y i n v e s t i g a t e d in o u r l a b o r a t o r y will b e p u b l i s h e d . A c o m p o u n d is classified as ' g e n o t o x i c ' or ' m u t a g e n i c ' if a t w o - f o l d i n c r e a s e o f m u t a t i o n f r e q u e n c y or a t w o - f o l d induction o f u m u o p e r o n a b o v e the b a c k g r o u n d level is reached. T h e e v a l u a t i o n o f a s u b s t a n c e ' s c a r c i n o g e n i c i t y w a s t a k e n f r o m : (1) the G e n e - T o x c a r c i n o g e n d a t a b a s e ; (2) the I A R C c a n c e r r e v i e w s ; (3) the N C I or N T P c a r c i n o g e n e s i s studies a n d / o r additional sources.

4. R e s u l t s a n d d i s c u s s i o n 4.1.

Comparison

between

umu

test a n d A m e s

test

data

M o s t o f the 4 8 6 c h e m i c a l s tested in the u m u test g a v e c o n s i s t e n t results w h e n i n v e s t i g a t e d in m o r e t h a n o n e l a b o r a t o r y ( T a b l e 1). In o u r o w n l a b o r a t o r y , 164 c o m p o u n d s were tested. A c o m p a r i s o n b e t w e e n u m u a n d A m e s test results was m a d e with t h o s e 2 7 4 c o m p o u n d s w h i c h were e x a m i n e d in b o t h test systems. A s s h o w n in T a b l e 2, the c o n c o r d a n c e a m o n g the t w o bacterial test s y s t e m s is 9 0 % , w i t h r e s p e c t to u n e q u i v o c a l test results. E i g h t y - s i x % o f the c h e m i cals w h i c h are f o u n d m u t a g e n i c in the A m e s test are

Table 2 Comparison of umu test and Salmonella~mammalian mutagenicity test using experimental results of 274 chemicals Ames test

umu test

+

-

+

Total 173 88 13 274

+

149

24

0

-

3

84

1

2 154

9 117

2 3

+ Total

f o u n d g e n o t o x i c in the u m u test, too. T h e A m e s test ( u s i n g at least 5 strains) was able to detect 9 7 % o f the u m u p o s i t i v e c o m p o u n d s . F o r 212 c o m p o u n d s tested in the u m u test, n o A m e s test data w e r e available. T w e n t y - f o u r c h e m i c a l s ( a c i d red 114, a c r y l o n i trile, 2 - a m i n o b i p h e n y l , o - a n i s i d i n e , b e n z o ( e ) p y r e n e , catechol, 1 - c h l o r o - 4 - n i t r o b e n z e n e , p - c h l o r o p h e n o l , c o b a l t ( I I ) c h l o r i d e , o - d i a n i s i d i n e , direct b l a c k 17, direct red 2, d i s u l f i r a m , f l u o r a n t h e n e , furan, h e x a b e n z o b e n z e n e , 8 - h y d r o x y q u i n o l i n e , m a n g a n e s e ( I I ) chloride, m e t h y l e n e c h l o r i d e , nitric oxide, p e r y l e n e , p o t a s s i u m chloride, p o t a s s i u m p e r m a n g a n a t e a n d s o d i u m azide) s h o w e d m u t a g e n i c i t y in the A m e s test, b u t n o r e s p o n s e in the u m u test. O n l y 6 o f t h e m are

Notes to Table 1: Genotoxicity and mutagenicity testing: +, - , +__,positive, negative, equivocal test result; *, strain TA102 (Ames test); * *, preincubation; • * *, hamster liver $9; * * * *, 30% $9 per assay; °, Strain NM2009 (umu-test); d. in the dark: n.d., not determined/no data available; ~', trade mark. Carcinogenicity studies: SP, P, positive; LP, limited positive; SE, some evidence; N/L, limited negative; N, negative; I, inadequate study or inconclusive test result; (m/r), mouse/rat; CAS Reg. No., Chemical Abstract Service Registry Number: EPA, Environmental Protection Agency; IARC, International Agency on Research of Cancer; NCI/NTP, National Cancer Institute/National Toxicology Program; summary response: +, - , +, positive, negative, equivocal. References: 1, Ashby et al., 1985; 2, Batzinger et al., 1977; 3, Bechtold et al., 1985; 4, Ben-Gurion, 1979; 5, Bootman et al., 1979; 6, Consolo et al., 1989: 7, Dahl, 1985; 8, Dayan et al., 1982; 9, Dean, 1978; 10, De Flora et al., 1984; 11, De M6o et al., 1991; 12, Dunkel et al.. 1984; 13, Dunkel et al., 1985; 14, E1-Bayoumy and Hecht, 1984; 15, Ferguson et al., 1988; 16, Fishbein et al., 1970; 17, Gocke et al., 1981; 18, Gold et al., 1990; 19, Haworth et al., 1983; 20, Hell et al., 1996; 21, Hirota and Yokoyama, 1981; 22, Isomura et al., 1984; 23, Jonger et al., 1978; 24, Kada and Ishidata, 1980; 25, Kosaka and Nakamura, 1988; 26, Kosaka and Nakamura, 1990; 27. Kosaka et al., 1985; 28. Levin et al., 1982; 29, Lijinsky et al., 1987; 30, Maron and Ames, 1983; 31, Matsukura et al., 1981; 32, McCann et al., 1975a; 33, McCann et al., 1975b; 34, McDaniels et al., 1990; 35, McMahon et al., 1979; 36, Mersch-Sundermann et al., 1988; 37, Moriya et al., 1983; 38, Mortelmans et al., 1986; 39, Nakagawa et al., 1987; 40, Nakamura et al., 1987: 41, Nakamura and Kosaka, 1989; 42, Nakamura et al., 1990; 43, Nesnow et al., 1987; 44, Ni et al., 1987; 45, Oda et al., 1985; 46, Oda et al.. 1988; 47, Oda et al., 1992; 48. Oda et al., 1993; 49, Oda et al., 1995; 50, Ong et al., 1987; 51, Pal et al., 1992; 52, Parodi et al., 1981; 53, Pool et al., 1979; 54, Prival et al., 1984; 55, Prival and Dunkel, 1989; 56, Quinto et al., 1984, 1995; 57, Rapson et al., 1980; 58, Reifferscheid et al., 1991; 59, Rinkus and Legator, 1979; 60, Rosenkranz et al., 1980; 61, RTECS, 1994; 62, Sakagami et al., 1988a; 63, Sakagami et al., 1988b 64, Sakagami et al., 1986: 65, Sakai et al., 1985; 66, Schweikl et al., 1994; 67, Seino et al., 1978; 68, Shimada et al., 1989; 69, Shimada and Nakamura, 1987; 70, Shirasu et al., 1981; 71, Stea et al., 1994; 72. Sugimura, 1985; 73, Sugimura et al., 1977a; 74, Sugimura et al.. 1977b; 75, Tennant et al., 1987; 76, Thomas and MacPhee, 1984: 77, Tokiwa et al., 1981; 78, Uehleke et al., 1977; 79, Ueno et al., 1978; 80, Ueno et al., 1991; 81, Venier et al., 1982; 82, Venitt and Crofton-Sleigh, 1981; 83, Wilcox et al., 1990: 84, Wild et al., 1991: 85, Yamazaki et al., 1992; 86, Yasunaka and Okada, 1985; 87, Ysern et al., 1990; 88, Zeiger et al., 1987; 89, Zeiger et al.. 1988; 90, Zeiger et al., 1992; 91, present study. xxx

G. Reifferscheid. J. Heil / Mutation Research 369 (1996) 129-145

known carcinogens, whereas benzo(e)pyrene, catechol, cobalt(II)chloride, disulfiram, fluoranthene, hexabenzobenzene, 8-hydroxyquinoline, perylene and sodium azide gave negative or inadequate results in carcinogenesis studies. The carcinogenicity of the remaining compounds has not yet been evaluated. On the other hand, 5 compounds (amphotericin B, bromobenzene, dimethylsulfoxide, harmane and onitroanisole) are scored genotoxic in the umu test, but were either not mutagenic in the Ames tester strains or gave inconclusive test results. In certain cases, special treatment of compounds is necessary to obtain a mutagenic response in the Ames test, such as, for example, using other preparations than rat liver $9 (activation of direct red 2 with hamster liver $9) or prolonged incubation time including preincubation steps (o-anisidine) or higher amounts of $9 (pyrene, o-toluidine: 30% $9). Neither of these test modifications have been used in the umu test yet to increase the rate of positive results. However, high amounts of $9 in the umu assay lead to considerable cytotoxicity (unpublished observation), which must be presumed for the Ames test as well. In a reversion assay, such as the Ames test, the target for mutagenicity is usually limited to a small number of already mutated sites in the DNA which provide the basis for the mutagenic specificity. To avoid an underestimation of the mutagenic potency of a compound, or even a loss of detection, a set of histidine-requiring strains must be used for mutagenicity testing, with each tester strain containing a different type of mutation in the histidine operon. A number of chemicals that have been tested in the umu test (e.g., cumene hydroperoxide, hydrogen peroxide, streptonigrin, bleomycin, t-butyl hydroperoxide, phenylhydrazine), which are known to generate oxygen radicals and previously have been shown to be mutagens and carcinogens, are not detectable as mutagens with the standard Salmonella tester strains TA1535, TA 1538, TA98 and TA100. Hence, a 'reduced' Ames test version (strains TA98 and TAI00 only, as it is planned to be introduced for the testing of environmental samples in Germany) will inevitably lead to a reduction of detectable compounds. The mere elimination of TAI02 from the set of Ames tester strains leads to a reduction of detectable umu genotoxins from 97 to 86%. Some mutagens are known to operate through a

141

SOS-independent pathway by provoking direct miscoding which leads to the formation of mutations. However, 149 out of the 173 Ames mutagens are detectable in the umu test as well. That means that approx. 86% of the genotoxins set lesions which induce a SOS response. Mutagenesis caused by the m ethylating agent N-methyl-N'-nitro-Nnitrosoguanidine (MNNG) is partly independent of the u m u C gene because O6-methylguanine, an important lesion caused by this agent, provokes direct mispairing (Toorchen and Topal, 1983), but, nevertheless, the umu operon is induced. On the other hand, MMS (methyl methane sulfonic acid) leads to the formation of 3-methyladenine, which stops replication (Larson et al., 1985) and induces a potent SOS signal. Other methylating agents (e.g., 7-methylguanine) generate DNA-adducts which neither cause errors in direct base-pairing nor block replication. These altered DNA bases are depurinated, thus generating the inducing signal for SOS response and error prone DNA repair. 4.2. C o m p a r i s o n between umu-, a n d carcinogenicity data

The results of the carcinogenicity studies of the 202 chemicals used in this evaluation are presented in Table 1. A chemical is regarded as 'carcinogenic' in the summary response if 'positive' or 'limited positive' evaluations prevail. Results being exclusively 'equivocal', 'inadequate' or 'inconclusive' are not considered in the comparison between umu test and carcinogenicity. The remaining 179 chemicals tested for genotoxicity and carcinogenicity comprise 149 carcinogens and 25 non-carcinogens. Five compounds have to be considered equivocal (butylated hydroxytoluene, hexachlorocyclohexane, methylbromide, naphthalene and pentachloronitrobenzene). The relations between genotoxicity and carcinogenicity are shown in Table 3. Ninety-three (63%) of the carcinogens are considered genotoxic ('sensitivity') and 54 (37%) nongenotoxic. In summary, 93% of the umu genotoxins are carcinogens ('positive predictivity') and 7% non-carcinogens. The proportion of non-genotoxins that are not carcinogenic is 72% ('specificity'), though only 25 non-carcinogens were examined. The agreement be-

142

G. Reifferscheid. J. Heil / Mutation Research 369 (1996) 129-145

Table 3 Comparison of umu test results and chemicals tested for rodent carcinogenicity Carcinogenicity +

umu test +

-

93

Total 149 25 5 179

54

2

7

18

0

0 100

5 77

0 2

-

_+ Total

+

tween carcinogenicity and genotoxicity ('concordance') is 65%. This agrees with earlier comparative studies which use results of long-term animal bioassays and Ames tests showing a concordance of about 62% (Tennant et al., 1987) and 58% (Zeiger, 1987), respectively. The results presented in this study clearly demonstrate that a chemical which induces the expression of the u m u operon can be regarded as a rodent carcinogen with a high degree of certainty. This elaborated extended data base proves the u m u test to be an important tool in genotoxicity assessment, using S a l m o n e l l a typhimurium TA1535/pSKI002 as primary and, if necessary, NM2009 as supplementary strain for special purposes, such as the sensitive detection of mutagenic aromatic amines.

Acknowledgements This study was supported by a grant from the Deutsche Bundesstiftung Umwelt (DBU 04343). The authors wish to thank Dr. Y. Oda for providing the u m u S a l m o n e l l a strains.

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