Review of the Salmonella typhimurium mutagenicity

Mutation Research 612 (2006) 58–76 www.elsevier.com/locate/reviewsmr Community address: www.elsevier.com/locate/mutres

Review

Review of the Salmonella typhimurium mutagenicity of benzidine, benzidine analogues, and benzidine-based dyes King-Thom Chung a,*, Ssu-Ching Chen b, Larry D. Claxton c a

Department of Biology, The University of Memphis, Life Science 201, Campus Box 526041, Memphis, TN 38152, USA b Department of Biotechnology, National Kaohsiung Normal University, Kaohsiung 831, Taiwan c Environmental Carcinogenesis Division, National Health and Environmental Effects Research Laboratory, Research Triangle Park, NC 27711, USA Received 30 June 2005; received in revised form 3 August 2005; accepted 3 August 2005 Available online 28 September 2005

Abstract We have reviewed the mutagenicity of benzidine analogues (including benzidine-based dyes), with a primary emphasis on evaluating results of the Salmonella/microsome mutagenicity assay. Many of these amines are mutagenic in tester strains TA98 and TA100 but require exogenous mammalian activation (S9) for activity. A few amines with halogen or nitro-groups in the structure are direct-acting mutagens. The addition of a sulfonic acid moiety to the molecule of benzidine reduced the mutagenicity of benzidine; whereas, methoxy, chloro, or methyl group additions did not. Complexation with a metal ion also decreased the mutagenicity. A substitution of an alkyl group on the ortho position next to an amine group also influenced the mutagenicity. Most carcinogenic benzidine analogues are mutagenic, and their metabolism to electrophiles that interact with DNA, leading to mutations, plays a central role in their carcinogenesis. # 2005 Elsevier B.V. All rights reserved. Keywords: Salmonella typhimurium; Benzidine analogues; Mutagenicity

Contents 1. 2. 3. 4. 5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . Mutagenicity of benzidine-based dyes . . . . . Mutagenicity of benzidine and its analogues . General observations concerning metabolism. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . Appendix . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .

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58 64 66 68 69 69 70 73

1. Introduction * Corresponding author. Tel.: +1 901 678 4458; fax: +1 901 678 4457. E-mail address: [email protected] (K.-T. Chung). 1383-5742/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.mrrev.2005.08.001

Benzidine, CAS number 92-87-5 (also known as [1,10 biphenyl]-4,40 -diamine; p-diaminodiphenyl; p,p0 -biani-

K.-T. Chung et al. / Mutation Research 612 (2006) 58–76

line; p,p0 -diaminobiphenyl; C.I. azoic diazo component 112; Fast Corinth Base B; 4,40 -bianiline; 4,40 -biphenyldiamine; 4,40 -diaminobiphenyl; 4,40 -diaminodiphenyl; 4,40 -diphenylenediamine; benzidin; benzidina; benzydyna; biphenyl-4,40 -diamino; 4,40 -biphenylenediamine; p,p0 -bianiline; C.I. 37225; 4,40 -diamino-1,10 -biphenyl; p,p0 -dianiline; NCI-C03361; RCRAwaste number U021; UN 1885; 40 -amino[1,10 -biphenyl]-4-ylamine), with the registered trade name Fast Corinth Base B, is a synthetic organic compound characterized as an odorless, white or slightly reddish crystalline solid. Benzidine and its congeners such as 3,30 -dimethylbenzidine (o-tolidine), 3,30 -dimethoxybenzidine (o-dianisidine), and dichlorobenzidine are the starting materials for the synthesis of azo dyes (referred to as benzidine-derived dyes or benzidine-based dyes) [1–5]. Benzidine-based dyes are widely used in textile, printing, leather, paper making, drug, and food industries [6–8]. Benzidine was also used in the rubber industry [4], in the manufacturing of plastics

59

[4], and in clinical laboratories [9]; however, benzidine is no longer used by these industries [10]. In the United States today, industries must use stringently controlled captive consumption approaches in which the compound is entirely made and used in the manufacture of other chemicals [10]. Examples of benzidine-based dyes are Direct Blue 6, Direct Brown 95, and Direct Black 38. Chemical structures of representatives of this group of dyes are shown in Fig. 1. In 1980, a NIOSH hazard review listed 236 direct dyes in commerce that contain a benzidine moiety [4]. At that time, 17 of 30 commercially important benzidine-based dyes were manufactured in the United States. By combining listings from NIOSH [4] and the EPA of presently available commercial dyes that contain a benzidine moiety, 229 related chemicals were identified (Table 1). Benzidine-based dyestuffs are manufactured by coupling tetrazotized benzidine with phenols and

Fig. 1. Chemical structures of some representative benzidine-based dyes.

Compound C.I. number (CAS No.)

Government TA1538 document Wo W

Pigment Yellow 12, C.I. 21090 Direct Red 28 or Congo Red, C.I. 22120 (573–58–0)

Neg Neg FR N

TA98 H

Aner

Wo

TA100 W

H

FR N

Acid Red 85, C.I. 22245 (3567–65–5)

FR N

Direct Blue 6, C.I. 22610 (2602–46–2)

FR N

H

Aner

Neg

Wan,N

Neg

HF,+

H

Aner

R,W+ BA,? Neg

Neg

1

[101] [55]

R,? BA,?

[102] 2 [54] [56] [45] [48]

Wan,Neg HF,+ HF,+ RF,w+

HF,+ BR,+

[43] Neg Neg

+

+

Neg Neg

Neg Neg

[47] U,+

[46] [103] NA

Neg

HF,+

Neg

HF,+

Neg Neg

HF,+

[3] +,N

Neg Neg

HF,+

Neg Neg Neg Neg Neg + Neg +

3

Neg Neg

[104] [3] [43] [39] [44] [105] [106]

Neg ?

[44]

5

HR,+

Neg Neg

Direct Red 2, C.I. 23500

W

[3] [43] Neg

Neg +

C.I. Direct Blue 14 or Direct Blue 2B, C.I. 22610 (2602–46–2) Direct Red 46, C.I. 23050 Direct Orange 6, C.I. 23365

Wo

REF. Com

Neg Neg

HF,+

Neg Neg

HF,+

HF,+ BR,+ HR,+

Neg Neg

[43] [43] Neg

Neg

R,+ BA,? Neg HF,+

Neg

R,+ BA,? Neg

[55]

[48]

4 6 NA 7


C.I. Direct Red 74, C.I. 22170 (8003–75–6)

W

Brc,+

Neg +

FR N

W o

Oth

HF Neg

Neg

C.I. Direct Red 17, C.I. 22150 (2769–07–5)

Aner

TA1535

60

Table 1 The Salmonella and associated genotoxicity of benzidine-based (including dimethylbenzidine, dimethoxylbenzidine, and dichlorobenzidine derivatives) dyes found in PubMed and Toxline cited references (listed in order by C.I. number)

RF,+ HF,+

Neg HF,+ BR,+

Neg +

HF,+

Neg +

HF,+

[3] [43]

Neg +

HF,+

[43]

+ Direct Red 39, C.I. 23630 Acid Red 114, C.I. 23635

+

Trypan Blue or Direct Blue 14, C.I. 23850

HF,+

Neg

Wan,Neg

R,w+ BA,+ Neg

HF,+ Neg + Neg + C.I. Direct Blue 53 or Evans Blue, C.I. 23860 (314–13–6)

Neg

+

R,+ BA,+

[55] +

[102] 9 [54] [56] [45] [48] [43] [88] 10

+

[102] 11 [45] [3] [44] 12

Wan,Neg HF,+ HF,+ ? Neg HR,+ RF,w+

HF,+ Neg Neg

Neg Neg

Neg Neg

HF,+ Neg Neg N

Neg Neg ?

?

HF,+ HF,+

Neg Neg Neg HF,+

Neg Neg Neg

Direct Violet 32, C.I. 24105

Neg +

HF,?+ BR,+

[43]

Direct Blue 8, C.I. 24140 Direct Blue 10, C.I. 24304

Neg + Neg +

HF,+ HF,+

[43] [43]

Direct Blue 15, C.I. 24400

Neg

R,+ BA,+ HF,+

Neg +

+

C.I. Direct Blue 218, C.I. 24401 Neg

FR N

Direct Brown 95, C.I. 30145 (16071–86–6)

FR N

[55] [3] [43] [107] 13

Wan,N

+

Neg Neg

HF,N

N

[54] [48]

+

Wan,Neg HF,+ RF,Neg HF,+

+

HF,+

[3]

+

HF,+

?+

Direct Brown 1.2, C.I. 30110 (2586–58–5)

R,+ BA,?

HF,+ +

C.I. Direct Blue 1 or Chloramin Sky Blue, C.I. 24410

Neg

Neg

[3]

[104] 15 [3] [43]

61

HF,+

14

[3]

+,N Neg +


Neg

[107] 8 [43]

Neg

Direct Blue 25, C.I. 23790

[3] [43]

Neg +

62

Table 1 (Continued ) Compound C.I. number (CAS No.)

Government TA1538 document Wo W

TA98 H

Aner

Wo

TA100 W

H

Aner

Neg Neg

W o

W

TA1535 H

Aner

Neg Neg

Wo

W

H

Oth

REF. Com

+

[39] [105] [106] [108] [109] [110]

16 NA 17 18 NA 19

+ Neg [104] [111] [54] [3] [43] + [39] [47] [105] [110] + [46] + [107]

20 NA

Aner

Neg ? +

Neg Neg

Neg Neg

Neg Neg

+

Wan,+

+

Wan,+ HF,+

+ Neg +

HF,+

Neg +

Neg Neg

Neg Neg

Neg + Neg + +

Neg Neg Neg Neg

Neg Neg

Neg

+

Direct Green 1, C.I. 30280 (3626–28–6)

FR N

Neg Neg Neg Neg

Neg ? Neg Neg

Direct Green 6, C.I. 30295 (4335–09–5)

FR N

Neg Neg

Neg Neg

Direct Brown 31, C.I. 35660 (2429–81–4)

FR N

+

Total Total Total Total

19 1 18 0

# # # #

(27) positive negative mixed results

Neg Neg

FR N

18 11 3 4

1 0 0 1

18 14 2 2

16 1 14 1

16 3 10 3

N +

15 10 0 5

13 0 11 2

NA 22 23 24

[112] 25 [46] 26 [47]

HF,+ 8 2 2 4

21

[3] 11 1 9 1

2 0 2 0

7 4 0 3

6 0 6 0

5 0 3 2

1 0 1 0

0 0 0 0

Compound/C.I. number/CAS No.: Common name(s) found in most references/Color Index Number/Chemical Abstract Service Number. Govern. Doc.: Government Documents reported by EPA and/or NIOSH as commercial dye (FR, EPA Federal Register notices; N, NIOSH report [4]). These reports change with time; however, they can be located upon the appropriate government web site. See the following Federal Register volumes: vol. 68 (2003) No. 227; vol. 68 (1998) No. 4; vol. 61 (1996) 195; vol. 60 (1995) No. 168. Also see the EPA Chemical Export Notification List (http://www.yale.edu/oehs/TSCA/EPA%20export%20notification%20chemical%20list.pdf). TA1538/TA98/TA100/TA1537 identifies the strain of Salmonella typhimurium reported. Results of other strains and assays are indicated in the ‘‘Oth’’ (Other) column and within the notes. Neg, nonmutagenic; +, mutagenic; ?, questionable. Com: Comments—numbers refer to notes within this footnote; NA, publication not available to the authors at the time of this publication [on order]. Wo, without an exogenous activation system; W, with a standard rat liver S9; H, hamster liver S9; Aner, anaerobic/reductive cleavage activation systems (as represented by BA, bacterial cell free extract; BR, bacterial reduction; HF, hamster/FMN system; RF, Rat/FMN system; R, riboflavin system; U, urine mutagenicity after in vivo exposure; Ug, urine mutagenicity of germ free animals; Wan, normal S9 system under anaerobic incubation); ML, mouse lymphoma system; S, SV40 S. typhimurium forward mutation assay. Notes: (1) Results not reliable due to solubility; (2) hamster hepatocyte DNA repair is w+, rat hepatocyte DNA repair is ?; (3) urine of rats in vivo, +; urine of germ free rats, N; (4) rat 90 day liver cancer; mice liver toxicity but no cancer; (5)TA1537W, N; TA1537wo, N; mouse lymphoma assay, N; (6) TA1537W, N; TA1537wo, N; mouse lymphoma assay, N; (7) TA100SuspensionW/wo, N;


Direct Black 38, C.I. 30235 (1937–37–7)


TA1537 Suspension W/wo, N; (8) S. typhimurium SV40 forward assay: W, +, wo, N; (9) hamster hepatocyte DNA repair: W, +, rat hepatocyte DNA repair: W, +; (10) TA1537: W/wo, N; (11) hamster hepatocyte DNA repair: +, rat hepatocyte DNA repair: w+; (12) TA1537: W/wo, mouse lymphoma: N; also, for all Salmonella assays: with S9 with both hamster and rat unless noted; (13) S typhimurium: SV40W, +; SV40H, +; (14) 1537H, N; (15) urine of rats in vivo, +; urine of germ free rats, N; (16) rat 90 day liver cancer; mice liver toxicity but no cancer; (17) TA1537S-W/wo, N; questionable positive is due to difference in two labs; S, suspension assay; (18) Urine of exposed animals, +; (19) TA1537W/wo, N; (20) urine of rats in vivo, +; urine of germ free rats, N; (21) rat 90 day liver cancer; mice liver toxicity but no cancer; (22) TA1537W/wo, N; (23) urine of exposed animals, +; (24) S. typhimurium: SV40W, +; SV40H, +; (25) TA1537W/wo, N; (26) urine of exposed animals, +. EPA and NIOSH benzidine-based dyes with C.I. number and CAS number (if known) for which no mutagenicity information was found: C.I. Direct Brown 62, C.I. 31720, 8003–56–3; C.I. Direct Black 29, C.I. 22580, 3626–23–1; C.I. Direct Blue 38, C.I. 30090, 1324–83–0; C.I. Direct Green 21, C.I. 31790, 8003–52–9; C.I. Direct Brown 13, C.I. 35710, 8003–82–5; C.I. Direct Brown 73, C.I. 35535, 6428– 43–9; C.I. Direct Violet 12, C.I. 22550, 2429–75–6, C.I. Direct Orange 2, C.I. 22380, 8005–97–8; C.I. Direct Black 83; C.I. 31850, 6837–80–5, C.I. Direct Brown 7, C.I. 30035, 6837–86–1, C.I. Acid Black 70, C.I. 30355, 8005–88–7; C.I. Direct Brown 56, C.I. 22040, 6486–31–3; C.I. Brown 165, C.I. 22045, 6486–32–4; C.I. Direct Orange 25, C.I. 22135, 6486–43–7; Paranil Bordeaux B, C.I. 22080; C.I. Direct Violet 4, C.I. 22555, 6472–95–3; Acid Red 85, C.I. 22245, 3567–65–5, C.I. Direct Brown 20, C.I. 30060, 1324–67–0; Direct Black 31; Acid Orange 45, C.I. 22195; 2429-80-3; Direct Brown 111; C.I. Direct Blue 48, C.I. 22565, 6459–89–8; Direct Dye, C.I. 30080; Direct Dye, C.I. 31793; C.I. Direct Brown 33, C.I. 35520, 1324–87–4; Direct Dye, C.I. 35065; C.I. Direct Red 13, C.I. 22155, 1937–35– 5; C.I. Direct Red 33,C.I. 22306, 6253–15–2; C.I. Direct Green 10, C.I. 30285, 6360–61–8; C.I. Direct Brown 75, C.I. 30325, 1324–84–1; Alkali Yellow R, C.I. 22390; C.I. Direct Orange 43, C.I. 22193; Diphenyl Brown RN, C.I. 22335; C.I. Direct Brown 14, C.I. 35715, 8002–97–9; Diazo Violet R, C.I. 22020; C.I. Direct Red 42, C.I. 22180, 6548–39–6; Direct Green 21:1; C.I. 22322; C.I. Direct Violet 85, C.I. 22520, 6507–84–2; Direct Black 4, C.I. 30245, 2429–83–6; C.I. Acid Black 66; C.I. 30275, 6360–59–4; C.I. Direct Green 7, C.I. 30330, 6360–64–1; Direct Gray R, C.I. 22545; C.I. Direct Red 10, C.I. 22145, 2427–70–1; C.I. Direct Yellow 24, C.I. 22010, 6486–29–9, C.I. Direct Brown 86,C.I. 22030, 6486–30–0; Direct Orange 8, C.I. 22130, 2429–79–0; Direct Red 1, C.I. 22310, 2429–84–7; Direct Orange 1, C.I. 22430, 6472–93–1; C.I. Direct Blue 230, C.I. 22455, 26527–65–7; C.I. Direct Violet 45, C.I. 22510, 6426–72–8; Direct Brown 154, C.I. 30120, 6360–54–9; C.I. Direct Brown 173, C.I. 30165, 6826–64–8; C.I. Direct Green 39, C.I. 30220, 6360–57–2; C.I. Direct Black 11, C.I. 30240, 6486–52–8; C.I. Direct Black 131, C.I. 30270, 6486–54–0; C.I. Direct Green 9, C.I. 30310, 6360–62–9; C.I. Direct Blue 51, C.I. 30340, 6360–65–2; Direct Dye, C.I. 31820; Direct Brown 2, C.I. 22311, 2429–82–5; Direct Dye, C.I. 35400; Direct Orange 1, mixture, 54579–28–1; C.I. Direct Orange 33, C.I. 22385, 13190–99–3; C.I. Direct Brown 43, C.I. 35700, 6471–44–9; Diamine Brown S, C.I. 22050; Brilliant Congo G, C.I. 22160; C.I. Direct Yellow 1, C.I. 22250, 6472–91–9; C.I. Direct Violet 3, C.I. 22445, 6507–83–1; C.I. Direct Violet 27, C.I. 22460, 6426–64–8; C.I. Direct Violet 17, C.I. 22465, 6426–65–4; C.I. Direct Violet 36, C.I. 22470, 6472–94–2; C.I. Direct Blue 19, C.I. 22485, 6426–68–2; C.I. Direct Blue 49, C.I. 22540, 6426–73–9; C.I. Direct Brown 171, C.I. 30040; Direct Brown 1, C.I. 30045, 3811–71–0; C.I. Direct Brown 79, C.I. 30050, 6483–77–8; C.I. Direct Brown 61, C.I. 30055, 6505–33–5; C.I. Direct Brown 17, C.I. 30100, 6661–48–9; Direct Dye, C.I. 30105; C.I. Direct Brown 68, C.I. 30125, 6449–85–0; Direct Dye, C.I. 30180; Direct Dye, C.I. 30200; Direct Dye, C.I. 30215; C.I. Direct Green 58, C.I. 30225, 110735–26–7; Leather Dye, C.I. 30255; Direct Dye, C.I. 30265; C.I. Direct Green 8, C.I. 30315, 5422–17–3; C.I. Direct Blue 11, C.I. 30350, 6451–04–3; C.I. Direct Brown 151, C.I. 31685, 10130–38–8; C.I. Direct Brown 57, C.I. 31705, 6360–28–7; C.I. Direct Black 40, C.I. 31760, 6449–81–6; C.I. Direct Brown 46, C.I. 31785, 8003–51–8; Direct Dye, C.I. 31795; Direct Dye, C.I. 31805; C.I. Direct Black 27, C.I. 31810, 6360–39–0; Direct Blue 2, C.I. 22590, 2425–73–4; Pyramidal Brown, C.I. 21060; Congo GR (A), C.I. 22000; Diazo Brown R Extra, C.I. 22035; Pyramine Orange 3G, C.I. 22060; Pyramine Orange RR, C.I. 22070; Oxamine Red B, C.I. 22095; Oxamine Orange G, C.I. 22100; Diazo Black R Extra, C.I. 22110; Glycine Red, C.I. 22125; Direct Dye, C.I. 22140; Direct Dye, C.I. 22165; Chlorazol Orange 2R, C.I. 22175; C.I. Direct Orange 102, C.I. 22190, 6528–39–8; C.I. Direct Red 60, C.I. 22200, 6486–49–3; C.I. Direct Red 43, C.I. 22205, 6486–50– 6; Zambesi Brown GG, C.I. 22210; Glycine Corinth, C.I. 22220; Para Green BBL, C.I. 22230; Direct Red 37, C.I. 22240, 3530–19–6; Cloth Orange, C.I. 22255; Brilliant Direct Orange G, C.I. 22260; Mordant Dye—Cloth Brown R, C.I. 22270; Palatine Chrome Red RX, C.I. 22275; C.I. Direct Red 18, C.I. 22280, 6548–26–1; Cloth Brown G, C.I. 22285; C.I. Direct Red 52, C.I. 22290, 6797–93–9; Ozamine Maroon, C.I. 22300; C.I. Direct Red 29, C.I. 22305, 6426–54–6; C.I. Direct Green 60, C.I. 22315, 6426–56–8; Diazol Brown MA, C.I. 22320; C.I. Direct Brown 60, C.I. 22325, 6426–57–9; Triazol Red 6B, C.I. 22330; C.I. Direct Brown 58, C.I. 22340, 6426–59–1; Direct Brown 59, C.I. 22345, 3476–90–2; C.I. Direct Red 84, C.I. 22360, 6459–86–5; Direct Orange 1N, C.I. 22370, 6459–87–6; Direct Orange 1N, C.I. 22375, 13164–93–7; C.I. Direct Red 59, C.I. 22420, 6655–94–3; C.I. Direct Violet 43, C.I. 22440, 6426–63–7; C.I. Direct Blue 16, C.I. 22475, 6426–66–0; Direct Violet 22, C.I. 22480, 6426–67–1; C.I. Direct Blue 58, C.I. 22490, 6426–69–3; Naphthamine Blue 3R, C.I. 22495; C.I. Direct Red 44, C.I. 22500, 2302–97–8; C.I. Direct Blue 42, C.I. 22505, 6426–71–7; Alkali Dark Brown G, V, C.I. 22530; Naphthamine Black RE, C.I. 22585; C.I. Direct Blue 64, C.I. 22595, 6426–74–0; Diamine Nitrazol Green RR, C.I. 22600; Naphthamine Blue 2B, C.I. 22605; C.I. Direct Black 15, C.I. 22620, 6426–75–1; C.I. Direct Blue 177, C.I. 22625, 6426–76–2; C.I. Direct Violet 38, C.I. 22630, 6426–77–3; Direct Dye, C.I. 22640; Direct Dye, C.I. 30065; C.I. Direct Brown 138, C.I. 30070, 6449–84–9; Direct Dye, C.I. 30075; Direct Dye, C.I. 30085; Direct Dye, C.I. 30115; Direct Dye, C.I. 30130; C.I. Direct Brown 5, C.I. 30135, 6844–77–5; Direct Brown 175, C.I. 30150; C.I. Direct Brown 21, C.I. 30155, 6442–05–3; Direct Dye, C.I. 30160; Direct Dye, C.I. 30170; Direct Dye, C.I. 30175; Direct Dye, C.I. 30190; Direct Dye, C.I. 30195; C.I. Direct Blue 43, C.I. 30205, 7273–59–8; Direct Dye, C.I. 30230; Direct Dye, C.I. 30250; C.I. Acid Black 69, C.I. Direct Black 41, C.I. 30260, 6486–53–9; C.I. Direct Green 12, C.I. 30290, 6486–55–1; Direct Dye, C.I. 30300; C.I. Direct Green 19, C.I. 30305, 6486–58–4; Direct Dye, C.I. 30335; C.I. Acid Black 94, C.I. 30336, 6358–80–1; C.I. Direct Black 14, C.I. 30345, 4656–30–8; Direct Dye, C.I. 30360; Direct Dye, C.I. 31695; C.I. Direct Brown 51, C.I. 31710, 4623–91–0; Direct Dye, C.I. 31715; C.I. Direct Brown 27, C.I. 31725, 6360–29–8; C.I. Direct Brown 54, C.I. 31735, 8003–50–7; C.I. Direct Brown 101, C.I. 31740, 8626–29–7; Direct Dye, C.I. 31745; C.I. Direct Brown 190, C.I. 31750; C.I. Direct Brown 159, C.I. 31755, 10214–11–6; Direct Dye, C.I. 31765; Direct Dye, C.I. 31770; C.I. Direct Green 22, C.I. 31775, 6860–33–4; Direct Green 22, C.I. 31775; Direct Dye, C.I. 31780; Direct Green 21, C.I. 31790; Direct Dye, C.I. 31800; Direct Dye, C.I. 31815; Direct Dye, C.I. 31830; Direct Dye, C.I. 31835; Direct Dye, C.I. 31840; C.I. Direct Brown 39, C.I. 35060, 6473–06–9; Direct Dye, C.I. 35070; C.I. Direct Black 34, C.I. 35075, 6473–08–1; Direct Dye, C.I. 35080; C.I. Direct Blue 131, C.I. 35085, 6661–39–8; Direct Dye, C.I. 35240; C.I. Direct Brown 70, C.I. 35530, 6428–42–8; Direct Dye, C.I. 35650; C.I. Direct Brown 215, C.I. 35720, 83606–72–8; Direct Dye, C.I. 35900; C.I. Direct Brown 25, C.I. 36030, 33363–87–0; Direct Dye, C.I. 36040; Direct Brown 74, C.I. 36300, 8014–71–3; Resin F Black WP, None; C.I. Direct Violet 88, C.I. 22046, 6358–33–4; C.I. Acid Red 323, C.I. 22238, 6358–34–5.

63

64


amines [1]. Some of these dyes are mutagenic and/or carcinogenic to animals [8,11–13]. Chung and Cerniglia [14] reviewed the mutagenicity of azo dyes and found that benzidine was a mutagenic moiety of many azo dyes. Benzidine can be generated from azo dyes through reduction by intestinal and environmental microorganisms [15–17]. Upon administration to various experimental animals, benzidine-based dyes typically undergo reduction of the azo bonds to produce benzidine and benzidine metabolites that later appear in the urine [18–20]. Benzidine was identified as a carcinogen for the human urinary bladder in 1973, and it was the primary etiological agent for occupational ‘‘aniline cancer’’ originally recognized in 1895 [6,21]. Hazardous waste sites are among the few remaining sources within the environment with the benzidine contamination. It has been found within 28 of the approximately 1585 U.S. Environmental Protection Agency (EPA) National Priority List (NPL) sites [10] (in surface water at 5 sites, groundwater at 10 sites, soil samples at 9 sites, and sediment samples at 4 of these 28 NPL sites). Benzidine ranks 25 out of 275 in the 1994 ‘‘Priority List of Hazardous Substances’’ of the Agency for Toxic Substances and Disease Registry (ATSDR). Although the production and use of benzidine was banned in the United States in the mid-1970s, and occupational exposures to benzidine were curtailed [10], the compound is still produced or imported for several specialty uses [22]. Also, exposures to benzidine continue in some countries [23]. In 1994, Fishbein [24] reported that benzidine is released into the environment on a large scale throughout the world. The contamination of benzidine-containing water sources occurs from the release of waste water discharged by the dye industry [22]. Therefore, a potential for exposure to benzidine and its metabolites does exist. There were many reports on the mutagenicity of benzidine-based dyes, benzidine, and its congeners [7,11,14,19]. Various laboratories have been involved in the mutagenicity testing of diverse benzidine analogues [3,25–28]. The Salmonella/microsome assay has been the most widely used mutagenicity test system [29–33]. In this review, we concentrate on results obtained from the Salmonella/microsome mutagenicity assay, and analyze the relationships between chemical structures and their mutagenic activities. The compounds analyzed are analogues that differ by specific, relative minor alterations in substituent groups in their structure. Such studies on structure–activity relationships may offer the prospect of identifying the critically important parameters in a molecular structure that influence their

biological activities [27,34–38]. This may lead to a better understanding of the general mechanisms involved in mutagenicity/carcinogenicity, facilitate the development of safe but useful compounds, and aid in the search for non-mutagenic alternatives to genotoxic compounds that are commercially important [37]. 2. Mutagenicity of benzidine-based dyes Since its first use in the late 1800s, benzidine has been an important intermediate in the production of dyes, especially azo dyes [39]. However, the production of benzidine-based dyes has decreased dramatically during the last century [39–41]. In spite of this, benzidine-based dyes and other mutagenic dyes are still produced and/or used in many parts of the world [42]. Although NIOSH and EPA documents list many potentially available benzidine-derived dyes, only a few are found in commercial use in the United States and Europe [42]. A PubMed/Toxline search found genotoxicity information for 27 benzidine-based dyes within 22 publications (Table 1). All 27 had been tested for bacterial mutagenicity using Salmonella typhimurium; however, the publications varied in indicator strains used and the type of protocol used (Fig. 2). Some of these compounds were also evaluated in other bioassays (hamster and/or rat hepatocyte DNA repair, the Salmonella mutagenicity of urine from exposed animals, whole animal cancer bioassays, and the mouse lymphoma assay; see comments in Table 1). However, the results of these other assays are very limited. For Salmonella assays, Table 1 shows that most benzidine-based dyes are non-mutagenic unless some form of exogenous activation system is used. Only Direct Blue 15 (mutagenic for TA1538) [43] and Direct Brown 31 (mutagenic for TA98) [3] showed mutagenic responses without exogenous activation. Seven compounds were tested in only one laboratory, which reported non-mutagenic responses when using rat S9. These dyes were Pigment Yellow 12 (TA1538) [3], C.I. Direct Blue 14 (TA1538, TA98, TA100, TA1535, TA1537) [44], C.I. Direct Blue 53 (TA1538, TA98, TA100, TA1535, TA1537) [3,44,45], C.I. Direct Red 17 (TA98, TA100) [46], C.I. Direct Blue 218 (TA98) [3], Direct Green 1 (TA98, TA100) [46,112], and Direct Green 6 (TA98, TA100) [47]. Of the 26 compounds for which we have results using some form of reductive metabolism, 21 compounds are mutagenic (see Table 1). Three compounds (Direct Green 6, Direct


65

Fig. 2. Mutagenicity (revertants/mg) of benzidine-based dyes by strain of Salmonella typhimurium and type of metabolic activation (for details, see Table 1). Values are the number of dyes reported for each category.

Green 1, and C.I. Direct Red 17) were not tested under reductive conditions. With the hamster/FMN reductive metabolism protocol, Pigment Yellow 12 was nonmutagenic with strain TA1538, and Direct Blue 218 was non-mutagenic with TA98. These results indicated that under oxidative conditions, bacterial metabolism alone cannot generate mutagenic metabolites for the majority of these dyes. Many of these dyes are mutagenic via the conversion by mammalian metabolism (i.e. S9), and can produce mutagenic metabolites after metabolism with a reductive and oxidative system (e.g. the use of hamster S9 with FMN). Most investigators indicated that the protocols that produce reductive or partially reductive conditions probably cleave azo bonds thereby releasing benzidine or other benzidine derivatives [3,14,16,48– 53]. Interestingly, anaerobic reductive cleavage alone may not be adequate for producing the mutagenic metabolites of some benzidine-based azo dyes; for example, three of four compounds (Direct Red 28, Trypan Blue, and C.I. Direct Blue 1) tested under strictly anaerobic conditions (with S9 present) did not produce a mutagenic response [54]. These three compounds, however, were mutagenic when tested under partially reductive conditions [3,43,45,48,54– 56].

Scientists have made a number of efforts to develop replacements for benzidine that are non-mutagenic themselves thus yielding non-mutagenic azo dyes (not referenced in the above discussion of commercial dyes). For example, Freeman et al. [57] demonstrated that the addition of nitrogen groups into the aromatic rings of benzidine yields diaminobipyridine and diaminophenylpyridin, which were non-mutagenic in the standard Salmonella assay. Also, the analogues to Congo Red and Direct Black 38 synthesized from these compounds were non-mutagenic in the standard assay. However, both the benzidine replacements and the new dye analogues were mutagenic in the Prival hamster S9/FMN Salmonella protocol [57]. Freeman et al. [57,58] showed that the addition of alkyl or alkoxy groups to ortho position relative to the amine groups or by adding of butoxy or propyl groups, one could synthesize compounds that were analogous to benzidine but not mutagenic in the standard assay. For example, studies showed that the nonmutagenic 2,20 -dimethyl-5,50 -dipropoxybenzidine [59] can be used to produce non-mutagenic azo pigments [60,61]. Bae and Freeman [1] synthesized a number of direct dyes that used 2,20 -dimethyl-5,50 -dipropoxybenzidine as a replacement for benzidine and found that the new dyes were all non-mutagenic in the standard Salmonella assay.

66


The above studies indicated that the mutagenicity of benzidine-based azo dyes is most commonly due to metabolic products of these dyes, especially to the benzidine or benzidine-derivative that is produced after reductive cleavage of the azo dye. Therefore, the mutagenic activity for benzidine-based dyes is best understood by examining the genotoxicity of benzidine congeners. 3. Mutagenicity of benzidine and its analogues Since the initial examination of the mutagenicity of benzidine and it analogues by Garner [34] using S. typhimurium TA1538, a number of investigators have examined these and other congeners of benzidine using a variety of strains and test conditions. Using quantitative summaries of studies where enough data were provided for the calculation of slope values (Appendices A–F). Fig. 3 demonstrates that small alterations in chemical structure can cause profound differences in mutagenic activity. The following discussion reviews these and other related studies in order to highlight what is known about the relative activity of these compounds. Garner [34] used the tester strain TA1538 in the presence or absence of liver enzyme preparation and found that 3,30 -dichlorobenzidine had some direct mutagenic activity; however, this activity was increased over 50-fold by the addition of liver preparation. The mutagenicity of 3,30 -dichlorobenzidine was approximately 10 times more active than that of benzidine, while the mutagenicity 3,30 -5,50 -tetrafluorobenzidine was approximately equipotent to the activity of benzidine. On the other hand, 3,30 -5,50 tetramethylbenzidine showed no mutagenic activity

either with or without a liver preparation. 3,30 Dimethoxybenzidine (o-dianisidine) had a slight mutagenic activity only in the presence of liver preparation [34]. Lazear and Louie [26] examined the mutagenic activity of benzidine and its analogues using strains TA98 and TA100 with mouse liver enzyme preparations, and found that benzidine, 4aminobiphenyl, 3,30 -dimethylbenzidine (o-tolidine), 3,30 -dimethoxybenzidine (o-dianisidine) and 3,30 dichlorobenzidine were mutagenic with both tester strains. 4-Aminobiphenyl produced both frameshift and base-pair substitution mutations, and 3,30 -dichlorobenzidine was only mutagenic towards TA98 in the absence of the mammalian liver enzyme system. They also found that the mutagenicity of hydrochloride salts of the 4-aminobiphenyl, benzidine, and 3,30 -dichlorobenzidine were less mutagenic than that of the parent compounds except for 3,30 -dimethylbenzidine. Prival et al. [3] showed that benzidine, o-tolidine (3,30 dimethylbenzidine), o-dianisidine (3,30 -dimethoxybenzidine), and 3,30 -dichlorobenzidine were mutagenic in the modified Salmonella assay in which the tested chemicals were preincubated with FMN and hamster liver S9. Reid et al. [62] tested the mutagenicity of benzidine, 3,30 -dimethoxybenzidine (o-dianisidine), 3,30 -dimethylbenzidine (o-tolidine), 3,30 -dichlorobenzidine, and their corresponding N-monoactylated and N,N0 -diacetylated derivatives with S. typhimurium strains TA98, TA100, TA1535, and TA1538. They found that the N-monoacetylated derivatives were more mutagenic than either the parent diamines or the N,N0 -diacetylated derivatives. The relative mutagenic potency of the parent amines for TA98 was 3,30 -dichlorobenzidine 3,30 -dimethoxybenzidine > benzidine > 3,30 -dimethylbenzidine. Cer-

Fig. 3. Mutagenicity (revertants/mg) of benzidine congeners in Salmonella typhimurium. Left-hand graph illustrates how modifications of benzidine affect mutagenicity. The graph on the right illustrates how benzidine mutagenicity compares to other 4-aminobiphenyl congeners. Note that the activity scale is logarithmic. Quantitative summary comparisons for these and other benzidine congeners can be found in Appendices A–F.


niglia et al. [53] also showed that the benzidine derivatives monoacetylbenzidine, 4-aminobiphenyl, and 4-acetylaminobiphenyl are mutagenic towards S. typhimurium TA98 following metabolic activation with rat or hamster S9. Wallace and Josephy [63] investigated the mutational specificity of benzidine employing tester strain TA98 using the modified Salmonella assay in which hydrogen peroxide-dependent peroxidation activation was used. They found that the most frequently observed spectra of mutation was deletion of two bases from a (CG)4 run, although the (CG)4 deletion was elevated in frequency among benzidine-induced revertants relative to spontaneous revertants [63]. Many other mutagenic spectra, including additions and deletions, were also observed. Only small frameshift types in mutation were observed among the benzidine-induced revertants, whereas some larger deletions were observed among the spontaneous revertants [63]. You et al. [64] examined the genotoxicity of benzidine and its 3,30 -diamino-, 3,30 -dimethyl-, 3,30 -dimethoxy-, 3,30 -difluoro-, 3,30 dichloro-, 3,30 -dibromo-, 3,30 -dicarbomethoxy-, and 3,30 -dinitro-derivatives together with 2-nitro- and 3nitrobenzidines using strains TA98, TA98/1,8-DNP6, and TA100. These derivatives pretreated with S9 mix were more mutagenic than benzidine. Among them, 3,30 -dinitro- and 3-nitro-benzidines had the greatest mutagenic activity. Similarly, Messerley et al. [65] investigated the mutagenic activities of benzidine, its hydrochloride salt, and other analogues (2-aminobiphenyl, 4-amino-biphenyl, 3,30 -diaminobenzidine, 3,30 -dichlorobenzidine, 3,30 -dimethoxybenzidine, 0 3,3 -dimethoxybenzidine dihydrochloride, and N,NN0 ,N0 -tetramethylbenzidine) in the Salmonella assay using strains TA98 and TA100 with and without S9 activation. With the exception of 3,30 -dichlorobenzidine in TA98, little or no mutagenicity was observed in these derivatives under these conditions without S9 activation. All compounds, except for N,N-N0 ,N0 tetramethylbenzidine, exhibited some mutagenic activity in TA100 with S9 activation. 3,30 -Dichlorobenzidine and 4-aminobiphenyl were significantly more mutagenic than the other compounds tested in the presence of metabolic activation. For benzidine and its 3,30 -disubstituted benzidine (dimethoxy-, diamino-, and dichloro-compounds), an increase in mutagenicity correlated to a decrease in basicity of the parent aniline in both TA98 and TA100. Savard and Josephy [66] demonstrated that the dihalogenated benzidines (i.e. 3,30 -dichorobenzidine, 3,30 -difluorobenzidine, and 3,30 -dibromobenzidine)

67

were directly mutagenic to strain TA98 and its acetylase-deficient strain, TA98/1,8-DNP6. The mutagenicity of these compounds increased greatly following the addition of hamster S9 mix. Sinsheimer et al. [28] examined the mutagenicity of a series of compounds including 4-aminobiphenyl, 4,40 -diaminobiphenyl (benzidine), 4-amino-40 -chlorobiphenyl, 4-amino-40 -nitrophenyl, and 4-amino-20 -methylbiphenyl, and found these compounds required S9 metabolic activation for mutagenicity in both TA98 and TA100 except the nitrocontaining 4-amino-40 -nitrobiphenyl. They also noticed that the substitution in the 40 -position of the biphenyl has a profound effect on the mutagenicity of the compound. You et al. [67] further determined the mutagenicity of 4amino-40 -substituted biphenyls including 4,40 -diaminobiphenyl (benzidine), 4-amino-40 -hydroxybiphenyl, 4amino-40 -methylbiphenyl, 4-aminobiphenyl, 4-amino40 -chlorobiphenyl, 4-amino-40 iodobiphenyl, 4-amino-40 cyanobiphenyl, and 4-amino-40 -nitrobiphenyl using strains TA98 and TA100 with and without S9 activation. The results showed that S9 was required for mutagenicity with the exception of 4-amino-40 -nitrobiphenyl. The nitro-derivative exhibits a relatively high mutagenic activity in TA98 without S9. It was also discovered that 3,30 -dichloro-benzidine-2HCL is directly mutagenic to TA98 and 4,40 -dinitro-2-biphenylamine is mutagenic to both TA98 and TA100. 2-Aminobiphenyl, 3-aminobiphenyl, and 3,30 -5,50 -tetramethylbenzidine were not mutagenic to either strain in the presence or absence of rat S9 mix. In the presence of rat S9 mix, 4aminobiphenyl, benzidine, 3,30 -dichlorobenzidine, 3, 30 -dimethoxybenzidine, 3,30 -4,40 -tetraaminobiphenyl, o-tolidine, N,N-N0 ,N0 -tetramethylbenzidine, and 4,40 dinitro-2-biphenyl were mutagenic to TA98; and 4-aminobiphenyl, 3,30 -dichlorobenzidine-2HCl, 3,30 dimeth-yoxybenzidine, and 4,40 -dinitro-2-biphenyl were mutagenic to TA100 [50]. Ioannides et al. [68] investigated the mutagenicity of three isomeric forms of aminobiphenyl (i.e. 2-, 3-, and 4-aminobiphenyls), and their N-hydroxy derivatives using S. typhimurium strains TA98 and TA100. 2Aminobiphenyl was reported to exhibit no mutagenicity, although some studies [65,69] contradicted that result. 3-Aminobiphenyl was not mutagenic, while 4aminobiphenyl exhibited strong mutagenicity in TA98 and TA100 in the presence of S9. N-Hydroxy-4aminobiphenyl was a potent direct mutagen towards both strains, and N-hydroxy-2-aminobiphenyl was only mutagenic to TA100. N-Hydroxy-3-aminobiphenyl displayed no mutagenicity in either strain. They also demonstrated 2-nitrosobiphenyl is a direct mutagen to TA100 but not to TA98, and 3-nitrosobiphenyl is a

68


direct mutagen to both TA98 and TA100 [68]. Similarly, Masson et al. [70] demonstrated that 4-nitrosobiphenyl was a direct mutagen to TA1538. They suggested that the lack of mutagenicity of 3-aminobiphenyl might be attributed to its N-hydroxyl metabolite not being a mutagen, and the lack of mutagenicity of the 2-aminobiphenyl might be attributed to the inability of the hepatic enzymes to convert it to its N-hydroxylate isomer. The presence of an amino group at the 2-position resulted in marked loss of planarity making it a poor substrate for the P450I family of proteins (enzymes), which readily catalyze the N-hydroxylation of many aromatic amines [68]. Although a thorough discussion is beyond the scope of this paper, we note that this group of compounds has been used by a number of investigators for examining specific structure activity relationships. Imamura et al. [71] analyzed the relationship between mutagenic activity and the electronic structure of 14 benzidine derivatives with least squares and cluster analysis. You et al. [64] tested the effect of ortho-substituents on the mutagenicity of benzidine derivatives using three strains (TA98, TA98/1,8-DNP6, and TA100) with S9 activation. El-Bayoumy et al. [72] found that for the methylated aryl amines (such as 3-methyl-4-aminobiphenyl), the methyl substituent located at ortho position to the amine moiety would cause a marked increase in mutagenicity. For example, 3-methyl-4-aminobiphenyl and 1-methyl-2-naphthylamine gave three-fold more mutation than 4-aminobiphenyl and 2-naphthylamine in S. typhimurium TA100. Likewise, Nussbaum et al. [73] reported that 3-methyl-4-aminobiphenyl induced a higher incidence of intestinal tumors than 4-aminobiphenyl in Wistar rats. Beland et al. [74] showed that arylamines with ortho alkyl substitutes tend to be more mutagenic and tumorigenic than analogues without an ortho alkyl substituent. They found that DNA-adducts containing alkyl groups ortho to the amine function had a greater percentage syn conformer about the glycosyl bond than those not bearing such groups. However, Ashby [75] found the synthesized 3,5-dimethyl-4aminobiphenyl was much less mutagenic than 4aminobiphenyl in TA98 with S9. This did not support the hypothesis of El-Bayoumy et al. [72] and Beland et al. [74]. Nevertheless, alkyl substitution on the ortho position to the amine moiety of the benzidine molecule would affect the potency of the mutagenicity of the molecule. The addition of a sulfonic acid group to the benzidine molecule may reduce the mutagenicity. Chung et al. [76] showed that 2-sulfo-p-phenylenediamine is not mutagenic to TA98. Ashby et al. [77] also showed that

4-aminobiphenyl-4-sulfonic acid is not mutagenic to TA1535, TA1537, TA1538 and TA98. The mutagenicity of benzidine–disulfonic acid is not known, but it is not a carcinogen in experimental animals [37,77]. Brown and DeVito in 1993 [15] pointed out that when the azo dyes were sulfonated on both sides of the azo linkage (giving two sulfonated aromatic amines upon azoreduction) they were not carcinogenic in any animal species. The lack of carcinogenic activity of the sulfonic acid derivatives may be associated with the increased water solubility conferred on them by the sulfonic acid substitute(s) [75,78,79]. Another contributing factor causing the decreased mutagenicity of benzidine derivatives appear to be the formation of a complex with a copper ion. Direct Blue 218, a copper complex of the mutagenic o-dianisidine dye Direct Blue 15, is not mutagenic [3]. Recent work of Edwards et al. [80] also showed that iron-complex formation of some azo dyes generally decreases bacterial mutagenicity. Thus, complexation with a metal may be an exciting new approach for designing a non-mutagenic azo dye. However, it is still too premature to make such a generalization. 4. General observations concerning metabolism It is also generally believed that initial metabolic activation involves N-oxidation subsequently leading to an electrophilic species, which can covalently interact with DNA or other macromolecules [81–84]. Morton et al. [85] proposed that the metabolic activation pathway of benzidine as: benzidine ! N-acetylbenzidine ! N,N0 -diacetylbenzidine ! N,N0 -diacetyl-N-hydroxybenzidine ! nucleic acid binding. Similarly, Josephy [86] suggested that the metabolic activation pathway is: benzidine ! N-acetylbenzidine ! N-hydroxy-N0 acetylbenzidine ! N-acetoxy-N0 -acetylbenzidine ! nitrenium ion ! DNA binding. Choudhary [22] summarized the principal features of several metabolic pathways currently thought to be involved or potentially involved in the metabolic activation of benzidine. The principal class of enzymes responsible for metabolic activation is the cytochrome P-450 system. The metabolism of 4-aminobiphenyl in the liver generally involves an initial N-oxidation to N-hydroxyaminobiphenyl by the hepatic cytochrome P-4501A2 [87]. N-Hydroxyaminobiphenyl then enters the circulation where it either becomes bound to hemoglobin or undergoes renal filtration into the urinary bladder lumen [88]. Upon reabsorption from the bladder epithelia, N-hydroxyaminobiphenyl is finally converted to a


reactive N-acetoxy derivative by N-acetyltransferase-1 (NAT-1). Metabolic activation of 4-aminobiphenyl to form DNA-adduct-creating metabolites is also reported to occur in human bladder microsomes by the hydroperoxidase activity of prostaglandin H synthase [89]. However, when 4-aminobiphenyl is inhaled into the lung, it is peroxidized by myeloperoxidase, and then it interacts with DNA to form DNA-adducts [90]. For benzidine, the major adducts were the C8-substituted deoxyguanosine derivatives [91,92]. G ! T transversions were identified as the major base-pair mutation at the first base of coden 61 in the c-Ha-ras gene mutational profile in chemically induced tumors in B6C3F I mice and in CD-1 mice [93,94]. Chen et al. [95] also demonstrated that G ! T transversions creates one of the major mutagenic spectra caused by 4,40 -dinitro-2-biphenylamine in Salmonella strain TA100. The metabolic activation of benzidine varies from species to species and organ to organ, and is subjected to the influence of various factors [96–100]. The detailed reviews of benzidine metabolism are available elsewhere [7,22] and are not included in this manuscript. The molecular structures and their mutagenic responses to Salmonella tester strains of these benzidine analogues are outlined in Table 1 and Appendices A–F. As shown by Shahin [37], a minor difference in substituent groups can lead to a major difference in mutagenicity.

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tion at the ortho position to the amine functional group affects the mutagenicity. However, the exact mechanism involved still needs further investigation. (3) The complexation of benzidine or benzidine congeners-based azo dyes with metal ions may produce non-mutagenic dyes or dyes with reduced mutagenic activity. This warrants further investigation. (4) An extended examination of structure–activity relationships is needed for this class of compounds. The factors involved in determining the minimum requirement for benzidine analogues, and the factors determining the potency of mutagenicity are still not exactly clear. It is still important to examine the structural factors affecting the mutagenicity of benzidine analogues. Such understanding of the structure–mutagenicity relationships will enable the development of non-mutagenic benzidine derivatives for wide application in industry. A minor difference in substituent groups can lead to major differences in the mutagenicity of benzidine, benzidine analogues, and benzidine-based dyes. A thorough understanding of how these features influence mutagenicity is key to unraveling the mechanisms involved in the metabolic activation of these carcinogenic amines. Such an understanding can also help in the design and manufacture of safe industrial compounds by developing a structure– activity strategy for creating non-mutagenic and non-carcinogenic compounds.

5. Conclusions Acknowledgements As the mutagenicity results are reviewed, the following observations appeared: (1) Metabolic activation is required for the mutagenicity of most benzidine analogues. The introduction of a nitro-group or halogen molecules into the molecules can convert them into direct mutagens; however, the effect of a halogen moiety is different from that of the nitro-group in causing the mutagenic activities of these analogues. The halogenated benzidines were mutagenic only to TA98 (frameshift mutations); whereas the nitrocompounds were mutagenic to both TA98 and TA100 (frameshift and base-substitution mutations). (2) Among all the benzidine analogues reported, only 3aminobiphenyl, N-hydroxy-3-aminobiphenyl, and 3,30 -5,50 -tetramethylbenzidine were not mutagenic in Salmonella strains. It seems that alkyl substitu-

The authors want to thank Helen R. Carlson for her skillful entry of data into the databases used for analysis, and to Thomas J. Hughes for his quality control efforts. This work was begun while Dr. Chung was a Resident Research Associate with Dr. Claxton under an Environmental Protection Agency—National Research Council cooperative agreement (Agreement Number 824072). The author would also like to thank Les Recio, Thomas J. Hughes, and Ron Rogers for their suggestions concerning this manuscript. This document has been reviewed by the National Health and Environmental Effects Research Laboratory in accordance with U.S. Environmental Protection Agency policy and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

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Appendix A. Mutagenic potencies, revertants/mg (revertants/micromole), of aminobiphenyls

Compound, molecular weighta

TA98, +S9b

TA98, S9

TA100, +S9

TA100, S9

TA1535, +S9

2-Aminobiphenyl, 169.222 3-Aminobiphenyl, 169.222 4-Aminobiphenyl, 169.222 3,30 -Diaminobenzidine, 214.267 3-Amino-4-methylbiphenyl, 183.249 4-Amino-3-methylbiphenyl, 183.249 4-Amino-2-methylbiphenyl, 183.249 4-Amino-40 -chlorobiphenyl, 203.667 4-Amino-40 -hydroxybiphenyl, 185.222 4-Amino-40 -methylbiphenyl, 183.249 4-Amino-40 -iodobiphenyl, 295.119

0.7 (118) Neg

Neg

2.0 (338) Neg

Neg

Neg

Neg

Neg

Neg

Neg

Neg

Neg

3.4 (575)

[72,69,28,67, 50,65] [64,50,65]

Neg

Neg

[72]

Neg

5.0 (916)

[72]

4-Amino-40 -cyanobiphenyl, 194.232 4-Amino-40 -nitrobiphenyl, 214.220 2,4-Acetamidobiphenyl, 211.259 4-Acetamidobiphenyl, 211.259 4,40 -Dinitro-2-biphenylamine, 259.218 4-(2-Phenylethyl)aniline, 197.276 a

35.2 (5956) 12.5 (2679)

Neg Neg

85.4 (14450) Neg 1.3 (279) 4.3 (788) 12.0 (2199) 7.9 Neg 13.5 (1448) (2474) 29.2 Neg 14.3 (5947) (2912) 0.6 Neg 0.2 (111) (37) 64.2 Neg 46.9 (11765) (8594) 28.5 Neg 10.4 (8411) (3069) 14.6 Neg 5.4 (2836) (1049) 48.1 12.0 (2571) 5.2 (10304) (1114) 0.4 (84.5) Neg 1.5 (317) 11.4 Neg 24.3 (2408) (5134) 2.8 118.0 (30588) 2.9 (726) (752) 0.6 Neg 1.2 (118) (237)

TA1535, S9

TA1538, +S9

0.1 (21)

TA1538, S9

References

[72,69, 50,65] [72,50]

Neg

[28]

Neg

[28]

Neg

[67]

Neg

[67]

Neg

[67]

Neg

[67]

Neg

[67]

Neg

[69]

Neg

[69]

38.7 (10032) Neg

[50] [28]

Molecular weight was taken from information on PubChem (http://pubchem.ncbi.nlm.nih.gov/). Values given are revertants/mg compound (calculated from the linear portion of the dose response using the model of Bernstein et al. [113] for positive compounds) and revertants/micromole (revertants/mg mg/micromole, rounded to integer) or Neg for non-mutagenic compounds. If the compound was not tested no indication is given. Positive results were determined by the significance of the slope rather than a two-fold rule. b

.


71

Appendix B. Mutagenic potencies, revertants/mg (revertants/micromole), of benzidine and methylbenzidines Compound, molecular weighta

TA98, +S9b

TA98, S9

Benzidine, 184.237 Benzidine dihydrochloride, 257.158 o-Tolidine, 212.29 3,30 ,5,50 -Tetramethylbenzidine, 240.343 N,N,N0 ,N0 -Tetramethylbenzidine, 240.343 N,N0 -Diacetyl-3,30 -dimethylbenzidine, 296.364

3.3 (608) 1.1 (283) 2.5 (531) Neg 0.2 (48) 1.0 (296)

Neg

TA100, +S9

0.3 (55) Neg 0.1 (26) 1.2 (255) 0.3 (64) Neg Neg Neg Neg Neg

TA100, TA1535, TA1535, TA1538, S9 +S9 S9 +S9

TA1538, S9

References

Neg

11.9 (2192)

[3,62,66,28, 64,67,50,65] [65]

Neg

3.5 (645)

Neg

0.7 (149) 1.7 (361) [3,62,64] [50] [50,65] 0.6 [62] (178)

Neg Neg Neg Neg

Neg Neg

a


.

Appendix C. Mutagenic potencies, revertants/mg (revertants/micromole), of methoxybenzidines Compound, molecular weighta

TA98, +S9b

o-Dianisidine, 244.289 3,30 -Dimethoxybenzidine hydrochloride, 280.760 3,30 -Dimethoxybenzidine dihydrochloride, 317.210 N-Acetyl-3,30 dimethoxybenzidine, 286.326 N,N0 -Diacetyl-3,30 -dimethoxybenzidine, 328.363 3,30 -Dicarbomethoxybenzidine, 300.309

8.4 (2052) 11.2 (3145) 9.6 (3045) 27.0 (7731)

a

TA98, S9

TA100, +S9

TA100, TA1535, TA1535, TA1538, TA1538, S9 +S9 S9 +S9 S9

0.5 (122) 1.6 (391) Neg 2.4 (674) Neg 2.4 (761) 4.1 (1174) 4.4 (1445) 0.6 (197) 123.0 (36938) Neg 3.5 Neg (1051)

Neg Neg Neg Neg

9.3 (2611) 9.3 (2950) 14.7 (4209) 4.1 (1346)

References

1.2 (293) [3,64,50,65] [62] [62,50] [62] [62] [64]


.

72


Appendix D. Mutagenic potencies, revertants/mg (revertants/micromole), of acetylbenzidines Compound, molecular weighta

TA98, +S9b

TA98, TA100, S9 +S9

TA100, TA1535, TA1535, TA1538, S9 +S9 S9 +S9

TA1538, References S9

N-Acetylbenzidine, 226.274 N,N0 -Diacetylbenzidine, 268.311 N-Acetyl-3,30 -dimethoxybenzidine, 286.326 N,N0 -Diacetyl-3,30 -dimethoxybenzidine, 328.363 N-Acetyl-3,30 -dichlorobenzidine, 295.163 N,N0 -Diacetyl-3,30 -dichlorobenzidine, 337.200

179.0 (40503) 13.6 (3649) 27.0 (7731)

84.7 (19165) Neg 4.1 (1174)

Neg Neg Neg

74.2 (16790) 23.7 (6359) 14.7 (4209)

[62] [62] [62]

4.4 (1445)

0.6 (197)

Neg

4.1 (1346)

[62]

316.0 (93272)

23.0 (6789)

Neg

113.0 (33353)

[62]

174.0 (58673)

14.6 (4923)

Neg

75.7 (25526)

[62]

a


.

Appendix E. Mutagenic potencies, revertants/mg (revertants/micromole), of nitrobenzidines Compound, molecular weighta

TA98, +S9b

TA98, S9

TA100, +S9

TA100, S9

2-Nitrobenzidine, 229.235 3-Nitrobenzidine, 229.235 3,30 -Dinitrobenzidine, 274.233

18.5 (4241) 3938.0 (902727) 544.0 (149181)

0.3 (69) 17.5 (4012) 1.9 (521)

1.8 (413) 129.0 (29571) 86.6 (23748)

Neg

[64]

1.4 (321) Neg

[64]

a

TA1535, +S9

TA1535, S9

TA1538, +S9

TA1538, S9

References

[64]


.


73

Appendix F. Mutagenic potencies, revertants/mg (revertants/micromole), of halogen-containing benzidines Compound, molecular weighta

TA98, +S9b

TA98, S9

TA100, +S9

3,30 -Dichlorobenzidine, 253.127 3,30 -Dibromobenzidine, 342.029 N-Acetyl-3,30 -dichlorobenzidine, 295.163 N,N0 -Diacetyl-3,30 -dichlorobenzidine, 301.200

184.0 (46574) 89.3 (30543) 316.0 (93272) 174.0 (52409)

9.2 (2329) 11.2 (3831)

5.3 (1342) 3.2 (1094) 23.0 (6789) 14.6 (4398)

TA100, S9

TA1535, +S9

TA1535, S9

TA1538, +S9

TA1538, S9

References

Neg

159.0 (40247)

[62,66, 64,50,65] [64]

Neg

113.0 (33353) 75.7 (22801)

[62]

Neg

Neg

[62]

a


.

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