Mutagenesfe vol.9 no.4 pp.295-299, 1994 Activation by caecal reduction of the azo dye D & C Red No. 9 to a bacterial mutagen Deborah Dillon, Robert Combes 1 and Errol Zeiger 2 ' 3 Inveresk Research International Ltd, Tranent EH33 2NE, Scotland, UK and 2 National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA 'Present address: FRAME, Eastgate House, 34 Stoney Street, Nottingham NG1 1NB, UK •'To whom correspondence should be addressed D & C Red No. 9 is a monoazo dye used for manufacturing printing inks, rubber and plastics, and as an additive in cosmetics and drugs. In an NTP carcinogenicity study in rats and mice it induced splenic sarcomas and liver nodules in male rats; no chemical-related tumours were induced in mice. On the basis of its contradictory responses in a range of in vitro tests and its inactivity in several in vivo genotoxicity assays, it has been suggested that the dye may act as a non-genotoxic carcinogen. We tested the dye in the Salmonella mutagenidty assay using several different protocols. The dye was not mutagenic when tested using the standard (aerobic) pre- incubation protocol. Variable responses were seen when the flavin mononucleotide (FMN) reduction protocol was used. A third protocol was provided by incubating the test compound overnight with a rat caecal preparation under anoxic conditions to reduce the azo bond. Ethyl acetate extracts of this incubation mixture, when tested in the standard preincubation protocol using induced rat liver S9, yielded dose-related mutagenic responses in TA100, and a weak response in TA98. The presumed major reduction product, l-amino-2-naphthol (1-A-2-N) was mutagenic to TA100, but not TA98, in standard protocols with S9. The results show that it is necessary to use a protocol in which D & C Red No. 9 is reduced in order to demonstrate the mutagenkity of this dye. The non-genotoxicity previously reported for D & C Red No. 9, may have been due to insufficient reductive cleavage. The carcinogenicity of this compound may, therefore, be a consequence of its genotoxicity, rather than a result of some non-genotoxic process. Introduction The monoazo dye, D & C Red No. 9 (Figure 1), has been used for many years in printing inks, polystyrene, rubber and enamels [International Agency for Research on Cancer (IARC), 1993]. In 1988, a special, certified grade of D & C Red No. 9 was provisionally listed by the US Food and Drug Administration (FDA) for use in externally and internally applied drugs and cosmetics (Food and Drug Administration, 1988; Dry Colour Manufacturers' Association, 1988). Acceptable uses included addition to lipsticks, dentifrices and mouthwashes, as well as in drugs intended for ingestion. However, the food and drug listings for all of these have since been terminated due to a reappraisal of the available toxicology (Food and Drug Administration, 1988; IARC, 1993). There was no evidence for tumour induction in mice in long-term carcinogenicity assays involving oral dosing of D & C Red No. 9. In two out of three studies with rats, the incidence of splenic sarcomas was significantly elevated in high dose male groups (National Toxicology Program, 1982; Weinberger et al., 1985). hi one of these studies, there was a non-significant increase also in hepatocellular nodules and carcinomas in males (National Toxicology Program, 1982). There was no indication of effects in female rats in any of these studies. It was concluded that there is limited evidence for the carcinogenicity of D & C Red No. 9 in rodents (Hart et al., 1986; IARC, 1993). D & C Red No. 9 was negative in most Salmonella mutagenidty assays (Brown et al., 1979; Muzzall and Cook, 1979; Miyagoshi et al., 1983), with the exception of a weakly positive response at precipitation dose levels (Zeiger et al., 1988). D & C Red No. 9 was non-genotoxic in mammalian cells in vitro, when tested for mutation induction at the tk locus in mouse lymphoma L5178Y cells (Myhr and Caspary, 1991), SCE and chromosomal aberrations in CHO cells (Ivett et al., 1989), and UDS in rat hepatocytes (Kornbrust and Barfknecht, 1985; Williams et al., 1989). The dye was inactive in two UDS assays in the liver, and in micronucleus assays in bone marrow of rats after oral administration (Kornbrust and Barfknecht, 1985; Westmoreland and Gatehouse, 1992). In view of the predominantly negative responses reported in genetic toxicity tests, it was suggested that D & C Red No. 9 behaves as a non-genotoxic carcinogen (Westmoreland and Gatehouse, 1992). We present data demonstrating that D & C Red No. 9 is reproducibly mutagenic in Salmonella using a protocol facilitating azo-reductive cleavage of the dye, such as can occur in the caecum in vivo. H,C HO Ba 2 + l-Amlno-2-naphthol H,C f V// VN=N OH NaO,S SO,Na Trypin Blue Fig. 1. Structures of D & C Red No. 9, l-amino-2-naphthol, and trypan blue. 295 at National Chung Hsing University Library on April 12, 2014 http://mutage.oxfordjournals.org/ Downloaded from
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Mutagenesfe vol.9 no.4 pp.295-299, 1994
Activation by caecal reduction of the azo dye D & C Red No. 9 toa bacterial mutagen
Deborah Dillon, Robert Combes1 and Errol Zeiger2'3
Inveresk Research International Ltd, Tranent EH33 2NE, Scotland, UK and2National Institute of Environmental Health Sciences, Research TrianglePark, NC 27709, USA
'Present address: FRAME, Eastgate House, 34 Stoney Street, NottinghamNG1 1NB, UK•'To whom correspondence should be addressed
D & C Red No. 9 is a monoazo dye used for manufacturingprinting inks, rubber and plastics, and as an additive incosmetics and drugs. In an NTP carcinogenicity study in ratsand mice it induced splenic sarcomas and liver nodules in malerats; no chemical-related tumours were induced in mice. Onthe basis of its contradictory responses in a range of in vitrotests and its inactivity in several in vivo genotoxicity assays,it has been suggested that the dye may act as a non-genotoxiccarcinogen. We tested the dye in the Salmonella mutagenidtyassay using several different protocols. The dye was notmutagenic when tested using the standard (aerobic) pre-incubation protocol. Variable responses were seen when theflavin mononucleotide (FMN) reduction protocol was used.A third protocol was provided by incubating the testcompound overnight with a rat caecal preparation underanoxic conditions to reduce the azo bond. Ethyl acetateextracts of this incubation mixture, when tested in thestandard preincubation protocol using induced rat liver S9,yielded dose-related mutagenic responses in TA100, and aweak response in TA98. The presumed major reductionproduct, l-amino-2-naphthol (1-A-2-N) was mutagenic toTA100, but not TA98, in standard protocols with S9. Theresults show that it is necessary to use a protocol in whichD & C Red No. 9 is reduced in order to demonstrate themutagenkity of this dye. The non-genotoxicity previouslyreported for D & C Red No. 9, may have been due toinsufficient reductive cleavage. The carcinogenicity of thiscompound may, therefore, be a consequence of its genotoxicity,rather than a result of some non-genotoxic process.
IntroductionThe monoazo dye, D & C Red No. 9 (Figure 1), has been usedfor many years in printing inks, polystyrene, rubber and enamels[International Agency for Research on Cancer (IARC), 1993].In 1988, a special, certified grade of D & C Red No. 9 wasprovisionally listed by the US Food and Drug Administration(FDA) for use in externally and internally applied drugs andcosmetics (Food and Drug Administration, 1988; Dry ColourManufacturers' Association, 1988). Acceptable uses includedaddition to lipsticks, dentifrices and mouthwashes, as well as indrugs intended for ingestion. However, the food and drug listingsfor all of these have since been terminated due to a reappraisalof the available toxicology (Food and Drug Administration,1988; IARC, 1993).
There was no evidence for tumour induction in mice in
long-term carcinogenicity assays involving oral dosing of D & CRed No. 9. In two out of three studies with rats, the incidenceof splenic sarcomas was significantly elevated in high dose malegroups (National Toxicology Program, 1982; Weinberger et al.,1985). hi one of these studies, there was a non-significant increasealso in hepatocellular nodules and carcinomas in males (NationalToxicology Program, 1982). There was no indication of effectsin female rats in any of these studies. It was concluded that thereis limited evidence for the carcinogenicity of D & C Red No. 9in rodents (Hart et al., 1986; IARC, 1993).
D & C Red No. 9 was negative in most Salmonella mutagenidtyassays (Brown et al., 1979; Muzzall and Cook, 1979; Miyagoshiet al., 1983), with the exception of a weakly positive responseat precipitation dose levels (Zeiger et al., 1988). D & C RedNo. 9 was non-genotoxic in mammalian cells in vitro, whentested for mutation induction at the tk locus in mouse lymphomaL5178Y cells (Myhr and Caspary, 1991), SCE and chromosomalaberrations in CHO cells (Ivett et al., 1989), and UDS in rathepatocytes (Kornbrust and Barfknecht, 1985; Williams et al.,1989). The dye was inactive in two UDS assays in the liver, andin micronucleus assays in bone marrow of rats after oraladministration (Kornbrust and Barfknecht, 1985; Westmorelandand Gatehouse, 1992).
In view of the predominantly negative responses reported ingenetic toxicity tests, it was suggested that D & C Red No. 9behaves as a non-genotoxic carcinogen (Westmoreland andGatehouse, 1992). We present data demonstrating that D & CRed No. 9 is reproducibly mutagenic in Salmonella using aprotocol facilitating azo-reductive cleavage of the dye, such ascan occur in the caecum in vivo.
H,C HOBa2 +
l-Amlno-2-naphthol
H,C
f V// VN=N OH
NaO,S SO,Na
Trypin Blue
Fig. 1. Structures of D & C Red No. 9, l-amino-2-naphthol, and trypanblue.
Materials and methodsChemicalsD & C Red No. 9 [5-cblon>-2^(24iydroxy-l-naphlhalenyI)azoH-niahyIbaizene-sulpbonic acid, barium salt; CASRN 5160-02-1; C.I. 15585; Figure 1] was fromRadian Corp., Austin, TX, USA, and was used without further purification. Thesample used was from the same batch as was used for the NTP carcinogenkntyassay (National Toxicology Program, 1982). l-Amino-2-naphrhol HC1 (1-A-2-N;CASRN 1198-27-2; Figure 1), FMN, NADPH and 2-aminoanthracene (positivecontrol) were from Sigma (Poole, Dorset, UK). NADP was from Randox(Crumlin, Co. Antrim, No. Ireland, UK). Trypan blue (positive control;CASRN 72-57-1; C.I. 23850; Figure 1) was from Aldrich (Wembley, Middlesex,UK). G-6-P-Dehydrogenase (G-6-PDH) was from Sigma. Ethyl acetate (98.5%pure) was from Rathbum Chemicals (Walkerburn, Scotland, UK).Tester strains
Salmonella typhimurium tester strains TA98 and TA100 were from ProfessorB.N.Ames (Berkeley, CA). The strains were stored in liquid nitrogen and cultureswere grown in 25 ml nutrient broth (Oxoid No. 2) in 125 ml Erlenmeyer flasksat 37°C for 16 h at 60 r.p.m., in a gyratory incubator.
Metabolizing systems
Liver S9 was prepared from Aroclor-induced male Fischer F344 rats, and contained25 mg protein/ml (Ames a al., 1975). Cofactors were added to toe S9 as in Ameset al. (1975) to produce S9 mix which was used at 30%. This S9 mix was usedin all experiments that employed non-reductive metabolic activation.
Mutagenicity assay
Liquid preincubation was conducted according to Maron and Ames (1983), butwith 30 min preincubation in a gyratory water bath at 100 r.p.m. FMN reductionwas based upon Prival and Mitchell (1982), but narrow bore tubes (0.9-1.1 cminternal diameter) were used with stationary incubation at 30°C (Robertson « al.,1982). S9 for FMN reduction contained: G-6-P (20 mM), NADP (4 mM), NADH(2 mM), FMN (2 mM), MgCl2-6H2O (8 mM), KC1 (33 mM), G-6-PDH (2.8units/ml), all in sodium phosphate buffer (0.1 M, pH 7.4) with rat liver S9 (10%v/v). Each tube contained S9 mix (0.5 ml), bacterial culture (0.1 ml), and testchemical in DMSO (0.1 ml). After incubation, 2.0 ml of top agar was addedbefore overlaying Vogel—Bonner agar plates (Maron and Ames, 1983).
In the caecal reduction protocol, 5 ml of DMSO (control) or dye (20 mg/mlin DMSO) was added to a capped tube containing caecal suspension [2 ml froma single Fischer F344 male rat (150-220 g)] in 73 ml brain-heart infusion broth(Reid et al., 1984). After overnight incubation at 30°C in an anaerobic workstation,the suspension was extracted with ethyl acetate and subjected to rotary evaporationand drying under N2. Initial experiments showed that centrifugation of thesuspension at 14 000 g for 10 min was unnecessary. Dried extracts were dissolvedin a minimum volume of DMSO and tested in the standard plate assay usingAroclor-induced rat liver S9 (composition as for liquid preincubation).
Concurrent positive controls were 2-aminoanthracene for preincubation, andtrypan blue for the FMN and caecal reduction protocols, respectively.
Table and figure legends include concentrations in ^mol/plate of the startingmaterials (D & C Red No. 9, DMSO, 1-A-2-N and trypan blue, respectively)which are equivalent to the top dose-level of each extract tested. Caecal extractdoses are calculated as II% dried extract/plate, as recovery of starting materialby the extraction process is not 100% efficient and is variable between experiments,and the extract contains components of the caecal preparation in addition to thedyes and their metabolites.
ChromatographyThin layer chromatography was performed using prelayered silica gel (Merck60 ?2M' 2 0 x 2 0 am) Plat" wi* 0 2 5 mm l a v e r thickness. The followingsolvent systems were used: (i) ethyl acetate:memanol:ammonium hydroxide:water(71:15:4:10 v/v); (ii) butanol:methylethylketone:ammonium hydroxide:water(50:30:10:10 v/v); and (iii) isoamyl alcohol:acetone:ammonium hydroxide:water(46:35:4:15 v/v). All solvents were of analytical grade. Spots were detected undervisible and UV (254 nm) light. For liquid chromatography (HPLQ, the followingequipment was used: Waters 510 or Philips 4100 pumps; Gilson 231 or Varian9090 autoinjectors; Pye Unicara PU4020 or Philips 4110 UV detectors; and PyeUnicam HP3396A or Trivector Trio data capture systems. Two differentchromatographic conditions were used: (i) column—hypersil or nucleosil ODS(5 /an), 250 x 4.6 mm, mobile phase—0.01 M tetraburylammoahim hydroxide:methanol:acetic acid (29:70:1 v/v), flow—1 ml/min; (ii) column—Lichrosorb RP8(10 fim) 250 x 4.6 mm, mobile phase A—0.01 M sodium perchlorate, mobilephase B—acetonitrile, programme—90%A to 47%A (linear) over 30 min, 47%Ato 90%A (linear) over 5 min, flow—2.5 ml/min. Solutions of reference standardswere prepared in DMSO at ~ 1 mg/ml.
ResultsD & C Red No. 9 was not mutagenic in strain TA98 in thestandard preincubation protocol or with FMN reduction (data not
Table I. Mutagenicity of D .&C Red1 No. 9strain TA100 using two different protocols
shown). In TA100, it produced negative or equivocal responsesin the standard preincubation and FMN procedures (Table I).The positive control, trypan blue, was mutagenic in TA100following FMN reduction (Table I).
Ethyl acetate extracts of an overnight incubation of D & C RedNo. 9 with the rat caecal preparation were mutagenic in TA100(Figure 2) but produced only a weak response in TA98 (Figure3). The different controls, extracted DMSO samples, wereinactive. The varying weights of extract added to the plateswere based on the amounts of non-polar material extracted byethyl acetate. This non-polar material included dye metabolitesand unmetabolized dye, as well as components of the caecalpreparations. As a result, it was not possible to quantify theamounts of dye and metabolites in the extracts.
Each experiment in Figure 2 was run with a different isolateof caecal flora. The differences in responses between experimentscould be a function of the makeup of the flora in the sample used,the number of appropriate organisms present, or the degree ofmetabolism achieved in the reaction tube.
The presumed reduction product, 1-A-2-N, was weaklymutagenic in strain TA100 in the standard preincubation assay;a slight, dose-related increase in mutant colonies was obtainedup to 160 jtg/plate, leading to a 1.6-fold increase (Table II). Nomutagenicity was seen in TA98, although 1-A-2-N was more tox-ic in this strain (data not given). Extracts prepared from an over-night caecal reduction of 1-A-2-N were mutagenic in TA100( —2.9-fold increase) and gave a marginal response in TA98(Table LT).
The authentic D & C Red No. 9 gave the following R{ valuesusing TLC with the respective solvent systems: 0.41 (i); 0.49(ii); 0.51 (iii). In most experiments, no spots corresponding toD & C Red No. 9 or 1-A-2-N were detectable in the ethyl acetateextracts of the overnight caecal incubates. HPLC retention times(min) at 502 nm for D & C Red No. 9 using the respectivesolvent systems were: 9.9 (i); 16.3 (ii). In most experiments only1 -4% of the parent dye was detectable by HPLC (100% nominalconcentration). TLC/HPLC of overnight caecal incubatestherefore suggested substantial, but incomplete, azo reduction.
DiscussionThe standard preincubation and FMN supplementation protocolswere not sufficiently rigorous to demonstrate the potentialmutagenicity of D & C Red No. 9. In the studies reported here,mutagenic responses were obtained only with extracts preparedfrom overnight incubations of the dye with rat caecal contentscomprising a mixed suspension of bacteria under reducing
Fig. 2. Mutagenicity of rat caecal incubates (circles, experiment 1; triangles, experiment 2): D & C Red No. 9 ( • . A ) and DMSO procedure control (O,A)in Salmonella strain TA100 (mean ± SD). Dose-levels are presented as /ig/plate (see text) and the highest doses tested are equivalent to the respective mol ofstarting substances/plate as follows. Experiment 1: D & C Red No. 9, 5.6 /imol/plate; DMSO, 1.6 /unol/plate. Experiment 2: D & C Red No. 9,13.1 /imol/plate; DMSO, 2.8 /imol/plate.
"Mean (triplicate plates) his+ revertams/plate ± SD.'TDMSO control extracted similarly to the 1-A-2-N-containing sample. The values in /ig/plate represent the amounts of substances from the caecal preparationthat were extracted by ethyl acetate.°Dose-levels are presented as /ig DMSO extract/plate (see text). The highest doses tested are equivalent to the respective mol of starting substances/plate:DMSO, 2.8 /imol/plate; 1-A-2-N, 7.5 /tmol/plate.
conditions. Evidence for reduction was provided by chromato-graphic analyses of the incubation productions, in which no, orlittle, original dye was detected.
The non-mutagenicity of the dye in standard protocols confirmsprevious experiments in which D & C Red No. 9 was non-mutagenic, or gave equivocal or weak positive responses at best(Brown et al., 1979; Muzzall and Cook, 1979; Miyagoshi et al.,1983; Zeiger et al., 1988). Indeed, it is noteworthy that thosedose levels at which a weak positive response was recorded,caused precipitation of dye on the plates (Zeiger et al., 1988).It is, thus, unclear whether the reported activity was due to thetest compound or to a low-level contaminant.
The need for azo reduction in order to detect D & C Red No. 9
mutagenicity is consistent with structure—activity considerations(Combes and Haveland-Smith, 1982; Rosenkranz and Klopman,1989). The parent molecule possesses a sulphonic acid group,a moiety suspected of being detoxifying for in vivo biologicalactivity of related and other chemicals. This group is substituenton only one ring system and, consequently, reductive cleavageof the dye would yield the unsulphonated molecule 1-A-2-N uponreductive cleavage. It was not possible to verify the presence ofthis reduction product as no suitable chromatographic methodcould be found for the authentic sample, probably due to thesusceptibility of the compound to autoxidation in air. There areother examples of azo dyes that are mutagenic only followingbacterial reduction provided either by caecal extracts or by
Fig. 3. Mutagenkity of rat caecal incubates of D & C Red No. 9 (A),trypan blue ( • ) ami DMSO procedure control ( • ) in Salmonella strainTA98 (means ± SD). Dose levels are presented as ^g/plate (see text) andthe highest doses tested are equivalent to the respective mol of startingsubstances/plate as follows. D & C Red No. 9, 5.6 jimol/plate; DMSO,1.6 fimol/plate; trypan blue, 0.135 junol/plate. At 1000 y.% extract/plateusing trypan blue, 165 ± 5 revertants/plate were recovered.
specific species under anaerobic conditions (Combes andHaveland-Smith, 1982; Reid et al., 1984; Levine, 1992). Thismode of activation is important because such conditions occurin the intestines of humans and laboratory animals.
1-A-2-N was marginally mutagenic both in the preincubationassay and after extraction from caecal incubates, under die sameconditions as those resulting in D & C Red No. 9 mutagenicity.Similar concentrations of extracts of the caecal reduction productsfrom D & C Red No. 9 and 1-A-2-N induced similar levels ofrevertants. It is feasible that, in the absence of toxicity, themaximum revertant yields for D & C Red No. 9 and 1-A-2-Nmay have been comparable. It is unlikely that the mutagenicityof reduced D & C Red No. 9 could be due to formation of2-amino-5-chloro-4-methylbenzenesulphonic acid (CLT acid) byreduction because this compound is non-mutagenic (Shimizuetal., 1985).
Our results widi 1-A-2-N contrast with two earlier studies inwhich it was reported to be non-mutagenic in Salmonella. In oneof these, 1-A-2-N was tested in TA1538 to a maximum dose of100 /ig/plate (Garner and Nutman, 1977). In the other study,1-A-2-N was tested in TA100 and other strains to a maximumdose level of 25 /tg/ml due to toxicity (Chung et al., 1981). Inthe studies reported here, 1-A-2-N was tested up to 200 /ig/platein TA100, and the maximum mutagenic effect was detected at150-160 /ig/plate (Table II).
Therefore, D & C Red No. 9 was activated by caecal reductionto a mutagen in TA100. One of the presumed major reductionproducts, 1-A-2-N, also was mutagenic in TA100 with andwithout caecal reduction. However, there is insufficient evidenceto attribute the activity of the parent dye to this metabolite.Firstly, the mutagenicity of 1-A-2-N was weak and needs furthersubstantiation in view of the negative data generated in otherlaboratories. Secondly, we were unable to confirm its presencein samples of D & C Red No. 9 following overnight caecal
incubation, despite evidence for loss of parent dye which suggestedextensive azo reduction. Thirdly, the sample of D & C Red No. 9used was 89.8% pure and the presence of potentially mutageniccontaminants cannot be discounted. These contaminants couldinclude unsulphonated D & C Red No. 9 which, upon reduction,would yield 5-chloro-m-toluidine (4-chloro-3-methylaniline).
It is of interest that several other azo dyes that can be reducedto 1-A-2-N, such as D & C Orange 4, D & C Red No. 2, SudanI, Sudan Yellow, Oil Red XO and Mercury Orange, have beenreported to be non-mutagenic in Salmonella using standardaerobic test procedures, probably due to lack of sufficient azoreduction (Chung etal., 1981; Combes and Haveland-Smith,1982; Levine, 1992). Other colours derived from 1-A-2-N provedto be mutagenic, eitfier because the dyes possess other structuralfeatures such as nitro and/or methyl groups (for example, CIPigment Red No. 3; D & C Orange 17) rendering the compoundsmutagenic, because a mutagenic compound is derived from theother reduction product (Levine, 1992; IARC, 1993), or becauseof tile presence of impurities.
Acid Alizarin Violet N, Orange II and Lithol Red are dyeswhich can be reduced to 1-A-2-N and which are weakly activeor mutagenic in Salmonella (Chung, 1983). The other cleavageproducts in each case, 5-hydroxyaniline-5-sulphonate, sulphanil-amide and 2-amino-l-naphdialene sulphonic acid respectively areconsidered non-mutagenic (Jung etal., 1992). Acid AlizarinViolet N was mutagenic only with chemical reduction in TA1535,TA1538 and TA98, but not in TA100 (Brown etal., 1978).Lithol Red and Orange II showed weak mutagenicity in TA1538with no data given for TA100. Attributing the mutagenicity ofthese dyes to 1-A-2-N is complicated, therefore, especially sinceeffects were detected only at high doses (500-5000 /ig/plate),increasing the possibility that impurities could be responsible.
Our data therefore imply that the carcinogenicity of D & CRed No. 9 may be due to the presence of an in vivo reductionproduct that is mutagenic. This conclusion contradicts the recentsuggestion that the dye behaves as a non-genotoxic carcinogen(Westmoreland and Gatehouse, 1992). These audiors noted thatD & C Red No. 9 was non-genotoxic in tile bone marrow andliver of rats when administered at doses up to 2000 mg/kg. Inthose studies the dye was administered orally and would, thus,have been exposed to gut microflora, with the concomitantpossibility of reductive azo cleavage. It is possible, however,that the tissues sampled for genotoxicity were not exposed tothe dye reduction products at levels sufficient to inducechromosome damage.
The data obtained imply that the non-genotoxicity previouslyreported for D & C Red No. 9, may have been due to insufficientreductive cleavage in the in vitro test conditions. Therefore, thecarcinogenicity of this dye batch used in the NTP studies maybe a consequence of the mutagenicity of a reduction product,rather than the result of a non-genotoxic process.
AcknowledgementsThe expert technical assistance of Margaret McCartney, Aileen Mitchell and SteveWilliams is gratefully acknowledged. This work was supported by contract no.NO1-ES-55127 with the NIEHS.
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Received on August 27, 1993; accepted on March 3, 1994
