Mutagenesis, Vol. 16, No. 2, 169-177,
March 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
The preparation of anthraquinone used in the National Toxicology Program cancer bioassay was contaminated with the mutagen 9-nitroanthracene
Butterworth Consulting, 4820 Regalwood Drive, Raleigh, NC 27613, USA and 1 Environmental Biocontrol, Intl, 3521 Silverside Road, Wilmington, DE 19810, USA
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Commercial anthraquinone (AQ) (9,10-anthracenedione) is produced by at least three different production methods worldwide: oxidation of anthracene (AQ-OX), Friedel–Crafts technology (AQ-FC) and by Diels–Alder chemistry (AQ-DA), with the final product varying in color and purity. AQ-OX begins with anthracene produced from coal tar and different lots can contain various contaminants, particularly the mutagenic isomers of nitroanthracene. AQ has been reported to be negative in a variety of genotoxicity tests including numerous Ames Salmonella mutagenicity assays. In addition, we report that AQ-DA is negative in the Salmonella–Escherichia coli reverse mutation assays, the L5178Y mouse lymphoma forward mutation assay, for inducing chromosomal aberrations, polyploidy or endoreduplication in Chinese hamster ovary cells, and in the in vivo mouse micronucleus assay. Further, a previous 18 month bioassay conducted with AQ administered to male and female B6C3F1 and (C57BL/6xAKR)F1 mice reported no induction of cancer. Thus, it was somewhat unexpected that in a long-term study conducted by the National Toxicology Program (NTP) AQ-OX induced a weak to modest increase in tumors in the kidney and bladder of male and female F344/N rats and a strong increase in the livers of male and female B6C3F1 mice. In the studies reported here, a sample of the AQ-OX used in the NTP bioassay was shown to be mutagenic in the Ames tester strains TA98, TA100 and TA1537. Addition of an S9 metabolic activation system decreased or eliminated the mutagenic activity. In contrast, the purified NTP AQ-OX as well as the technical grade samples AQ-FC and AQ-DA were not mutagenic in the Ames test. The chemical structure of AQ does not suggest that the parent compound would be DNA reactive. Therefore, a mutagenic contaminant was present in the NTP bioassay sample that is either directly mutagenic or can be activated by bacterial metabolism. Analytical studies showed that the primary contaminant 9-nitroanthracene (9-NA) was present in the NTP AQ-OX at a concentration of 1200 p.p.m., but not in the purified material. The 9-NA and any other contaminants that might have been present in the NTP AQ-OX induced measurable mutagenicity at 9-NA concentrations as low as 0.15 µg/plate in tester strain TA98, indicating potent mutagenic activity. On the basis of revertants per microgram, 9-NA was more potent than benzo[a]pyrene (B[a]P) and was about equally as potent as the 2-nitrofluorene run concurrently as positive controls. TD50 quantitative carcinogenicity potency estimates indicate that a carcinogen of a potency in the range between B[a]P and dimethylnitrosamine would be required to produce the observed carcinogenic response at the levels of the contaminants found in the test sample. While recognizing that there are limitations in extrapolating mutagenic potency to potential carcinogenic potency, these estimates do indicate that it is plausible that the 9-NA contaminant might have been responsible for all of the tumor induction observed in the NTP study. In fact, in the absence of reliable cancer data, the genetic toxicology profile indicates that AQ would not be a genotoxic carcinogen. Thus, no conclusion as to the carcinogenic activity of AQ can be made at this time.
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The importance of source and purity
Anthraquinone (AQ) (9,10-anthracenedione) is used to enhance the efficiency of the Kraft Process for the production of paper, thus reducing the demand for trees to be cut down (Cofrancesco, 1992
). AQ is the active ingredient in the most effective and non-harmful bird repellent used, for example, for keeping birds from airport runways or areas where they would conflict with the human population (Ballinger and Price, 1996; Cummings et al., 1997; Ballinger et al., 1998; Dolbeer et al., 1998).
In assessing the potential biological activity of preparations of AQ, it is critical to be aware of how the preparation of interest was manufactured and the potential contaminants inherent with the different synthesis processes. AQ is produced in large quantities by at least three different production methods in various parts of the world (Cofrancesco, 1992). The oxidation of anthracene to yield AQ is the oldest known production process and is now practiced primarily in Europe. AQ from the oxidation process (AQ-OX) involves the oxidation of anthracene derived from coal tar. The quality of the AQ-OX produced is dependent on the number of contaminating high-boiling mutagenic and carcinogenic polycyclic aromatic hydrocarbons (PAHs), which co-distill with anthracene, found in the starting material. Profiles of contaminants from this process can differ substantially. Of particular concern is the observation that the mutagenic nitroanthracenes are often seen in AQ-OX preparations, sometimes at concentrations >2500 p.p.m. (US EPA, 1977; ICI, 1978a, 1978b).
Benzene and phthalic anhydride undergo the Friedel–Crafts reaction to yield o-benzoylbenzoic acid, which is treated with concentrated sulfuric acid to yield AQ. This is the most prevalent production method employed in China and India. AQ produced by the Friedel–Crafts process (AQ-FC) is substantially free of the PAH contaminants and nitroanthracenes that can be found in AQ-OX.
Production of AQ by the Diels–Alder reaction (AQ-DA) between 1,4-naphthoquinone and 1,3-butadiene is practiced primarily in Japan. Because this process involves shifts between the aqueous and organic phases, contaminants are easily removed and AQ-DA is particularly clean and free of contaminants.
To our knowledge, all AQ used commercially in the US is either AQ-FC or AQ-DA, rather than AQ-OX. Interestingly, the reagent grade material supplied to research laboratories is often AQ-OX. The National Toxicology Program (NTP) recently completed a cancer bioassay with AQ showing that it exhibited weak to modest carcinogenic activity (NTP, 1999). The material employed in that bioassay was from the oxidation process, AQ-OX, and contained an unidentified peak by GC analysis at a level of 0.12% (Battelle, 1993; NTP, 1999). One purpose of the studies presented here was to identify and quantify that and other potential contaminants in the material used by the NTP.
Genotoxicity
A large number of mutagenicity assays have reported that neither AQ nor its metabolites exhibit genotoxic activity. Negative results in the Ames Salmonella bacterial mutagenicity assay have been reported by seven independent laboratories (Brown and Brown, 1976; Anderson and Styles, 1978; Gibson et al., 1978; Salamone et al., 1979; Sakai et al., 1985; Tikkanen et al., 1983; National Cancer Institute, 1987). AQ is negative in the Syrian hamster embryo (SHE) cell transformation assay (Kerckaert et al., 1996). AQ is also not mutagenic in a line of human B-lymphoblastoid cells that constitutively express cytochrome P4501A1 (Durant et al., 1996). An 18 month bioassay conducted with AQ administered to male and female B6C3F1 and (C57BL/6xAKR)F1 mice reported no induction of cancer (Innes et al., 1969). In that study, AQ composition was confirmed by infrared spectroscopy, gas chromatography and thin-layer chromatography. No contaminants were reported (Innes et al., 1969).
Thus, it was somewhat unexpected when the NTP reported that AQ induced a weak to modest increase in tumors in the kidney and bladder of male and female F344/N rats and a strong increase in the livers of male and female B6C3F1 mice (NTP, 1999). In contrast to the numerous papers documenting a lack of AQ mutagenic activity noted above, two papers reported that AQ was mutagenic in the Ames Salmonella mutagenicity assay. The pattern of activity of AQ reported was, however, unusual in that mutagenic activity was seen without metabolic activation, and that addition of an S9 metabolic activation system reduced or eliminated the response (Liberman et al., 1982; Zeiger et al., 1988). The chemical structure of AQ does not suggest that the parent compound would be a DNA reactive mutagen. Therefore, it appeared that a mutagenic contaminant was present in the positive Ames test samples that was either directly mutagenic or could be activated by bacterial metabolism. Similarly, there is one report of weak induction of micronuclei in SHE cells, but the material used was the NTP AQ-OX (Gibson et al., 1997) and, as noted below, several other micronuclei assays are negative.
In fact, the problem of contamination of AQ-OX with nitroanthracenes producing a mutagenic preparation has been well documented (US EPA, 1977; ICI, 1978a, 1978b). For example, for a TSCA submission, six samples of AQ were submitted to the Ames Salmonella mutagenicity assay (US EPA, 1977). Only one was positive, and that was without metabolic activation. It was concluded that the mutagenic activity came from contamination of the sample with 9-nitroanthracene (9-NA). When that sample was purified and retested, it showed no mutagenic activity.
It is clear that samples of technical grade AQ can vary widely in how they are produced, and the types and amounts of impurities. Another purpose of these studies was to determine the mutagenic activity of the actual material used in the NTP bioassay and to contrast that with other preparations of AQ. A sample of the archived NTP AQ-OX was generously provided by the NTP. The activity of this material was contrasted in the Ames Salmonella mutagenicity assay with purified NTP AQ-OX as well as samples of AQ-FC and AQ-DA. In addition, results from several additional genotoxicity assays are reported using the AQ-DA material.
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Chemicals
A sample of the AQ-OX powder employed in the NTP 2-year toxicology and carcinogenesis studies (NTP, 1999) was generously provided by Cynthia Smith of the NTP and Donna Browning, NTP Chemical Custodian (Battelle, Columbus, OH). This sample was designated as NTP AQ-OX. The bright yellow powder obtained from the NTP was labeled Anthraquinone, Battelle Task Identifier: 5-064-SHIP-211, lot: 5893, CAS: 84–65-1. The technical report stated that the sample had been analyzed by the NTP and was found to be ~99% AQ and noted an impurity at a concentration of 0.12% (Battelle, 1993
; NTP, 1999). A portion of the NTP AQ-OX was purified by 2x recrystalization from ethanol. This sample was designated as purified NTP AQ-OX.
A sample of technical grade AQ-FC typical of that in commercial use was obtained from Environmental Biocontrol, Intl. (Wilmington, DE). This sample was designated as AQ-FC.
Samples of technical grade AQ-DA typical of that in commercial use were obtained from Environmental Biocontrol, Intl (Wilmington, DE). These were used over a period of several months for the various genotoxicity assays presented here. High quality control standards and analytical verification indicated that these samples were very uniform. These samples were designated as AQ-DA.
Analytical techniques including high-performance liquid chromatography (HPLC) and GC-MS were performed by DCV Group (Wilmington, DE) and Covance Laboratories (Leesburg, VA) to verify that the main component in all preparations was AQ, that purification did not alter the parent compound and to identify potential contaminants (Table I). GC-MS analysis was conducted to identify impurities. US EPA Methods 610, 625 and 8270 in the Code of Federal Register 40 identify 16 PAH and 72 additional toxic or carcinogenic substances of concern in environmental samples. Analyses were done using the conditions and looking for the target toxic compounds specified in these standard procedures. Quantitation of contaminants was based on HPLC analysis.
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Bacterial mutagenicity assays
Samples NTP AQ-OX, purified NTP AQ-OX, AQ-FC and AQ-DA were submitted to Covance Laboratories (Vienna, VA) to test for mutagenic activity in the Salmonella–Escherichia coli/Mammalian-Microsome Reverse Mutation Assay with a confirmatory assay. Industry accepted standard protocols and published procedures were followed in compliance with Good Laboratory Practice regulations (Ames et al., 1975
; Brusick et al., 1980; Maron and Ames; 1983). The assay tested for the ability to induce reverse mutations at the histidine locus in the genome of specific Salmonella typhimurium tester strains (Ames test), and the tryptophan locus in an E.coli tester strain both in the presence and absence of an exogenous metabolic activation system (Ames et al., 1975; Brusick et al., 1980; Maron and Ames; 1983). The activation system was a microsomal enzyme preparation derived from Aroclor-induced rat liver (S9). Dose levels were based on a toxicity range-finding study. The top doses demonstrated test article precipitate on the plates. No appreciable toxicity was observed with any of the samples. The experiments were repeated independently to confirm initial results. Criteria for a positive response were at least a 2-fold increase in the mean revertants per plate of at least one tester strain over the mean revertants per plate of the appropriate vehicle control for tester strains TA98, TA100 and WP2uvrA and/or at least a 3-fold increase for tester strains TA1535 and TA1537.
L5178Y thymidine kinase (TK)+/– mouse lymphoma forward mutation assay
A sample of AQ-DA was submitted to Covance Laboratories (Vienna, VA) to test for mutagenic activity in the L5178Y TK+/– mouse lymphoma forward mutation assay. Industry accepted standard protocols and published procedures were followed in compliance with Good Laboratory Practice regulations (Amacher et al., 1980; Clive et al., 1987). The objective of the assay was to evaluate the ability of AQ to induce forward mutations at the TK locus in the mouse lymphoma L5178Y cell line. The test article formed a suspension in dimethylsulfoxide at concentrations >1.56 mg/kg and was insoluble in medium above ~25 µg/ml. Assays were run with and without a rat liver S9 metabolic activation system. Range-finding studies showed AQ to be non-toxic at nominal doses up to 500 µg/ml with and without metabolic activation. The testing limit for the mutation assays was set at 50 µg/ml, which is about twice the solubility limit in medium. Two independent assays were conducted each with and without metabolic activation. The criterion for a positive response is induction of a mutation frequency that is at least two times that of the control mutant frequency for that given experiment. Colony sizing was not done because no positive responses were observed with AQ.
Chromosomal aberration induction in Chinese hamster ovary (CHO) cells
A sample of AQ-DA was submitted to Covance Laboratories (Vienna, VA) to test for the ability to induce chromosomal aberrations in CHO cells with and without metabolic activation. Industry accepted standard protocols and published procedures were followed in compliance with Good Laboratory Practice regulations (Evans, 1962). A dose range-finding study was conducted to select experimental doses. Solubility considerations determined the highest dose to be 50 µg/ml. Replicate cultures of CHO cells were incubated with up to 50 µg/ml AQ-DA with and without metabolic activation, with a 20.0 h harvest in an initial trial and with 20.0 and 44.0 harvests in the confirmatory trials. No visual signs of cytotoxicity were observed in the cultures analyzed. A test article was considered positive for inducing chromosomal aberrations if a significant increase was observed compared to controls at a level of P < 0.01
In vivo bone marrow mouse micronucleus assay
A sample of AQ-DA was submitted to Covance Laboratories (Vienna, VA) for evaluation in the in vivo mouse micronucleus assay. The objective of this whole animal assay was to evaluate the ability of AQ-DA to induce micronuclei in bone marrow polychromatic erythrocytes (PCE) of Crl:CD-1 (ICR) BR mice. Industry accepted standard protocols and published procedures were followed in compliance with Good Laboratory Practice regulations (Heddle et al., 1991; Salamone and Mavournin, 1994; Schmid, 1976). In the dose selection study the test article was suspended in corn oil and dosed by oral gavage at up to 5000 mg/kg. Based on lack of significant toxicity, 5000 mg/kg was selected as the highest dose for the micronucleus studies. In the micronucleus assay, AQ-DA was suspended in corn oil and dosed by oral gavage at 1250, 2500 and 5000 mg/kg. Five males and five females were randomly assigned to each dose per harvest time group. Animals were killed ~24, 48 and 72 h after dosing with the test article for extraction of the bone marrow. A response was judged positive if it was significantly greater than the corresponding vehicle control at a level of P < 0.01.
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Quantitative analysis
In every case the main component of the test material was confirmed to be AQ (Table I
). Thus, purification of the NTP AQ-OX did not alter the main AQ constituent. The NTP report on AQ (Battelle, 1993; NTP, 1999) noted an extra peak on the GC trace in the AQ-OX at a level of 1200 p.p.m., but did not identify the contaminant at that time. The same peak was observed also at 1200 p.p.m. in the analytical studies reported here and was identified as 9-NA. The purification procedure removed the 9-NA from the NTP AQ-OX (Table I).
Bacterial mutation assays
All samples were run in the same laboratory under identical conditions. The NTP AQ-OX sample was mutagenic in a dose-dependent manner in strains TA98, TA100 and TA1537 (Table II). Mutagenic activity was reduced or eliminated by addition of an S9 rat liver microsome metabolic activation system. In contrast, no mutagenic activity was observed with the purified NTP AQ-OX (Table III). Because AQ is not mutagenic, all the mutagenic activity in the NTP AQ-OX sample can be ascribed to the 9-NA. Mutagenic activity of the 9-NA is seen at concentrations as low as 0.15 µg/plate, indicating potent activity (Table II). In tester strain TA98, the induced revertants over controls per microgram of chemical for the concurrently run positive controls are 137 revertants/µg for benzo[a]pyrene (B[a]P), and 175 revertants/µg for 2-nitrofluorene. The 9-NA with 173 revertants/µg is more potent than B[a]P and equal in potency to 2-nitrofluorene (Table II).
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Neither AQ-FC nor AQ-DA showed any mutagenic activity either with or without metabolic activation in the Ames tester strains or in WP2uvrA (Tables IV and V
).
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L5178Y TK +/– mouse lymphoma forward mutation assay
No cytotoxicity was observed in any of the trials in the L5178Y mutation assays. Mutant frequencies of treated cultures varied randomly with dose toxicity and no increases above the minimum criteria for a positive response were induced (Table VI
). AQ-DA was therefore evaluated as negative with and without metabolic activation at the TK locus in L5178Y mouse lymphoma cells under the conditions used in this study.
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Chromosomal aberration induction in CHO cells
CHO cells were incubated with up to 50.0 µg/ml of AQ-DA with harvest times of 20 and 44 h (Table VII
). No significant increase in cells with chromosomal aberrations, polyploidy or endoreduplication was observed at the concentrations analyzed. AQ-DA was considered negative for inducing chromosomal aberrations in CHO cells with and without metabolic activation.
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In vivo bone marrow mouse micronucleus assay
In the micronucleus assay, AQ-DA was suspended in corn oil and dosed by oral gavage at 1250, 2500 and 5000 mg/kg. No bone marrow toxicity was observed as a decrease in the polychromatic erythrocyte: normochromatic erythrocyte (PCE:NCE) ratio. AQ-DA did not induce a significant increase in micronuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the mouse bone marrow micronucleus test (Table VIII
).
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Genetic toxicology
The lack of mutagenic or genotoxic activity in a variety of assays in numerous laboratories indicates that AQ is not a DNA-reactive genotoxic carcinogen (Brown and Brown, 1976
; Anderson and Styles, 1978; Gibson et al., 1978; Salamone et al., 1979; Sakai et al., 1985; Tikkanen et al., 1983; National Cancer Institute, 1987; Kerckaert et al., 1996; Durant et al., 1996). Those assays are strengthened by the studies presented here in which we report that purified NTP AQ-OX, AQ-FC and AQ-DA are negative in the expanded Ames mutagenicity test, that AQ-DA is negative in the L5178Y mouse lymphoma forward mutation assay, that AQ-DA does not induce chromosomal aberrations, polyploidy or endoreduplication in CHO cells, and that it is negative in the in vivo mouse micronucleus assay (Tables III–VIII).
The report of direct-acting genotoxic activity in two Ames tests (Liberman et al., 1982; Zeiger et al., 1988) was unexpected because the data were in conflict with so many other reports of negative mutagenic activity. In both of those reports the AQ was obtained from Aldrich Chemical Co. and the label purity was listed as only 97% (Liberman et al., 1982; Zeiger et al., 1988). No analysis of the identity of the remaining 3% of non-AQ material was done. The problem of contamination with nitroanthracenes producing genotoxic activity in preparations of AQ-OX has been described (US EPA, 1977; ICI, 1978a, 1978b). In fact, the NTP AQ-OX was contaminated with 1200 p.p.m. 9-NA (Table I). The NTP AQ-OX was mutagenic in strains TA98, TA100 and TA1537, while the purified NTP AQ-OX was not. The structure of AQ does not suggest direct DNA reactivity, yet the NTP AQ-OX was mutagenic without added metabolic activation. Taken together these data indicate that AQ is not genotoxic, rather that the NTP sample of AQ-OX contains the mutagenic contaminant 9-NA.
Cytogenetics
The NTP report described a mouse peripheral blood micronuclei assay test from the 14 week range-finding study that preceded the cancer bioassay (NTP, 1999). The tentative conclusion was that the data showed that AQ-OX exhibited weak activity in that assay. However, the doses used were up to four times the maximum tolerated dose used in the bioassay, no response was seen in the female animals, and the response in the males was judged as positive only with a highly non-conservative trend test. In contrast, AQ-OX was negative in a bone marrow micronucleus assay (NTP, 1999). AQ-DA was also negative in a mouse bone marrow micronucleus assay in the studies reported here (Table VIII) and a chromosomal aberration assay in CHO cells (Table VII). Taken together, the weight of evidence of the data indicates that AQ does not induce cytogenetic damage.
Cancer studies
The observation of a mutagenic contaminant confounds any interpretation of the NTP bioassay with AQ. A previous bioassay with males and females in two strains of mice had been conducted with AQ. That study was not done following contemporary bioassay standards and needed to be repeated. Nevertheless that bioassay did not show carcinogenic activity with AQ (Innes et al., 1969). Looking at the data as a whole strongly indicates the possibility that the observed tumors in the NTP bioassay cancer were the result of a mutagenic contaminant.
Pathological evaluations of tissues from the NTP study suggest that there may have been some degree of cell death and regenerative cell proliferation in some target tissues. For example, in the rat kidneys from AQ-treated animals hyaline droplet accumulation, nephropathy, transitional epithelium hyperplasia and mineralization were observed. Centrilobular hypertrophy and focal necrosis were seen in the male B6C3F1 mice livers, and centrilobular hypertrophy and focal fatty degeneration were seen in the female B6C3F1 mice livers (NTP, 1999). In follow up studies in F-344 rats, AQ-induced cell proliferation was noted in the urinary bladder (National Institute of Environmental Health Sciences, 1999). If 9-NA or other mutagenic contaminants were present, even a small amount of regenerative cell proliferation would act synergistically to enhance the mutagenic and carcinogenic responses (Columbano et al., 1981).
Contaminants as confounders of the cancer bioassay
The best way to evaluate the plausibility as to whether the contaminant in the NTP AQ-OX produced the tumor response in the NTP study would be to do a potency calculation based on the carcinogenic potency of the 9-NA contaminant. Unfortunately, no cancer data are available for 9-NA. One way to estimate potency is to extrapolate comparisons of genotoxic potency to potential carcinogenic potency. Although it is recognized that there are limitations to such comparisons, a reasonable estimate of plausibility can usually be made. Another way to address the plausibility question is to ask how potent a carcinogen the contaminant would have to be in order to produce the observed response.
Mutagenic potency comparisons
Both 9-NA and 2-nitroanthracene (2-NA) are mutagenic with the 2-NA isomer being substantially more potent (Fu et al., 1986). 9-NA is present in the NTP AQ-OX at a level of 1200 p.p.m. Therefore, the amount of the contaminant present on each Ames test plate is easily calculated (shown in Table II). Since AQ is not mutagenic, all the mutagenic activity can be ascribed to the contaminants. Table II shows that mutagenic activity can be seen in strain TA98 without metabolic activation at a level of only 0.15 µg 9-NA per plate. As a first approximation it is reasonable to assign this mutagenic activity to the primary contaminant 9-NA. Accordingly, on the basis of induced revertants per microgram in tester strain TA98, 9-NA with 173 revertants/µg was more potent than B[a]P with 137 revertants/µg and was as potent as the 2-nitrofluorene with 175 revertants/µg run concurrently as positive controls (Table II).
Cancer potency calculations
A critical question is whether it is plausible that a contaminant could be a significant contributor to the carcinogenic activity observed in the NTP bioassay. The Handbook of Carcinogenic Potency and Genotoxicity Databases defines a valuable parameter to rank cancer potency, the Tumor Dose 50 (TD50) (Gold and Zeiger, 1997). The TD50 is the tumorigenic dose-rate for 50% of experimental animals, or the dose-rate that will halve the probability of remaining tumor free at the end of a standard lifespan. For a given target site, if there are no tumors in control animals, then the TD50 is that chronic dose rate in mg/kg body weight/day, which would induce tumors in half the test animals at the end of a standard lifespan study. This parameter is useful because it is analogous to the LD50 and the units are understandable as mg/kg/day. The TD50 for key target sites in the NTP AQ cancer study were estimated from a least squares linear fit of the dose–response tumor data and are presented in Table IX. Knowing that the percentage of contaminating 9-NA was 0.12%, and with the assumption that the 9-NA was responsible for inducing all the tumors, the resulting theoretical TD50 for 9-NA was calculated and is also presented in Table IX. The TD50 for AQ is in the range 310–940 for rats and is about 380 for mice. The AQ TD50 potency in rats is in the range of butylated hydroxyanisole. If the 9-NA were responsible for all the tumorigenic activity, the TD50 values would be in the range 0.37–1.1 for rats and would be about 0.45 for mice. Such values indicate that 9-NA would have to be in the potency range of B[a]P or 2-acetylaminofluorene in rats or dimethylnitrosamine or 3-nitro–3-hexene in mice (Gold and Zeiger, 1997). In fact, 9-NA has a greater mutagenic potency than B[a]P (Table II). These data indicate that it is plausible that the 9-NA contaminant was responsible for all of the tumor induction observed in the NTP study.
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In fact, in the absence of reliable cancer data, the genetic toxicology profile indicates that AQ would not be a genotoxic carcinogen. Thus, no definitive conclusion can be drawn at this time as to whether AQ itself might exhibit carcinogenic activity.
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Acknowledgments |
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We acknowledge the generous gift from Cynthia Smith (NTP) and Donna Browning (Battelle) of a sample of the AQ from their archives that was used in the NTP Cancer bioassay. Genetic toxicology studies were conducted by Michael Mecchi, Timothy Lawlor, Hemalatha Murli, James Ivett and Maria Cifone of Covance Laboratories (Vienna, VA).
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2 To whom correspondence should be addressed. Tel: +1 302 695 5761; Fax: +1 302 695 5763; Email: ebiken{at}aol.com
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Received on October 6, 2000; accepted on November 10, 2000.
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