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" From Arey (1998a). See also Gupta et al. (1996) for the mutagenicities of the nitronaphthalenes and methylnitronaphthalenes are essentially those found in Gupta et al. (1996). See this paper for experimental details.

6 Values in parentheses obtained by Harger et al. (1992) using standard plate assay.

" From Arey (1998a). See also Gupta et al. (1996) for the mutagenicities of the nitronaphthalenes and methylnitronaphthalenes are essentially those found in Gupta et al. (1996). See this paper for experimental details.

6 Values in parentheses obtained by Harger et al. (1992) using standard plate assay.

al., 1986), personal exposure to ETS (Kado et al., 1991), and vapor-phase mutagens (Kado et al., 1992). Similarly, it has been applied to 2-nitronaphthalene, which is the product of a nighttime atmospheric reaction of NO, and the daytime OH-initiated reaction of naphthalene. This nitronaphthalene is present at relatively high levels in ambient air and is a human cell as well as a direct bacterial mutagen (Arey, 1998a, and references therein; see Section F.2).

The large increase in the mutagenic activity of 2-nitronaphthalene determined earlier with the standard assay, 0.2-0.9 rev/nmol (Rosenkranz and Mermelstein, 1985a, 1985b) compared to 680 rev/nmol with the microsuspension assay (Arey, personal communication), is important in studies of ambient air. Other examples of the application of a microsuspension modification to the Ames assay include ambient air particles (Kado et al., 1986; Atkinson et al., 1991; Zinbo et al., 1992; Matsumoto et al., 1998), ambient vapor-phase and particulate mutagens (Kado et al., 1992; Harger et al., 1992; Watanabe et al., 1995), vapor-phase mutagens in ambient air (Gupta et al., 1996), laboratory studies of the photooxidations of 2- to 4-ring PAHs (Sasaki et al., 1995), vapor-phase mutagens in diesel exhaust (Hsieh et al., 1993), personal exposure to environmental tobacco smoke (Kado et al., 1991), and diesel particles (Bagley et al., 1992).

d. The Salmonella typhimurium Reversion Assay for Gas-Phase Mutagens

Until the early to mid-1980s, research on the mutagenicity of respirable POM focused almost exclusively on the particulate phase. Another aspect of tropo-spheric chemistry with significant health implications is the application of the Ames bacterial assay (with or without the microsuspension modification) to the detection and identification of mutagenic vapor-phase PAHs and PACs. For example, Harger and co-workers (1992) reported that the mutagenicities of concurrently collected samples of vapor-phase and particle-phase organics in southern California (Claremont, California) ambient air were comparable.

As discussed in Section A.4, there are a variety of approaches to separating the gas and particle phases that allow their contributions to the total mutagenic activity to be determined independently. These include, for example, a combination of a TIGF filter and three PUF plugs, discussed earlier and shown in Fig. 10.4 (Arey et al., 1989a), or a combination of a filter for particles and XAD-2 resin to trap the gas-phase species (e.g., see Alfheim et al, 1985; Pyysalo et al., 1987; and Tuominen et al., 1988). Such approaches have been applied to studying the gas-phase mutagenic PAHs and PACs in ambient air, in environmental chamber studies of 2- to 4-ring PAHs and of VOC-NOx systems, and in wood smoke and automobile exhaust (see, for example, the following: for ambient air, Claxton (1985), Wong et al. (1991), Arey et al. (1992), Harger et al. (1992), Kado et al. (1992), and Gupta et al. (1996); for environmental chamber photooxidations of 2- to 4-ring PAHs, Sasaki et al. (1995); for a variety of VOCs Shepson et al. (1985, 1986, f987), Kleindienst et al. (1985a, 1985b, 1990), and Kleindienst (1994); for wood smoke and automobile exhaust, Kleindienst et al. (1986, 1992) and Hsieh et al. (1990, 1993)).

e. Accuracy and Precision

Both intra- and interlaboratory mutagenic potencies reported in the literature for a given chemical (e.g., a common reference standard, 2-nitrofluorene), or complex mixture (e.g., extracts of particulate POM), can vary widely. Given their widespread international use, knowing the accuracy and precision of the mutagenic potencies obtained from plate incorporation assays is essential if they are to be used to correlate mutagen levels in primary emissions and ambient air with changes in parameters such as fuel composition and operating conditions or with meteorological conditions, season of the year, gaseous pollutant levels (e.g., VOCs, NOx, and 03), and emission inventories.

Some of the experimental variability associated with this assay were addressed, for example, by Belser et al. (1981) and Maron and Ames (1983), who recommended procedures to improve the accuracy and precision of the assay. These included concurrent assays conducted with "positive controls," i.e., reference chemicals of known mutagenicities such as 2-nitrofluorene for strains TA98 and TA100 with and without activation; spontaneous reversion rate (SRR) controls; checks for toxicity; and attention to such parameters as uniformity of the soft agar thickness, cell density, and temperature uniformity during incubation. For examples of the effectiveness of these and other measures used to improve accuracy and precision, see, Fitz et al. (1983), Ball et al. (1984), Hsieh et al. (1990), Claxton et al. (1992a, 1992b), Lewtas et al. (1992), May et al. (1992), Greenberg et al. (1993), and Gupta et al. (1996).

International collaborative studies have also been carried out to investigate the accuracy and precision of such bioassays when applied to complex mixtures (e.g., Claxton et al., 1992a, 1992b; Lewtas et al., 1992; May et al., 1992). The conclusions from one of the major studies (Claxton et al., 1992b) are representative of such intercomparisons:

• The intralaboratory variance of the bioassay results ranged from 16 to 88%, depending on the Standard

Reference Material used and the particular bioassay conditions, such as tester strain and metabolic acti vation; interlaboratory variance, i.e., reproducibility, ranged from 33 to 152%.

• About 55-95% of the total variation for the three environmental samples studied was due to between-laboratory variability.

• The variation in the mutagenic potency of the control compounds was similar to that of the environmental samples; the exception was 1-nitropyrene, for which the reproducibility ranged from f27 to 132%.

f. Some Mutagenic Potencies in the Salmonella typhimurium Assay

Table 10.16 contains the direct mutagenicities for a number of nitroarenes of atmospheric interest determined in one laboratory (strains TA98, TA98NR, and TA98/1,8-DNP6; rev/^g, -S9) (Winer and Atkinson, 1987; Arey et al., 1988b) using the Salmonella typhimurium plate incorporation assay and the quality control measures described by Belser et al. (1981) and Maron and Ames (1983). Zielinska and co-workers (1988b) have also determined the mutagenic activities in these strains for a series of nitrofluoranthenes.

The genotoxicity, mutagenicity, and carcinogenicity of nitroarenes, as well as their occurrence and modes of formation, have been critically reviewed by Rosenkranz and Mermelstein (1985a, 1985b) and Tokiwa and Oshini (1986). Specific activities (in rev nmol"1) of some 80 nitroarenes (plate incorporation assay) on strain TA98, without (— S9) and with (+ S9) microsomal activation, are cited by Rosenkranz and Mermelstein (1985a), whereas Tokiwa and Oshini give the direct activities (rev nmol-1, — S9) on strains TA98 and TA100 (plate incorporation assay) for 53 nitroarenes.

More recently, Einesto et al. (1991) determined the mutagenicities on strains YG1021 and YG1024 (vide supra) and strains TA98, TA98NR, and TA98/1,8-DNP6 for 18 different chemicals, including 2-nitrofluorene, f-nitropyrene, and 1,8-dinitropyrene (all — S9). Additionally, mutagenicities of 12 chemicals, including BaP ( + S9) and 2-nitronaphthalene ( — S9), were determined on strains YG1026 and YG1029 vs TA100, TA100NR, and TA100/l,8-DNPh (see the paper for a description of their assay procedures). As seen in Table 10.19, 2-nitrofluorene is significantly more sensitive on strains YG1021 and YG1024 than on TA98, 1,8-dinitropyrene is significantly more sensitive on strain YG1024 than TA98, 2-nitronaphthalene ( — S9) is more sensitive on strains YG1026 and YG1029 than on TA100, and the promutagen BaP (+ S9) is about the same on all strains.

Direct mutagenicities (rev/nmol; — S9) for both the plate incorporation and microsuspension assay are given in Table 10.20 for four isomeric nitrofluorenes and four nitrophenanthrene lactones (Arey et al., 1992; Atkinson

TABLE 10.19 Mutagenic Activities of Several Nitroarenes ( — S9) and Benzo[a]pyrene ( + S9) Assayed with Strains TA98 and TA100 and Several Derivative Strains "~c

Mutagenic activity (rev nmol ' )

Compound MW S9 mix TA98 YG1021 YG1024

TABLE 10.19 Mutagenic Activities of Several Nitroarenes ( — S9) and Benzo[a]pyrene ( + S9) Assayed with Strains TA98 and TA100 and Several Derivative Strains "~c

Mutagenic activity (rev nmol ' )

Compound MW S9 mix TA98 YG1021 YG1024

2-Nitrofluorene

0 0

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