Document OEzJ453Yn7xDov0mK9ZGqbJnK

EVALUATION OF THE GENETIC ACTIVITY OF INDUSTRIALLY PRODUCED CARBON BLACK C. j. Kirwin, J .V. LeBlanc, W. C.Thomas Medical Department, Phillips Petroleum Company, Bartlesville, Oklahoma S. R. Haworth, P. E. Kirby, A. Thilagar EG&G Mason Research Institute, Rockville, Maryland J.T. Bowman Utah State University, Logan, Utah 0. J. Brusick f Litton Bionetics, Rockville, Maryland Commercially produced oil furnace carbon black (Chemical Abstract Service Registry No. 1333-86-4) has been evaluated by five different assay for genetic activity. These were the Ames Salmonella typhimurium reverse mutation test, sister chromatid exchange test in CHO cells, mouse lymphoma test, cell transformation assay in C3Hf 70Ti cells, and assay for genetic effects in Drosophila rnelanogaster. Limited cellular toxicity was exhibited but no significant genetic activity was noted. INTRODUCTION Carbon black is a generic term applied to a large family o f colloidal, nongraphitic carbons made under carefully controlled manufacturing conditions (Johnson and Eberline, 1978). This finely divided material is produced from gas or oil by thermal decomposition when brought in direct or indirect contact with heat from the products of combustion of fuel and air. Temperatures of decomposition vary up to a maximum of about 1650C. The decomposition is most often controlled by quenching with water, after which the reaction gases are usually filtered to remove the carbon black product. Carbon black is made by three different processes: channel, thermal, and furnace. It is no longer made by the channel process in the United Requests for reprints should be sent to C. J. Kirwin, Phillips Petroleum Company, 6-57 AB, Bartlesville, Oklahoma 74004. 913 Journalof Toxicology and Environmental Health, 7:973-989, 1981 Copyright 0 1981 by Hemisphere Publishing Corporation 0098-4108/81/050973-17S2.25 914 C.I. K l R W l N ET AL. States, but some operations s t i l l exist in other countries. The furnace process is the preferred method of manufacture and, with heavy oil used as feedstock, accounts for over 97% of carbon black production in Western nations. Unfortunately, many involved in biological evaluation do not dis tinguish carbon black from soot (Sax, 1975; Eisenstadt, 1979; NIOSH, 1978; Falk e t al., 1958; Kaden e t al., 1979). Carbon black and soot differ in quantity and chemical content of inorganic ash, particulate makeup, and organic extract (carbon black has less). Organic extract and aqueous extract are chemical!y different in carbon black and soot (D. Rivin, personal communication). On the other hand, the Chemical Abstract Service has assigned the number 1333-86-4 to carbon black, considering it an identifiable classification of materials, but no attempt has been made to classify soot. The definitions of carbon black and soot in standard technical dictionaries and encyclopedias indicate that they are synonymous but technically this is unwarranted (Johnson and Eberline, 1978; Hampel and Hawley, 1976; Considine, 1976; Lapedes, 1974; Smith and Bean, 1964). Occupational exposure of carbon black production workers has been studied in epidemiologic surveys over 40 yr (Ingalls, 1950; Ingalls and Risquez-lribarren, 1961; Robertson and Ingalls, 1980). No increased incidence of cancer mortality has been noted. Animal studies with various types of carbon black were reviewed by Rigdon (1975) and NlOSH ( 1978). No tumor-forming potential in animals was described for carbon black. All available evidence indicates that carbon black is not tumorigenic and does not present a carcinogenic hazard to employees involved in its production. Nevertheless, because of the increased emphasis in recent years on the possible hazards of all chemicals, a study of the mutagenic potential of carbon black was undertaken. There is a general lack of information on genetic activity for industrial carbon black. METHODS AND MATERIALS A single sample of N-339, oil furnace carbon black, was used. Properties of the sample are shown in Table 1. The polynuclear aromatic hydrocarbon (PAH) content was determined by suspending carbon black in benzene for 48 h. The extract was analyzed by gas chromatographymass spectrometry. PAHs having a molecular weight above 330 are not included in the quantitative value. The PAH content i s approximately 20% of the total toluene extractables. The cyclohexane procedure was not as efficient as the toluene procedure for total extractables. All the short-term procedures employed were considered to be standard methods. In the mammalian microsome plate test five tester strains of Salmonella typhimurium were used: TA98, TA100, TA1535, TA1537, and TA1538. The procedure (Ames e t ai., 1975) was conducted with a nonactivated system and an activated system, using Aroclor 1254 GENETIC ACTIVITY OF CARBON BLACK 975 TABLE 1. Characterization Tests of N-339 Carbon Black Test Units Nitrogen surface area CTAsPurface area Iodine number Dibutyl phthalate (DBP) absorption Compressed DBP Tint strength Heat loss at 125C Volatiles at 950C Ash at 550C PH Toluene discoloration Toluene extractables Cyclohexane extractables Polynuclear aromatic hydrocarbons 100 m2/g 100 m'/g 90 mg/g 124 cm3/ l o 0 g 104 cm31100 g 109% 0.76 wt % 1.80 wt % 0.61 wt % 7.5 84% 0.15 wt % 0.06 w t % 294 ppm aCetyltrimethylammonium bromide. Procedure ASTM D3037-A ASTM 03765 ASTM DlSlO ASTM D2414 ASTM D3493 ASTM 03265 ASTM 01509 Phillips method ASTM'D1506 ASTM D1512 ASTM 01618 48 h (Phillips) 48 h (Phillips) Gas chromatography- mass spectoscopy (Phillips) induced liver microsomal enzymes from Sprague-Dawley male rats. All control and test compound dilutions were plated in triplicate. In the activated system, the S9 mix was prepared from Aroclor 1254 (500 mg/kg) induced liver microsomal enzymes from Sprague-Dawley male rats. Liver was homogenized in 0.15 M KCI a t 3 ml per gram of wet liver and centrifuged at 9000 X g for 10 min. The supernatant, referred to as the S9 fraction, was decanted and stored in liquid N,. A l - m l portion of the microsomal enzyme reaction mixture (S9 mix) added to the soft agar overlay contained the following: s9 0.4 M MgCl2 1.65 M KCI 0.04 M NADP 0.05 M glucose-6-phosphate 1.00 M NaH, PO4, pH 7.4 H20 Total 0.05 ml 0.02 ml 0.02 ml 0.10 ml 0.10 rnl 0.10 ml 0.61 ml 1.00 ml Chinese hamster ovary (CHO) cells were used to evaluate sister chromatid exchange. The procedure (Brusick, 1980) was conducted with a nonactivated system and an activated system, using liver microsomal enzymes from Fischer 344 male rates. Due to i t s insolubility, the N-339 carbon black was handled in dimethyl sulfoxide (DMSO) as a stock suspension of 0.1 g/ml. Four replicate cultures per dose were employed in t h i s assay. The CHO biopsy cells were obtained from the American Type 976 C. J. KlRWlN ET AL, Culture Collection (Rockville, Md.; Repository NO. CCL61). Cells were grown in Ham's F12 medium supplemented with 10% fetal calf serum, split back to 3 X 10' cells per 75-cm2 plastic flask, and fed 24 h before treatment with 10 ml fresh medium. Approximately l o 6 cells were treated with growth medium with the test material and then incubated at 37OC for 2 h on a rocker. Exposure was terminated by washing the cells twice with saline. After addition of 5-bromo-2'-deoxyuridine t o a final concentration of 20 pM, incubation was continued in the dark for 24 h. Colcemid a t a final concentration of 2 X l o - ' M was added for the final 3 h of incubation. Metaphase cells were collected by mitotic shake-off. These cells were swollen with 0.075 M KCI, hypotonic solution, then washed 3 times with methanol and acetic acid ( 3 : l )fixation, dropped onto slides, and air-dried. For the activated system, Fischer 344 rat livers were induced with Aroclor 1254. Five days later, the animals were killed by decapitation, bled, and the livers were excised and placed in ice-cold 0.25 M sucrose buffered with Tris at pH 7.4. A collection o f livers were washed with buffered sucrose and homogenized. The homogenate was centrifuged for 10 min a t 9000 X g , and the S9 supernatant acted as the core reaction mixture in NADP (Na salt) (2.4 mg/ml) and isocitric acid (4.5 rnglml). The S9 preparation contained 24.2 mg protein per milliliter and the aryl hydrocarbon hydroxylase (AHH) specific activity was 0.45, which is an indirect measure o f the cyctochrome P-450 and P-448 activity of the preparation. The S9 preparation, which included 0.15 pI S9 and 0.60 ml cofactors, was used at 0.75 ml per 10-ml test volume. Slides were stained for 10 min with Hoechst 33258 ( 5 pg/mI) in phosphate buffer (pH 6.8) and exposed to ultraviolet light. They were then stained with 10% Giemsa for 10 min, rinsed in deionized water, and a i r-dried. Results for the solvent control and the test material or positive control were compared by Student's t-test and the standard error (SE) of the mean. The mouse lymphoma assay was a modification of the procedure of Clive and Spector (1975). Toxicity was evaluated to determine the optimum dose levels for mutagenesis assay o f carbon black. Test material was sterilized by suspending it in a single glass centrifuge tube with 0.5 ml acetone for each dose level; the same volume o f acetone was used for the two control tubes. The acetone was evaporated in a hood for 4 d. Test cultures were started by adding 4.0 ml of Fischefs medium supplemented with streptomycin and Pluronic S68 (FOP)t o tubes not receiving metabolic activation and 4.0 ml S9 plus cofactcrs to those receiving metabolic activation. A 6-ml portion of a L5178Y TK+/- cell suspension (clone 3.7.2~ from D. Clive, Burroughs Wellcome, Research Triangle Park, N.C.) con- taining 1 X l o 6 cells per milliliter was then added to each tube, and the tube was gassed with 5% CO,. The tubes were placed on a Bellco roller drum at 25 rpm for a 4-h exposure period. The preparation was conducted under GENETIC ACTIVITY OF C A R B O N BLACK 977 amber lights and kept in darkness during the exposure period. Because o f the difficulty of separating the insoluble test material from the cells, the 4-h contact time was extended. Cells were counted with hemacytometers. Cell population density was determined 24, 48, and 72 h after compound exposure by making 1:lO dilutions o f 1-ml samples of 0.1% trypsin, incubating a t 37C for 10 min to disperse the cells, and counting them in a hemacytometer. In preparation o f cells before use in the assay, the growing and cleansing procedures of Clive and Spector ( 1975) were employed to reduce the frequency of spontaneously occurring TK-/- cells. Dose levels for the assay were chosen so that the highest nonactivated dose approximated an LD90. To establish the background level of TK-/- colonies, two control tubes containing acetone solvent only were run. The concentrations of ethylmethane sulfonate e (EMS) and 7,12-dimethylbenz[a] anthracene (DMBA) were used as positive controls for direct-acting mutagens and promutagens, respectively. The 4-h exposure conditions were as described above; however, a t the end of the exposure period the cells were washed twice with 10 ml Fischer's medium supplemented with streptomycin, 10% horse serum and Pluronic S68 (F,,P) and centrifuged a t 1000 X g for 10 min. After the supernatant was decanted, the cells were resuspended in 20 ml Flop, gassed with 5% CO, in air, and placed on a roller drum at 37C. After initial exposure to the test material, the cells were incubated for 3 d with an adjustment each day to yield a cell population of 0.3 X l o 6 cells per milliliter. A t the end of this expression period, cells were placed in selective medium containing 1.O pg/ml trifluorothymidine (TFT) and 0.35% noble agar. The selective cloning medium kills the TK+/- cells and allows growth of discrete colonies of TK-/- cells. The general cloning preparation included two flasks per control and two flasks per test concentration for both activated and nonactivated systems. One flask was labeled selective medium and one was labeled viable count (VC). Each flask was warmed to 37"C, filled with 100 ml cloning medium, and shaken until used. From each culture 3 X l o 6 cells were removed, centrifuged, and resuspended in 2 ml medium. This suspension was placed in a selective medium flask. A 5 X dilution was made by adding 1 ml selective medium to 9 ml F,,P, adding 1 ml of this to 4 ml F,,P, and adding 1 ml of that dilution to the appropriate VC flask containing 100 ml cloning medium. After the dilution, 1 ml stock solution of TFT (100 pg/ml) was added to the selective medium flask, and both the selective medium flask and the VC flask were placed on a shaker a t 125 rpm a t 37C. After 15 min the flasks were removed and 33 ml cell suspension was placed in each of three appropriately labeled petri dishes. The plates were incubated a t 37C in a humidified 5% COz atmosphere for 10 d. The activated test system was derived from Sprague-Dawley rats. The 978 C. 1. KlRWlN ET AL. S9 mix for the L5178Y mouse lymphoma assay contained 15.49 mg/ml DL-isocitric acid, 6.0 mg/ml NADP, 0.25 ml S9 supernate per milliliter of mix, and 0.75 mi FOP per milliliter of mix. The concentrations in the cultures during exposure were Isocitric acid NADP S9 supernatant FOP 6.2 mg/ml 2.4 mg/ml 0.1 ml/ml 0.3 ml/ml The supernatant was prepared by homogenizing rat liver induced with Aroclor 1242 and 1254 suspended in 3 ml 0.25 M sucrose per gram of liver. The homogenized material was centrifuged a t 9000 X g for 10 min and the microsomal enzyme fraction stored a t -195C. The cell transformation study was conducted with modifications of the procedure of Bertram (1977) and Reznikoff e t al. (1973a, 1973b). C3H/lOT1/2 CL8 cells were obtained a t the sixth passage from c. Heidelberger (McArdle Laboratory for Cancer Research, University of Wisconsin). Stock inoculum was examined for typical growth characteristics and freedom from spontaneous transformation before use. Stock cultures were grown in antibiotic-free Eagle's basal medium (BME) containing 10% heat-inactivated fetal bovine serum (HI FBS). Cultures were subpassaged weekly at a 1:5 split ratio and maintained in a humidified incubator a t 37C in an atmosphere of 5% COz in air. Cells in late logarithmic phase were trypsinized with 0.1% trypsin for 4 min and plated at a concentration of 1 X l o 3 cells per petri dish in 5 ml BME with 10% HIFBS, penicillin (50 U/ml), and streptomycin (50 pg/mI). A second series of replicate cultures containing 200 cells per plate were set up from the same cell suspension for toxicity determinations. Cell toxicity was determined by suspending the test material in 0.5 ml acetone and adding the desired dose to tissue culture medium. The material was tested in duplicate with 200 cells per plate grown in 5 ml BME with 10% HIFBS for 24 h. After 18 h o f exposure, the cells were washed, refed with medium, and incubated for 10 d. Toxicity plates were then washed, fixed for 20 min with absolute methanol, air-dried, and stained with Giemsa stain for 30 min. The number of colonies per plate was determined from: Cloning efficiency = average no. of colonies per no. of cells per plate plate x 100 At 24 h after seeding the cells, the transformation cultures were treated with appropriate concentrations of test material suspended in acetone. Solvent control cultures received an equal amount o f solvent. Twelve cultures per dose were used. A t 18 h after treatment with the test compound, the GENETIC A C T I V I T Y OF CARBON BLACK 979 plates were refed with growth medium and reincubated. This refeeding schedule was repeated weekly for 35 d after removal of the test material. All plate cultures were washed, fixed, stained, examined microscopically and macroscopically, and scored for transformation as type II and type III foci. Various Drosophila melanogaster stocks were used to conduct assays for the detection of point mutations, chromosomal aberrations, and chromosome loss. Mosaics Males with X chromosomes carrying five copies of the white-ivory ( w ' ) mutant and marked with the recessive mutants yellow body (y) and echinus eyes (ec) were mated to attached -X females with normal red eyes, yellow body, and forked bristles (4. The stock was placed on bakers' yeast for egg-laying periods o f up to 15 h and then removed. Three days later larvae were collected by washing through a 60-mesh screen. These larvae were treated with the test substance at a concentration of 1% in the food until pupation. The flies were scored for mosaic spots after eclosion. Mutagenic activity is detected by a significant increase in the frequency of red mosaic spots in the eyes of the males. Y Chromosome Loss Treated males that carry a duplication of the gene for normal (red) eyes on the Y chromosome and a mutant allele white ( w ) on the X chromosome were fed with 1% test material for 6-h and then mated to white-eyed ( w / w ) females. Male offspring are expected ~ to carry the genes for red eyes. The occurrence of a white-eyed male signals loss o f the Y chromosome from the spermatozoon. Dominant Lethal Treated Canton-S males were mated with Canton-S females in nylon net cages on Welch's grape juice solidified with 2% agar. After an egg-laying period of not more than 12 h, the plates were stored a t 25C for approximately 30 h. Any genetic change that blocks development prior to hatching from the egg may be termed a dominant lethal mutation. The agar plates were scored to determine the fraction from which larvae were not hatched. Bithorax Test of Lewis (Lewis, 1954; Mendelson, 1976). The occurrence o f rearrangements with a breakpoint between the centromere of chromosome 3 and the locus o f bithorax ( b x ) can be determined by scoring the offspring of treated bithorax-34e males and Ultrabithorax (Ubx) females. These Ubx/bxMe offspring, which have their halteres modified to form a second set of wings, develop a band o f hairy tissue, the metanotum, between the thorax and abdomen if a chromosomal aberration between bx and the centromere has been induced. Sex-linked Lethals Females homozygous for the X-chromosome balancer Basc were mated to Canton-S (wild-type) males that had been fed 1% test material. All males from this parental cross carry the Basc X chromosome. All daughters carry one Pasc chromosome and a treated X from their fathers. In the F,, individual daughters were mated to their brothers. If the treatment applied to the parental males induced a lethal in a sperm, the F, female that inherited that chromosome will produce no c II -- 982 i i i 9w m7 ws 0-0 OWN 1"? 000 OWN ??? 000 s woo- I moo c-c y? - Ww Ow I - 0 - I-w 6WW 000 s - 9W I - t - 0-- r-wm 9095 -00 WWWIOWW wwwmwI- NNN hw wl (wYIw-wwmo o0 NNN OW-m-0 w6wwmm NNN 983 984 C. 1. KlRWlN ET AI.. induce any significant increase in mutant frequencies in the L5178y mouse lymphoma assay. This was true for both nonactivated and sg activated preparations. Results are presented in Tables 6 and 7. The cloning data for 35,000 pg/ml carbon black in Table 6 include average colonies per plate that are less than lower or higher dose levels. This is probably due to loss o f cells in the supernate during the cloning process, not to toxicity. It does not affect the mutant frequency value, since the reduction is evident in both the selective medium plates and the V c plates, and the mutant frequency is based on the ratio of the average number o f colonies in these plates. The reduction in the average number of colonies per plate for the V c plates does, however, affect on the total compound toxicity for the culture treated with 35,000 pg/ml. The cloning growth, expressed as percent of control, should probably be around 100 rather than 40%, which would change the percent growth from 7 to approximately 18%. The positive control for the nonactivated system was EMS and that for the activated system was DMBA. Because of the wide spread o f doses in the first evaluation, a second assay was conducted with a narrower range of doses. The results are presented in Table 8. These data corroborate those of the first evaluation. The compound did not cause significant increases in mutant frequencies of TABLE 7. Mouse Lymphoma, L5178/ TK+/- Locus Cells, Mutant Frequency, Activated Observations Acetone control N-339 carbon black (rglml) 10,000 15,000 Acetone control DMBA (wdml) 2.5 5.0 Cell counts ( x ~ O - ~ ) Day 1 Day 2 Day 3 Suspension growth Total Percent Cloning data vc Selective media Cloning vc Percent Percent growth Mutant frequency Induced mutant frequency 0.86 1.26 1.25 52.2 100 204 233 237 59 74 67 225 100 100 0.6 - 0.64 1.10 0.95 24.7 47 257 263 271 74 76 71 264 117 56 0.6 0.0 0.40 0.06 0.61 0.6 1 183 198 1a7 72 58 66 189 a4 1 0.7 0.1 0.62 1.37 1.06 33.3 100 128 131 133 88 90 86 131 100 100 1.4 - 0.25 0.62 1.oa 6.2 19 154 138 128 260 213 257 140 107 20 3.5 2.1 0.25 ,0.62 1.01 5.7 17 125 117 115 295 2aa 298 119 91 15 4.9 3.5 mc y cmy cwy Odd vm mw0m0 Odd c 0000; I -22 omcy '9?7 0-- I5 r P )PIP- : ? ? v! '-0 2 i 985 988 C. J. KlRWlN ET AL. Bertram, 1. 5. 1977. Effects o f serum concentration on the expression o f carcinogen-induced transformation in the C3H/lOT1/2 CL8 cell line. 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Quantitative and qualitative studies of chemical transformation of cloned C3H mouse embryo cells sensitive to postconfluence r inhibition of cell division. Cancer Res. 33:3239-3249. Reznikoff, C. A., Brankow, D. W., and Heidelberger, C. 1973b. Establishment and characterization of a cloned line of C3H mouse embryo cells sensitive to postconfluence inhibition of division. Cancer Res. 33:3231-3238. CQ H. 1975. Tissue reaction to foreign materials. Crit. Rev. Toxicol. 3:435-476. ertson, J. M. D. and Ingalls, T. H. 1980. A mortality study in United States carbon black wor ers 1935-1 974. Arch. Environ. Health 35:181-186. Sax, N. 1. 1975. Dangerous Properties of Industrial Materials, 4th ed., p. 519. New York: Van Nostrand Reinhold. Seelig, M. G. and Benignus, E. L. 1936. Coal smoke soot and tumors of the lung in mice. Am. 1. Cancer 28:96-111. ; Skopek, T. R., Liber, H. L., Kaden, D. A., e t al. 1979. Mutation of human cells by kerosene soot. i /. Nptl. Cancer Inst. 2: 309-3 12. Smith, W. R. and Bean, D. C. 1964. Carbon (carbon black). I n Kirk-Oihmer Encyclopedia o f Chemical Technology, 2d ed., eds. A. Standen, H. F. Mark, j. J. McKetta, and 0. Othmer, vol. 4. New York: Wiley. Steiner, P. E. 1954. The conditional biological activity of the cancinogens in carbon black, and its elimination. Cancer Res. 14:103-110. Stevens, W. L. 1942. Accuracy of mutation rates. /. Genet. 43:301-307. Sweitzer, C. W. 1952. Colloidal carbon-invaluable soot. /. Chem. Educ. 29:493-502. Todd, R. G. 1970. Direct identification of polycyclic aromatic hydrocarbons from carbon black. Dissertation. Univ. of Oklahoma, Oklahoma City, Okla. Von Haam, E. and Mallette, F. S. 1952. Studies on the toxicity and skin effects of compounds d .+. used in the rubber and plastics industries. Arch. Ind. Hyg. Occup. Med. 6:237-242. Received May 20, 1980 Accepted November 10, 1980