4
Genotoxicity and Carcinogenicity

The carcinogenic potential of beryllium and beryllium compounds has been assessed by various agencies in the last decade. The International Agency for Research on Cancer (IARC 1993) classifies beryllium and beryllium compounds as carcinogenic in humans, the U.S. Environmental Protection Agency (EPA 1998a,b) considers them probable human carcinogens, and the National Toxicology Program (NTP 1999, 2005) lists them as reasonably expected to be carcinogens. As noted in Chapter 1, EPA has performed a dose-response analysis of the cancer data to estimate an air unit risk of 2.4 ×10−3 per μg/m3. This chapter examines the literature used in the previous assessments, more recent reviews, and relevant new studies. First, information on the genotoxic potential of beryllium and beryllium compounds is presented. The literature on carcinogenicity, including the epidemiologic literature and animal bioassays, is then reviewed.

GENOTOXICITY

Compounds of beryllium have tested positively in nearly 50% of the genotoxic studies conducted without exogenous metabolic activation, but were nongenotoxic in most bacterial tests. Beryllium chloride, beryllium nitrate, beryllium sulfate, and beryllium oxide have been shown to be nongenotoxic in the Ames plate incorporation assay and assays with Escherichia coli pol A, E. coli WP-2 uvr A, and Saccharomyces cerevisiae (Table 4-1) (reviewed in EPA 1998a,b; ATSDR 2002; Gordon and Bowser 2003). Beryllium sulfate also did not induce unscheduled DNA synthesis in primary rat hepatocytes (Williams et al. 1982, 1989), was not mutagenic when injected intraperitoneally into adult mice in a S. typhimurium host-mediated assay (Simmon et al. 1979a), and failed to increase the incidence of micronucleated polychromatic erythrocytes in bone marrow (Ashby et al.1990). Lung tumors in F344/N rats treated with beryllium sulfate did not have mutations of the p53 or c-raf-1 gene, but weak mutations were detected in the K-ras gene (Nickell-Brady et al. 1994).

Positive genotoxic results have been reported for beryllium sulfate in the B. subtilis rec assay (Kada et al. 1980; Kanematsu et al. 1980) and the E. coli rec assay (Dylevoi 1990), for beryllium nitrate in the B. subtilis rec assay (Kuroda et al. 1991), and for beryllium chloride in the B. subtilis rec assay with spores (Kuroda et al. 1991), the E. coli forward-mutation assay (Zakour and Glickman 1984), and the Photobacterium fischeri assay (Ulitzur and Barak 1988). Gene mutations have been observed in



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Health Effects of Beryllium Exposure: A Literature Review 4 Genotoxicity and Carcinogenicity The carcinogenic potential of beryllium and beryllium compounds has been assessed by various agencies in the last decade. The International Agency for Research on Cancer (IARC 1993) classifies beryllium and beryllium compounds as carcinogenic in humans, the U.S. Environmental Protection Agency (EPA 1998a,b) considers them probable human carcinogens, and the National Toxicology Program (NTP 1999, 2005) lists them as reasonably expected to be carcinogens. As noted in Chapter 1, EPA has performed a dose-response analysis of the cancer data to estimate an air unit risk of 2.4 ×10−3 per μg/m3. This chapter examines the literature used in the previous assessments, more recent reviews, and relevant new studies. First, information on the genotoxic potential of beryllium and beryllium compounds is presented. The literature on carcinogenicity, including the epidemiologic literature and animal bioassays, is then reviewed. GENOTOXICITY Compounds of beryllium have tested positively in nearly 50% of the genotoxic studies conducted without exogenous metabolic activation, but were nongenotoxic in most bacterial tests. Beryllium chloride, beryllium nitrate, beryllium sulfate, and beryllium oxide have been shown to be nongenotoxic in the Ames plate incorporation assay and assays with Escherichia coli pol A, E. coli WP-2 uvr A, and Saccharomyces cerevisiae (Table 4-1) (reviewed in EPA 1998a,b; ATSDR 2002; Gordon and Bowser 2003). Beryllium sulfate also did not induce unscheduled DNA synthesis in primary rat hepatocytes (Williams et al. 1982, 1989), was not mutagenic when injected intraperitoneally into adult mice in a S. typhimurium host-mediated assay (Simmon et al. 1979a), and failed to increase the incidence of micronucleated polychromatic erythrocytes in bone marrow (Ashby et al.1990). Lung tumors in F344/N rats treated with beryllium sulfate did not have mutations of the p53 or c-raf-1 gene, but weak mutations were detected in the K-ras gene (Nickell-Brady et al. 1994). Positive genotoxic results have been reported for beryllium sulfate in the B. subtilis rec assay (Kada et al. 1980; Kanematsu et al. 1980) and the E. coli rec assay (Dylevoi 1990), for beryllium nitrate in the B. subtilis rec assay (Kuroda et al. 1991), and for beryllium chloride in the B. subtilis rec assay with spores (Kuroda et al. 1991), the E. coli forward-mutation assay (Zakour and Glickman 1984), and the Photobacterium fischeri assay (Ulitzur and Barak 1988). Gene mutations have been observed in

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Health Effects of Beryllium Exposure: A Literature Review TABLE 4-1 Genotoxicity Studies of Beryllium Compounds Assay or End Point Species Compound With Activation Without Activation Reference In vitro Plate incorporation assay Salmonella typhimurium BeSO4 Negative Negative Rosenkranz and Poirier 1979; Simmon 1979a; Simmon et al. 1979; Dunkel et al. 1984; Arlaukas et al. 1985; Ashby et al. 1990 S. typhimurium Be(NO3)2 Negative Negative Arlaukas et al. 1985; Kuroda et al. 1991 S. typhimurium BeO Negative Negative Kuroda et al. 1991 S. typhimurium BeCl2 Negative Negative Kuroda et al. 1991 Eschericia coli WP-2 uvrA BeSO4 Negative Negative Dunkel et al. 1984 Rec assay Bacillus subtilis BeSO4 — Positive Kada et al. 1980; Kanematsu et al. 1980 B. subtilis BeCl2 — Positive Kuroda et al. 1991 B. subtilis BeCl2 — Negative Nishioka 1975 B. subtilis Be(N03)2 — Positive Kuroda et al. 1991 B. subtilis BeO — Negative Kuroda et al. 1991 E. coli BeSO4 — Positive Dylevoi 1990 DNA modification E. coli pol A+/A− BeSO4 — Negative Rosenkranz and Poirier 1979 Bioluminescence test Photobacterium fischeri BeCl2 — Positive Ulitzur and Barak 1988 Recombogenic activity Saccharomyces cerevisiae BeSO4 Negative Negative Simmon 1979b Host-mediated assay S. cerevisiae BeSO4 — Negative Simmon et al. 1979 S. typhimurium BeSO4 — Negative Simmon et al. 1979 Chromosomal aberration Swine lymphocytes BeCl2 — Positive Vegni-Talluri and Guiggiani 1967 Syrian hamster embryo cells BeSO4 — Positive Larramendy et al. 1981 Human lymphocytes BeSO4 — Positive Larramendy et al. 1981 Chinese hamster ovary cells BeSO4 — Negative Brooks et al. 1989 Cytogenetic assay Chinese hamster lung cells BeSO4 Negative Negative Ashby et al. 1990 Sister chromatid exchange assay Chinese hamster V79 cells BeCl2 — Positive Kuroda et al. 1991 Chinese hamster V79 cells Be(NO3)2 — Positive Kuroda et al. 1991 Chinese hamster V79 cells BeO — Negative Kuroda et al. 1991 Syrian hamster embryo cells BeSO4 — Positive Larramendy et al. 1981 Human lymphocytes BeSO4 — Positive Larramendy et al. 1981 Human lymphocytes BeSO4 — Negative Andersen 1983 Mouse macrophage P388D1 cells BeSO4 — Negative Andersen 1983 DNA repair Rat hepatocytes BeSO4 — Negative Williams et al. 1982, 1989

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Health Effects of Beryllium Exposure: A Literature Review Assay or End Point Species Compound With Activation Without Activation Reference Transformation assay Syrian hamster embryo cells BeSO4 — Positive DiPaolo and Casto 1979 Rat respiratory epithelial cells Rocket exhaust residue — Mixed Steele et al. 1989 Rat respiratory epithelial cells BeO (low fired) — Positive Steele et al. 1989 Rat respiratory epithelial cells BeO (high fired) — Mixed Steele et al. 1989 BALB/c-3T3 cells BeSO4 — Positive Keshava et al. 2001 DNA damage Rat respiratory epithelial cells Rocket exhaust residue — Mixed Steele et al. 1989 Rat respiratory epithelial cells BeO (low fired) — Positive Steele et al. 1989 Rat respiratory epithelial cells BeO (high fired) — Mixed Steele et al. 1989 Mutation of HGPRT gene Chinese hamster ovary K1-BH4 cells BeSO4 — Positive Hsie et al. 1979 Chinese hamster V79 cells BeCl2 — Positive Miyaki et al. 1979 Mutation of lacI gene E. coli BeCl2 — Positive Zakour and Glickman 1984 Mutation of K-ras gene Rat lung tumors Be — Weak positive Nickell-Brady et al. 1994 Mutation of p53 gene Rat lung tumors Be — Negative Nickell-Brady et al. 1994 Mutation of c-raf-1 gene Rat lung tumors Be — Negative Nickell-Brady et al. 1994 In vivo Transformation assay Syrian hamsters (embryo cells evaluated after maternal exposure) BeSO4 — Positive DiPaolo and Casto 1979 Micronucleus assay CBA mice BeSO4 — Negative Ashby et al. 1990 Note: Be = beryllium, BeCl2 = beryllium chloride, Be(NO3)2 = beryllium nitrate, BeO = beryllium oxide, BeSO4 = beryllium sulfate. SOURCE: Adapted from ATSDR 2002. mammalian cells cultured with beryllium chloride (Vegni-Talluri and Guiggiani 1967; Hsie et al. 1979; Miyaki et al. 1979) and beryllium sulfate (Larramandy et al. 1981; Brooks et.al. 1989); beryllium nitrate has resulted in clastogenic alterations (Kuroda et al. 1991). Overall, mutation and chromosomal-aberration assays of beryllium compounds have yielded somewhat contradictory results. Although the bacterial assays have been largely negative, the mammalian test systems exposed to beryllium compounds have shown evidence of mutations, chromosomal aberrations, and cell transformations. Further studies would confirm the mutagenic or genotoxic properties of the various beryllium compounds.

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Health Effects of Beryllium Exposure: A Literature Review CARCINOGENICITY Epidemiologic Studies Several studies and reviews are available on cancer in relation to beryllium exposure in humans. Two worker cohorts involved in beryllium extraction, production, and fabrication have been extensively studied and are the primary basis of conclusions drawn to date on cancer in humans. One cohort is in Lorain, Ohio, and the other in Reading, Pennsylvania. The original study (Mancuso 1979) reported a lung-cancer standardized mortality ratio (SMR) for the two plants combined of 1.42 (95% confidence interval [CI], 1.1-1.8). The study involved 1,222 workers at the Ohio plant and 2,044 workers at the Pennsylvania plant who had been employed for at least 3 months during 1942 through 1948. No analysis by job title or by exposure category was performed, and the excess-lung-cancer finding was limited to workers who were employed for less than 5 years. The exposures of the workers were often at high concentrations. For example, a study at the Lorain plant in 1947-1948 by the U.S. Atomic Energy Commission measured beryllium at concentrations ranging from 411 μg/m3 in the mixing area to 43,300 μg/m3 in the breathing zone of alloy operatives (Zielinski 1961). Control limits at U.S. plants were introduced in 1949 (Wagoner et al. 1980). Mancuso (1980) reanalyzed the same two cohorts but expanded the period of employment of the study cohorts to 1937 through 1948 and used workers at the rayon plant for comparison purposes. The comparison between the two types of industrial workers found a significant relative SMR for lung cancer of 1.40 for the beryllium-worker cohort. Wagoner et al. (1980) expanded the cohort in the Pennsylvania plant to include workers employed during 1941 through 1967. The group of 3,055 workers was found to have a lung-cancer SMR of 1.25 (95% CI, 0.9-1.7). When the analysis was adjusted for latency, there was a significant SMR of 1.68 for the group that had a latency of 25 years or longer. However, there was no relationship with duration of employment. The results of a 1968 medical survey of smoking histories of the workers showed that differences in smoking habits were sufficient to increase the cancer risk among beryllium workers by 14%. However, if the working population’s risk is compared with lung-cancer mortality in the county where the plant was instead of using the U.S. rates, the SMR is underestimated by 19% (Wagoner et al. 1980). The National Institute for Occupational Safety and Health (NIOSH) conducted a retrospective cohort mortality study of seven beryllium production facilities that included the Pennsylvania and Ohio cohorts previously studied by Mancuso and Wagoner. In the study, Ward et al. (1992) developed a cohort of 9,225 male workers who had worked for at least 2 days during 1940 through 1969 and were followed through 1998. The SMR for lung cancer was 1.26 (95% CI, 1.12-1.42) on the basis of 280 lung-cancer deaths. The researchers also observed an SMR for nonmalignant respiratory disease of 1.48 (95% CI, 1.21-1.80). Ward et al. (1992) reported that SMR increased with latent period, with a significant SMR of 1.46 for a latent period greater than 30 years among the workers at the combined seven plants. The SMR for lung cancer was a significant 1.42 for those hired before 1950 and less than l for those hired during 1960 through 1969. IARC (1993) has provided a detailed description and critique of the cohort studies. It pointed out that the risk of lung cancer was consistently higher in plants in which there was an excess mortality from nonrespiratory disease. IARC also concluded that the association between lung-cancer risk and beryllium exposure did not appear to be confounded by smoking. A second line of investigation is embodied in the Beryllium Case Registry, which was established in 1952 to follow the clinical aspects and complications of people with beryllium-related diseases, including both chronic beryllium disease (CBD) and acute beryllium-related pneumonitis. The data were analyzed first by Infante et al. (1980) and more recently by Steenland and Ward (1991). In the Steenland and Ward study, the cohort consisted of 689 people who entered the registry during 1952 through 1980 and were followed through 1988. The researchers reported an SMR of 2.00 (95% CI, 1.33-2.89) on the basis of 28 observed lung-cancer deaths. The lung-cancer SMR was greater among people who had acute beryllium pneumonitis (SMR, 2.32) than among those who had CBD (SMR, 1.57); the former was

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Health Effects of Beryllium Exposure: A Literature Review statistically significant. IARC (1993) concluded that the studies of cases in the Beryllium Case Registry provided indirect evidence that beryllium, rather than smoking, explained the increase in lung cancer under the assumption that people with acute pneumonitis were unlikely to smoke more than workers with CBD. With respect to other cancer end points, Carpenter et al. (1988) conducted a nested case-control study of cancers of the central nervous system among workers at facilities in Oak Ridge, Tennessee. There were 72 male and 17 female deaths due to central-nervous-system cancer. Using job titles, the investigators considered the potential exposure to each of 26 chemicals, including beryllium. There was a weak association with exposure to beryllium with an odds ratio (OR) of 1.5 (95% CI, 0.6-3.9). The authors concluded that their study did not support the hypothesis that occupational exposures to the chemicals they studied appreciably increased the risk of cancer of the central nervous system. IARC (1993) noted that there was an increasing risk of cancer of the central nervous system with longer duration of employment in jobs with greater exposure to beryllium. On the basis of the studies described above, IARC concluded that there is sufficient evidence in humans of the carcinogenicity of beryllium and beryllium compounds. That was based on the cohort studies, which showed A large number of lung-cancer cases with a stable estimate of the SMR. Consistency among locations. A greater excess of lung cancer among workers hired before 1950, when exposures were greater. The highest lung-cancer risk at the plant that had the greatest proportion of acute beryllium pneumonitis cases in the Beryllium Case Registry. High lung-cancer risks at plants with the greatest risk of pneumoconiosis and other respiratory diseases. A greater lung cancer risk observed in the Beryllium Case Registry cohort. Increasing risks with increasing latency. IARC pointed out the following limitations: Absence of individual exposure measurements. Relatively low excess lung-cancer risks. Absence of any mention of exposures to other lung carcinogens in the workplace. A series of letters and papers issued after the IARC report raised concerns and objections about the basis of its conclusions. Some raised concerns about the IARC procedures, the information available to the IARC working group, and possible conflicts of interest (Kotin 1994a,b; Vainio and Kleihues 1994). Others questioned the validity of the Ward et al. study. Questions were raised about the dataset used to estimate background lung-cancer rates, how to combine data from multiple plants, and how to adjust for cigarette-smoking (MacMahon 1994; Levy et al. 2002). Levy et al. (2002) have reported that making alternative adjustments and comparisons to address those issues resulted in no statistical association between beryllium exposure in the workers and lung cancer. Since the IARC evaluation in 1993, there have been two additional studies. Sanderson et al. (2001b) conducted a nested case-control study of plant workers at the Reading, Pennsylvania, facility. The cohort of 3,569 male workers was the same cohort in the Ward et al. study in 1992. The lung-cancer cases numbered 142 on the basis of a followup of the cohort through 1992, each of which was age- and race-matched to five controls. In addition to assessment of beryllium exposures, the potential for confounding by smoking was evaluated. The cases had lower lifetime exposures to beryllium. However, when a 10-year lag and a 20- year lag were applied, the exposure metrics were higher for cases. Furthermore, significant positive trends with the log of exposure metrics were observed, and the authors concluded that smoking did not confound the exposure-response analysis.

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Health Effects of Beryllium Exposure: A Literature Review Methodologic concerns have been raised about the Sanderson et al. study. Deubner et al. (2001c) suggested that concomitant exposure to acid mists and vapors was a possible confounder, noted difficulties with the adjustment for tobacco-smoking, and raised issues about the selection of control subjects. The study results were reanalyzed with different methods for summarizing exposure histories and for matching controls to cases (Levy et al. 2007). Each alternative method resulted in lower exposure ORs that were nonsignificant. Brown et al. (2004) published a study of lung cancer and internal doses of plutonium among workers at the Rocky Flats plant in Colorado. The case-control study obtained information on smoking histories and on cumulative exposures to four lung carcinogens: asbestos, beryllium, hexavalent chromium, and nickel. In their analysis, none of the exposures to the four carcinogens was significantly associated with lung-cancer mortality. Animal Studies This section focuses on studies of inhalation exposure to beryllium and its compounds and the later development of neoplasms in laboratory animals (see Table 4-2). Lung neoplasms have been found in rats and monkeys exposed to beryllium compounds via inhalation. Albino Sherman and Wistar rats (male and female) were exposed via inhalation to an aqueous aerosol of beryllium sulfate tetrahydate (which contained beryllium at 35.7 μg/m3) for 8 h/day, 5.5 days/week, for 6 months (Schepers et al. 1957). The rats were observed for 18 months after exposure. Lung neoplasms (18 adenomas, five squamous-cell carcinomas, 11 papillary adenocarcinomas, and seven alveolar-cell adenocarcinomas) were observed in the treated rats but not in the control rats. A study by Vorwald and Reeves (1959) reported the development of lung neoplasms in Sherman rats (number and sex not reported) exposed via inhalation to beryllium sulfate at 6 and 54.7 μg/m3 for 6 h/day, 5 days/week, for up to 18 months. The neoplasms observed were primarily adenomas and squamous-cell cancers. A study by Reeves et al. (1967) exposed male and female Sprague-Dawley rats to beryllium sulfate at 34.25 μg/m3 for 7 h/day, 5 days/week. The mean particle size of the beryllium sulfate aerosol was 0.118 μm. Exposure lasted up to 72 weeks. After 13 months of exposure, all the exposed rats developed alveolar adenocarcinomas; the control rats had no lung neoplasms. The neoplasia was preceded by a proliferative response that progressed from hyperplasia to neoplasia. In another study in which particle size was calibrated, Charles River CD rats were exposed to beryllium sulfate at 35.16 μg /m3, with a mean particle size of 0.21 μm, for 35 h/week (Reeves and Deitch 1969). The exposure durations were 800, 1,600, and 2,400 h. The lung- tumor incidence in young rats exposed for 3 months (86%, 19 of 22 rats) was the same as that in older rats exposed for 18 months (86%, 13 of 15 rats). However, older rats that were exposed to beryllium sulfate for 3 months had fewer lung neoplasms than rats that were exposed when they were younger. The pulmonary neoplasms were typically observed after a latency of 9 months. Preneoplastic lesions were described as epithelial hyperplasia at 1 month, metaplasia at 5 to 6 months, and anaplasia by 7 to 8 months. Male and female rhesus monkeys (Macaca mulatta) were exposed to beryllium sulfate at 35 μg/m3 for 6 h/day, 5 days/week (Vorwald 1968). Exposure was often interrupted for considerable periods to prevent the monkeys from developing acute beryllium pneumonitis (four monkeys died of acute chemical pneumonitis during the first 2 months of the study). The longest exposure was for a total of 4,070 h, and most of the exposure periods occurred during the first 4.5 years of the study. A 6-month exposure occurred 2.5 years after the initial 4.5-year exposure period. The authors reported that pulmonary anaplastic carcinomas (adenomatous and epidermoid patterns) were observed in eight of 12 monkeys; the first tumor was observed in a monkey that had been exposed for 3,241 h. The neoplasms metastasized to mediastinal lymph nodes and other areas of the body. Lung tumors were observed in male white random-bred rats exposed to beryllium fluoride (at 0.4 or 0.04 mg/m3) or beryllium chloride (at 0.2 or 0.02 mg/m3) for 1 h/day, 5 days/week, for 4 months

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Health Effects of Beryllium Exposure: A Literature Review TABLE 4-2 Inhalation Carcinogenicity Studies of Beryllium Reference Species Route Dose Findings Acute Exposures Sanders et al. 1978 Rat Inhalation 1.0-91 μg of Be from BeO (single, alveolar deposition) Particle size: 1.10 ± 0.17 μm (GSD, 2.17 ± 0.17 μm) Alveolar half-life of Be in lungs was 325 d; 1 of 184 rats had lung tumors after 2 years Groth et al. 1980 Rat Intratracheal Be at 0.5 or 2.5 mg/m3 as passivated metal (Be-Cr), alloys (Al, Cu, Ni, Cu/Co) Particle size: 1-2 μm Lung adenomas, adenocarcinomas found in 2 of 21 and 9 of 16 treated with Be, 7 of 20 and 9 of 26 treated with Be-Cr, 1 of 21 and 4 of 24 treated with Be-Al, respectively; no tumors with other alloys Litvinov et al. 1983 Rat Intratracheal BeO at 0.036, 0.36, 3.6, or 18 mg/kg (low- and high-fired) Malignant epithelial lung tumors found;. after low-fired BeO, 0 of 76, 0 of 84, 2 of 77, 2 of 103, respectively; after high-fired BeO,: 3 of 69, 7 of 81, 18 of 79, 8 of 26 Nickell-Brady et al. 1994 Rat Inhalation Be at 410, 500, 830, 980 mg/m3 (single exposure; lung burdens, 110, 40, 360, 430 μg) Particle size: 1.4 μm (GSD, 1.9 μm) 64% of rats developed lung tumors (primarily adenocarcinomas) after 14 months Short-term and Subchronic Exposures Schepers et al. 1957 Rat Inhalation Be at 35.7 μg/m3 as BeSO4 (8 h/day, 5.5 days/week for up to 6 months) Lung-cancer rates higher in exposed rats than in controls Vorwald and Reeves 1959 Rat Intratracheal 4.5 mg of Be as BeO, 0.1071 mg of Be as BeSO4 (three injections over 3 weeks) Lung tumors began to appear after 8 months; percentage of rats affected not specified in paper Ishinishi et al. 1980 Rat Intratracheal 1 mg of BeO (low-fired) (once a week for 15 weeks) 1 adenocarcinoma, 1 squamous-cell carcinoma, 4 adenomas Chronic Exposures Dutra et al. 1951 Rabbit Inhalation BeO at 1, 6, 30 μg/L (5 h/day, 5 days/week, for 9-13 months) Particle size: 0.285 μm (mean), 0.11-1.25 μm (range) 6 of 9 rabbits developed osteosarcomas after 1 year

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Health Effects of Beryllium Exposure: A Literature Review Reference Species Route Dose Findings Vorwald and Reeves 1959 Rat Inhalation Be at 0.0547 mg/m3 as BeSO4, at 0.006 mg/m3 as BeO (6 h/day, 5 days/week, for various durations up to 18 months) Lung tumors began to appear after 9 months; percentage of rats affected not specified, but later report (Vorwald et al. 1966) describes incidence of cancer as “almost 100%” in “large number” of surviving rats Reeves et al. 1967 Rat Inhalation Be at 34.25 μg/m3 (mean) as BeSO4 (7 h/day, 5 days/week, for 72 weeks) Particle size: 0.118 μm All rats developed lung tumors (adenocarcinomas) by 13 months Vorwald 1968 Monkey Inhalation Be SO4 at 35 μg/m3 (6 h/day, 5 days/week, with various interruptions and variable durations up to 4,070 h) 8 of 12 monkeys had pulmonary anaplastic carcinomas (adenomatous and epidermoid patterns); first tumor observed after 3,241 h of exposure Reeves and Deitch 1969 Rat Inhalation BeSO4 at 35.16 μg/m3 (35 h/week for 800, 1,600, 2,400 h) Particle size: 0.21 μm (mean) 19 of 22 young rats and 13 of 15 older rats developed lung tumors after 3 and 18 months, respectively; at 3 months, older rats had fewer lung neoplasms than younger rats Wagner et al. 1969 Rat, hamser, squirrel monkey Inhalation Bertrandite dust at 15 mg/m3 (Be at 210 μg/m3) or beryl ore at 15 mg/m3 (Be at 620 μg/m3) (6 h/day, 5 days/week, for up to 23 months) Particle size: bertrandite, 0.27 μm (mean); beryl ore, 0.64 μm (mean) 18 of 19 rats exposed to beryl ore had lung tumors (bronchial alveolar cell tumors, adenomas, adenocarcinomas, or epidermoid tumors); no increased incidence of tumors in rats from dust or in other species from either compound Litvinov et al. 1975 Rats Inhalation BeF2 at 0.04 or 0.4 mg/m3or BeCl2 at 0.02 or 0.2 mg/m3 (1 h/day, 5 day/week, for 4 months) Lung tumors found in treatment groups Litvinov et al. 1984 Rats Inhalation BeO or BeCl2 at 0.8, 4, 30, or 400 μg/m3 (1 h/day, 5 days/week, for 4 months) Malignant lung tumors found in 3 of 44, 4 of 39, 6 of 26, 8 of 21 in BeO group and in 1 of 44, 2 of 42, 8 of 24, 11 of 19 in BeCl2 group, respectively

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Health Effects of Beryllium Exposure: A Literature Review Reference Species Route Dose Findings Finch et al. 1998b Mouse (p53+/− knockout and p53+/+ wild-type) Inhalation 15 or 60 Be μg (60 μg achieved over 3 days) Particle size: 1.4 μm (mean) (GSD, 1.8 μm) 4 of 28 p53+/− mice in high-dose group developed lung tumors; no lung tumors found in low-dose group or in p53+/− mice Mode-of-Action Studies Skilleter et al. 1991 Rat hepatic BL9L cells In vitro 50 μM BeSO4 BeSO4 inhibited cell division during G1 phase of cell cycle, but expression of c-myc was maintained in serum-stimulated cells Nickell-Brady et al. 1994 Rat Inhalation Be at 410, 500, 830, 980 mg/m3 (single exposure; lung burdens, 110, 40, 360, 430 μg) Particle size: 1.4 μm (GSD, 1.9 μm) Analysis of p53 and c-raf-1 genes in neoplasms did not indicate genetic alterations; weak evidence of mutation of K-ras gene Swafford et al. 1997 Rat primary lung tumors and cells lines from Nickell-Brady et al. (1994) study     Aberrant methylation status of p16INK4a , leading to loss of expression Joseph et al. 2001 Mouse BALB/c-3T3 cells Transformed cells injected into nude mice, cell lines derived from resulting tumors BeSO4 at 50-200 μg/mL Analyses of gene expression indicate that cell transformation and tumorigenesis are associated with upregulated expression of genes related to cancer (such as c-fos, c-jun, c-myc, R-ras) and downregulation of genes involved in DNA synthesis, repair, recombination (such as MCM4, MCM5, PMS2, Rad23, DNA ligase I) Keshava et al. 2001 Mouse BALB/c-3T3 cells In vitro BeSO4 at 50-200 μg/mL Results show that cells transformed by BeSO4 are potentially tumorigenic; transformation might involve gene amplification of K-ras, c-jun; some transformed cells have neoplastic potential because of genomic instability Belinsky et al. 2002 Rat Inhalation 40, 110, 360, 430 μg of Be (single dose; mean lung burdens) Tumors induced in part through inactivation of p16 and ER genes Misra et al. 2002 Mouse peritoneal macrophages In vitro 1-5 nM BeF2 Phosphorylation increased for kinases MEK1, ERK1, p38 MAPK, JNK; increases also seen in NF-κB, CREB transcription factors, c-fos, and c-myc Note: Al = aluminum, Be = beryllium, BeCl2 = beryllium chloride, BeF2 = beryllium fluoride, BeO = beryllium oxide, BeSO4 = beryllium sulfate, Co = cobalt, Cr = chromium, Cu = copper,; GSD = geometric standard deviation, Ni = nickel.

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Health Effects of Beryllium Exposure: A Literature Review (Litvinov et al. 1975). The first neoplasms were observed after 16 months in rats exposed to beryllium fluoride at 0.4 mg/m3 and beryllium chloride at 0.2 mg/m3. Neoplasms also developed in the lungs of rats exposed at the lower concentrations, but not in the lungs of the control rats. Litvinov et al. (1984) exposed female albino rats to beryllium oxide or beryllium chloride at 0.8, 4, 30, and 400 μg/m3 for 1 h/day, 5 days/week, for 4 months. Malignant lung neoplasms developed in a dose-related manner after exposure to either beryllium oxide or beryllium chloride, but none was found in the controls. The carcinogenicity of two beryllium ores, bertrandite and beryl, was evaluated in male squirrel monkeys (Saimiri sciurea), male Charles River-CD rats, male Greenacres Controlled Flora (GA) rats, and male Golden Syrian hamsters (Wagner et al. 1969). The rats and hamsters were exposed to bertrandite or beryl at 15 mg/m3 for 6 h/day, 5 days/week, for 17 months, and the monkeys were exposed for 23 months. Beryllium from bertrandite was present in the test atmospheres at 210 μg/m3 and from beryl at 620 μg/m3; the geometric means of the particles were 0.27 μm and 0.64 μm, respectively. In the beryl-exposed rats, squamous metaplasia or small epidermoid tumors were identified in the lungs of five of 11 rats killed after 12 months of exposure and 18 of 19 rats after 17 months of exposure. Eighteen of the rats had bronchiolar alveolar-cell tumors, nine had adenocarcinomas, seven had adenomas, and four had epidermoid tumors. Although granulomatous lesions were observed in the bertrandite-exposed rats, no neoplasms were identified in the rats exposed for 6, 12, or 17 months. Neither neoplasms nor granulomas developed in the control rats. Atypical proliferations were observed in the lungs of hamsters 12 months after exposure to bertrandite or beryl. The lesions were reported to be larger and more adenomatous in the beryl group after 17 months. No pulmonary lesions occurred in the control hamsters. No tumors were observed in either the bertrandite- or beryl-treated monkeys. The carcinogenicity of beryllium metal has also been investigated. In one study, male and female F344/N rats were received a single, nose-only exposure to a beryllium metal aerosol at 500 mg/m3 for 8 min, at 410 mg/m3 for 30 min, at 830 mg/m3 for 48 min, or at 980 mg/m3 for 39 min (Nickell-Brady et al. 1994). The latent period for development of neoplasms was about 14 months; tumor incidence was 64% over the lifetime of the rats. Most of the neoplasms were adenocarcinomas, although multiple tumor types were observed. In another study of beryllium metals (Groth et al. 1980), lung adenomas and adenocarcinomas were observed in nine of 16 female Wistar rats that received a single intratracheal instillation of 2.5 mg of beryllium metal, nine of 26 rats treated with 2.5 mg of passivated beryllium metal, and four of 24 rats given 2.5 mg of beryllium-aluminum alloy. No neoplasms were observed in the lungs of the controls. Pulmonary neoplasms developed in inbred albino rats that were given single intratracheal deposits of beryllium oxide (fired at high and low temperatures) at 0.036, 0.36, 3.6, or 18 mg/kg (Litvinov et al. 1983). The neoplasms were adenomas, adenocarcinomas, and squamous-cell carcinomas. Wistar rats received intratracheal instillations of 1 mg of beryllium oxide (low-fired) once a week for 15 weeks (Ishinishi et al. 1980). An adenocarcinoma, a squamous-cell carcinoma, and four adenomas were observed in the lungs of 30 beryllium-treated rats and no neoplasms in the 16 controls. SUMMARY Genotoxicity studies of beryllium have yielded conflicting results that appear to be somewhat compound dependent. The committee will critically evaluate the literature in its next report and consider how the information should be factored into the carcinogenic assessment of beryllium. There is evidence from controlled studies that exposure to beryllium can cause lung cancer in both sexes of rats, and one study reported lung tumors in monkeys. Epidemiologic studies have reported increases in lung-cancer risk in two worker cohorts exposed to beryllium. Those studies were instrumental in forming the basis of the current cancer classifications by such agencies as the International Agency for Research on Cancer, the U.S. Environmental Protection Agency, and the National Toxicology Program. In its second report, the committee will focus on assessing the collective evidence in characterizing the carcinogenic potential of beryllium and estimating carcinogenic risks.