Page 10: Guidelines for Canadian Drinking Water Quality: Guideline Technical Document – Benzene

Part II. Science and Technical Considerations - Continued

9.0 Health effects

9.1 Effects in humans

9.1.1 Acute toxicity

Acute exposure to high levels of benzene affects the central nervous system, causing dizziness, nausea, vomiting, headache, and drowsiness. Exposure to levels between 50 and 150 ppm by inhalation over 5 hours can reportedly result in headaches, lethargy, and weakness, although exposure to 25 ppm for 8 hours produced no acute clinical effects (IPCS, 1993; Paustenbach et al., 1993). Inhaling benzene at 20 000 ppm for 5-10 minutes, at 7500 ppm for 30 minutes, or at 1500 ppm for 60 minutes may cause death or severe toxicity in humans (Holliday and Englehardt, 1984; IPCS, 1993). Individuals who have died from sniffing glue containing benzene reportedly had blood concentrations from 1 to 65 mg/L, with death resulting from pulmonary haemorrhaging and inflammation, renal congestion, cerebral oedema, or a combination of these (IPCS, 1993). ATSDR (2007) estimates a lethal oral dose of benzene in humans to be about 125 ppm.

9.1.2 Subchronic and chronic toxicity

Subchronic and chronic exposure to benzene leads to numerous adverse effects, including damage to bone marrow, changes in circulating blood cells, immunological effects, and cancer (see Section 9.1.5). The most commonly reported non-cancer effects from chronic exposure to inhaled benzene include blood disorders, such as aplastic anaemia, pancytopenia, thrombocytopenia, granulocytopenia, and lymphocytopenia. The effects of benzene exposure on several blood cell lineages suggest that benzene and/or its metabolites target the bone marrow or early progenitor cells (IPCS, 1993; ATSDR, 2007).

A study by Lan et al. (2004) of 250 shoe workers in China exposed to benzene found a highly significant dose-dependent decrease in colony formation of progenitor cells with increasing benzene exposure. With a greater proportional decrease in progenitor cell colony formation than the proportional decrease in the levels of differentiated white blood cells and granulocytes, Lan et al. (2004) suggested that early progenitor cells are more sensitive to the haematotoxic effects of benzene than mature blood cells. This is in agreement with other earlier findings in both humans and animals (Smith et al., 2000; Abernathy et al., 2004).

In a group of 44 healthy Chinese workers, Rothman et al. (1996a, 1996b) reported that early biomarkers of exposure to relatively low levels of benzene included significantly depressed numbers of total red blood cells, white blood cells, absolute lymphocyte count, platelets, and haematocrit. The workers were exposed to benzene in the workplace (median 8-hour timeweighted average [TWA] of 31 ppm; minimal exposure to other solvents) for an average of 6.3 years. Age- and gender-matched workers with no history of occupational exposure to benzene served as controls. The authors reported significantly depressed absolute red blood cells and platelets among the 22 workers whose mean 5-day benzene exposure levels did not exceed 31 ppm (median 8-hour TWA of 13.6 ppm); of these, a subgroup of 11 workers with a median 8-hour TWA of 7.6 ppm also showed a significantly decreased absolute lymphocyte count.

9.1.3 Genetic toxicity

Benzene is reported to be clastogenic in humans, with effects including aneuploidy, ploidy, micronuclei, chromosomal deletions, translocations, and rearrangements (IARC, 1982; ATSDR, 2007). Most cytogenetic studies have looked at the blood lymphocytes of exposed workers and report increased structural (chromatid and/or chromosome breaks) and/or numerical chromosomal aberrations in mitogen-stimulated peripheral lymphocytes (ATSDR, 2007). Benzene exposure in humans has also been shown to result in the types of chromosomal aberrations that are common with certain leukaemias, such as acute myelogenous leukaemia and myelodysplastic syndromes (Smith and Zhang, 1998). Aberrations include specific gains or losses in chromosomes, translocations, deletions, and inversions, most commonly associated with chromosome 5, 7, 8, 9, 21, or 22.

Lymphocytes in Chinese workers occupationally exposed to benzene have been shown to contain higher frequencies of specific chromosomal alterations such as chromosome 9 hyperdiploidy, translocations between chromosomes 8 and 21, and aneusomies of chromosomes 8 and 21 (Zhang et al., 1996; Smith and Zhang, 1998). Significant increases in the rates of monosomy for chromosomes 5 and 7 (p < 0.001 and p < 0.0001, respectively) and increases in the frequencies of trisomy and tetrasomy of chromosomes 1, 5, and 7 have also been reported (Zhang et al., 1998). Many of these chromosomal alterations have also been observed in vitro in human cells treated with benzene metabolites. Zhang et al. (1994) and Stillman et al. (1997) found doserelated increases of aneuploidy of chromosomes 5 and 7 in human haematopoietic cells treated with hydroquinone or 1,2,4-trihydroxybenzene. Zhang et al. (1994) reported trisomy and tetrasomy of chromosomes 7 and 9 in a human cell line treated with hydroquinone or 1,2,4-benzenetriol. Exposure of human lymphocytes to hydroquinone resulted in hyperdiploidy in chromosome 9 (Eastmond et al., 1994).

9.1.4 Developmental and reproductive toxicity

Studies are limited regarding the effects of maternal exposure to benzene. Abnormal menstruation and excessive blood loss during childbirth have been reported in women occupationally exposed to benzene (OEHHA, 1997). These reports, however, are limited, since the comparison groups were exposed to different environments that were not described, the methods were poorly described, and co-exposure to other solvents associated with employment in rubber and/or leather factories likely occurred. More definitive studies with accurate assessment of benzene-specific exposure are needed.

9.1.5 Carcinogenicity

There are numerous studies that report increased cancer rates from occupational exposure to benzene (Bond et al., 1986; Wong, 1987; Hayes et al., 1996; Schnatter et al., 1996; Rushton and Romaniuk, 1997). Reviews of benzene carcinogenicity due to occupational exposure have been published by IARC (1982), IPCS (1993), and ATSDR (2007).

The Ohio Pliofilm (rubber hydrochloride) cohort represents a good published set of data for assessing human cancer risks from exposure to benzene, since it has the fewest reported coexposures to other possible carcinogenic substances in the workplace that could impact a risk analysis for benzene, and the Pliofilm workers were exposed to a wider range of estimated benzene concentrations than were workers in other cohort studies (U.S. EPA, 1998b). Rinsky et al. (1981) was the first to extensively study the Pliofilm cohort, which included 748 male workers in three facilities in Ohio who were exposed to benzene during employment between 1940 and 1949 and were followed until the end of 1981. Benzene exposure levels were estimated to range from 100 ppm in 1941 to 10 ppm (8-hour TWA) in 1949. A statistically significant increase in mortality due to malignancies of the lymphatic and haematopoietic tissue (standardized mortality ratio [SMR] = 330; p < 0.01) was reported, with seven of the deaths due to leukaemia (SMR = 560; p < 0.001). Workers exposed for longer than 5 years had an SMR due to leukaemia of 2100. Rinsky et al. (1987) subsequently updated and expanded the Ohio cohort study to include individuals who had worked at least 1 day between 1940 and 1965, with person-years at risk starting in 1950. The updated cohort was composed of 1165 white males followed through 1981, which included an additional 6.5 years of follow-up from the earlier study, as well as individual estimates of personal exposure. Duration of employment and personal exposure estimates during that time of employment were used to generate risk estimates based on grouped data. Once again, a strong positive trend in leukaemia mortality was seen with increasing exposure to benzene; a statistically significant increase was observed for all lymphatic and haematopoietic cancers (15 deaths) compared with that expected in the general population (SMR = 227, 95% confidence interval [CI] = 127-376). For total leukaemia deaths (nine deaths), the SMR was 337 (95% CI = 159-641). An increased risk of multiple myeloma (four deaths) was also reported (SMR = 398, 95% CI = 110-1047). Analyses by other authors (Paustenbach et al., 1993; Paxton et al., 1994) with expanded periods of follow-up and altered exposure estimates have yielded slightly different results; however, the differences fall within the same range of uncertainty.

A large retrospective cohort study of benzene-exposed workers in China by Yin et al. (1987) examined 28 460 exposed workers from 233 factories and 28 257 control workers from different industries. Thirty leukaemia cases were identified (23 acute, 7 chronic) in the exposed workers compared with four cases in the unexposed controls (SMR = 574, p < 0.01). Exposure estimates at the time of the survey ranged from 3 to 313 ppm, with the majority of exposures in the range of 16-157 ppm. In 1994, the cohort was expanded by Yin et al. (1994) to include 74 828 benzene-exposed workers (since 1949) and 35 805 controls from 712 factories located in 12 Chinese cities. Dosemeci et al. (1994) described the exposure assessment, which included job title and assignment to individual work units reflecting exposures of individual workers. Yin et al. (1996) reported the overall cancer findings among the expanded benzene-exposed and control worker cohorts. An increased incidence in the benzene-exposed group compared with controls was observed for leukaemia (relative risk [RR] = 2.6, 95% CI = 1.3-5.0), malignant lymphoma (RR = 3.5, 95% CI = 1.2-14.9), and lung cancer deaths (RR = 1.4, 95% CI = 1.0-2.0). Among leukaemia cases, incidence of acute myelogenous leukaemia was increased in the benzeneexposed group (RR = 3.1, 95% CI = 1.2-10.7). Significant increases were also reported for aplastic anaemia and myelodysplastic syndromes.

9.1.6 Mode of action for carcinogenesis

Benzene biotransformation results in the generation of several metabolites (see Section 8.0) that can induce cytotoxicity through different metabolic mechanisms (Smith, 1996; Ross, 2000; Snyder, 2000). These reactive metabolites include quinones that can bind to cellular macromolecules (including DNA), tubulin, histones, and topoisomerase II. Benzoquinones and other benzene metabolites can cause oxidative DNA damage, lipid peroxidation in vivo, formation of hydroxylated deoxyguanosine residues, and strand breaks in the DNA of bone marrow cells, implicating a role for reactive oxygen species and covalent binding in benzeneinduced toxicity. The formation of DNA double-strand breaks by reactive oxygen species and other mechanisms can lead to increased mitotic recombination, chromosomal translocations, and aneuploidy (Smith, 1996). Genetic events such as these can result in proto-oncogene activation, tumour suppressor gene inactivation, gene fusions, and other changes in stem cells that can ultimately result in leukaemia.

9.2 Effects in experimental animals

9.2.1 Acute toxicity

Animals exposed to a one-time high dose of benzene have displayed narcotic effects and death. Oral LD50 values for rats fall in the 300-8100 mg/kg bw range. An LC50 of 10 000 ppm for short-term exposure to benzene in air was reported for rats, mice, rabbits, and guinea pigs (IPCS, 1993; Paustenbach et al., 1993).

9.2.2 Subchronic and chronic toxicity

Subchronic and chronic exposure of experimental animals to benzene has resulted in haematological effects similar to those observed in humans following occupational exposure. Lymphocytopenia, anaemia, leukopenia, and changes in bone marrow morphology and cellularity have been consistently reported by many authors (Snyder et al., 1978, 1984; Cronkite et al., 1985; Ward et al., 1985; Aoyama, 1986; Li et al., 1986; NTP, 1986; ATSDR, 2007). A 2- year study by the U.S. National Toxicology Program (NTP, 1986) reported haematological effects in rats and mice (both sexes), which included lymphoid depletion of the splenic follicles (rats) and thymus (male rats), bone marrow haematopoietic hyperplasia (mice), lymphocytopenia, and associated leukocytopenia (rats and mice). Several of these effects occurred at the lowest exposure level (25 mg/kg bw per day). In animals, lymphocyte levels generally appear to fall the most in the shortest time, whereas granulocytes appear to be the most resistant of the circulating cells; anaemia does not appear to occur as frequently as lymphocytopenia (ATSDR, 2007).

In 2007, the U.S. NTP exposed groups of 15 male and 15 female haploinsufficient p16Ink4a/p19Arf mice to 0, 25, 50, 100, or 200 mg benzene/kg bw per day in corn oil by gavage 5 days per week for 27 weeks. Males exposed to 25 mg benzene/kg bw per day or greater and females exposed to 50 mg benzene/kg bw per day or greater displayed black, brown, or grey pigmentation of the feet. Thymus weights of all dosed groups of males were significantly decreased. At weeks 13 and 27, dose-related decreases in haematocrit, haemoglobin, and erythrocyte count values in all dosed males and in the 100 mg/kg bw per day or greater females were reported. Decreased leukocyte counts, primarily lymphocyte counts, resulted in a doserelated leukopenia in males and females. In males, segmented neutrophil counts were also decreased. In the bone marrow, significantly increased incidences of minimal to mild atrophy were observed in the 100 and 200 mg/kg bw per day male dose groups compared with the vehicle controls; a significantly increased incidence of lymphoid follicle atrophy in the spleen was also observed in these dose groups. The incidence of haematopoietic cell proliferation was significantly increased in the 200 mg/kg bw per day dose group males. The 100 and 200 mg/kg bw per day dosed males also displayed significantly increased incidences of atrophy of the thymus and lymph nodes (mandibular, mediastinal, and mesenteric atrophy); a significantly increased incidence of atrophy of the mediastinal lymph node was also seen in the 100 mg/kg bw per day dosed females. The incidences of skin pigmentation were significantly increased in all dosed groups of males and in females dosed with 50 mg/kg bw per day or greater.

9.2.3 Genetic toxicity

Benzene has also been shown to be genotoxic in animals. In vitro studies have shown benzene to exhibit mixed results, with positive findings reported for gene mutations in bacteria and inhibition of DNA or RNA synthesis in mammalian cells. Benzene metabolites such as phenolic, quinone, epoxide, and aldehyde species cause mutations in bacteria, as well as sister chromatid exchanges, micronuclei formation, DNA strand breaks, DNA adducts, and oxidative DNA damage in mammalian cells. In vivo, benzene induces chromosomal aberrations in lymphocytes (mice) and in bone marrow cells (rats and hamsters) and increases the incidence of micronuclei in bone marrow (mice and hamsters), peripheral erythrocytes (mice), and lymphocytes (rats). Other genotoxic effects include gene mutations and polyploidy in mouse lymphocytes, as well as sister chromatid exchanges in the mouse fetus, liver, bone marrow, and rat and mouse lymphocytes. Sperm head abnormalities have also been observed in benzene-exposed male mice (ATSDR, 2007).

9.2.4 Developmental and reproductive toxicity

Benzene has not been found to be teratogenic in animals, although embryotoxic and fetotoxic effects have been reported at airborne concentrations as low as 47 ppm in rats (a level found not to be toxic to the dams) (Tatrai et al., 1980). Haematological effects are also reported in mice exposed to low levels of benzene in utero (Keller and Snyder, 1986). Administration of 20 ppm benzene to pregnant Swiss Webster mice for 6 hours per day on gestational days 6-15 caused reductions in the levels of erythroid progenitor (CFU-E) cells of the fetuses, whereas 5 and 10 ppm benzene caused enhancement of these colony-forming cells. In 2-day-old neonates, CFU-E numbers in the 5 ppm group returned to control values, but the 10 ppm neonates showed a bimodal response by litter. Granulocytic colony-forming cells were enhanced in neonates exposed in utero to 20 ppm benzene. Some of the mice exposed to 10 ppm prenatally were reexposed to 10 ppm as adults. Their haematopoietic progenitor cell numbers were depressed compared with controls exposed for the first time as adults. In a follow-up study by Keller and Snyder (1988), pregnant Swiss Webster mice exposed to 5, 10, or 20 ppm benzene for 6 hours per day on gestational days 6-15 showed no significant changes in erythrocyte and leukocyte counts, haemoglobin analysis, and the proliferating pool of differentiating haematopoietic cells in 16-day fetuses. In 2-day neonates, however, exposure in utero to all concentrations of benzene exhibited a reduced number of circulating erythroid precursor cells, and, at 20 ppm, increased numbers of hepatic haematopoietic blast cells and granulopoietic precursor cells accompanied by decreased numbers of erythropoietic precursor cells were observed. Six-week-old adult mice exposed in utero to 20 ppm of benzene had a similar pattern of enhanced granulopoiesis. However, this effect was not clearly evident in 6-week-old adult mice exposed in utero to 5 or 10 ppm.

9.2.5 Carcinogenicity

A 2-year study by the NTP (1986) exposed F344 rats and B6C3F1 mice (50 animals per sex per group) orally (by gavage) to benzene in corn oil 5 days per week for 103 weeks. Female rats and mice were exposed to 0, 25, 50, or 100 mg/kg bw per day, and males were exposed to 0, 5, 100, or 200 mg/kg bw per day. Female rats in the mid- and high-dose groups had significantly higher incidences of cancer of the oral cavity, Zymbal gland (an auditory sebaceous gland that opens into each external ear canal; not found in humans), and uterus; in males, an increased incidence of cancers of the oral cavity, Zymbal gland, and skin was observed. Female mice were reported to have significant dose-related increases in the rate of cancer of the Zymbal gland, ovary, mammary gland, Harderian gland, and lung. In male mice, a dose-related increase in the rate of cancer of the Zymbal, preputial, and Harderian glands and lungs was also observed.

Numerous other studies have shown benzene to be carcinogenic in rats and mice. Maltoni et al. (1982, 1983, 1985, 1989) reported that benzene administered (by stomach tube) to 13-week-old Sprague-Dawley rats at 0, 50, or 250 mg/kg bw in olive oil, 4-5 times per week for 52 weeks, resulted in dose-related increases in the incidence of Zymbal gland carcinomas in female rats only. In another study by Maltoni et al. (1989), 7-week-old male and female Sprague-Dawley rats orally exposed (by stomach tube) to 0 or 500 mg benzene/kg bw in olive oil 4-5 times per week for 105 weeks displayed significantly higher incidence (related to controls) of Zymbal gland and oral cavity carcinomas (males and females), nasal cavity and skin carcinomas (males), and cancer of the forestomach (females). Wistar rats, Swiss mice, and RF/J mice (50 animals per sex per group) orally exposed to 0 or 50 mg benzene/kg bw in olive oil 4-5 times per week for 104, 78, and 52 weeks, respectively, showed an increased incidence in cancer compared with controls (Maltoni et al., 1989). Wistar rats displayed an increased incidence of cancers of the Zymbal gland (males) and oral cavity (females); Swiss mice had an increased incidence of cancers of the Zymbal gland (males), mammary gland (females), and lung tissue (males and females); and RF/J mice were found to have a higher incidence of pulmonary tumours (males and females) and mammary gland carcinomas (females). Maltoni et al. (1982, 1983, 1985, 1989) also assessed the carcinogenic potential of benzene through inhalation studies using pregnant Sprague-Dawley rats and their offspring. Exposure to 0, 200, or 300 ppm of benzene for 15 or 104 weeks also showed increased incidences (compared with controls) of Zymbal gland cancers and mammary gland tumours in adults, with significantly higher incidences of Zymbal gland cancers and non-significant increases in cancers of the oral and nasal cavity, mammary gland, and liver also reported in the offspring. In another experiment by Maltoni et al. (1989), Sprague-Dawley rats were exposed to benzene in utero (via dams exposed to 0, 200, or 300 ppm) from day 12 of gestation and during lactation. Slight increases in the incidences of Zymbal gland carcinoma, oral cavity carcinoma, hepatoma, and leukaemia were reported (Maltoni et al., 1989).

Leukaemia and lymphoma have been reported in several other studies investigating benzene-mediated effects following inhalation and oral exposure. In a series of studies by Cronkite et al. (1984, 1985, 1989) and Cronkite (1986), C57BL/6 and CBA/Ca mice were exposed to 300 ppm benzene in air 6 hours daily, 5 days per week, for 16 weeks at variable intervals mimicking patterns of human occupational exposure to benzene. A significant increase in both leukaemia and lymphoma was reported in both strains of mice, as well as solid tumours (mammary and hepatoma) for CBA/Ca mice. Cronkite et al. (1989) reported a higher incidence of leukaemia in male and female CBA/CA mice exposed to 300 and 3000 ppm for 16 weeks; exposure to 3000 ppm, however, did not shorten the latency or increase the incidence compared with the 300 ppm treatment group. In a study by Farris et al. (1993), 125 male CBA/Ca mice were exposed to 300 ppm benzene for 6 hours daily, 5 days per week, for 16 weeks and sacrificed after 18 months; controls (sham-exposed, n = 125) were treated with filtered air. Significant increases in incidences of malignant lymphoma were observed in addition to preputial gland squamous cell carcinoma, lung adenoma, carcinoma of the Zymbal gland and forestomach squamous cells, as well as increased granulocytic hyperplasia of the bone marrow and spleen.

As part of an ongoing effort to determine the carcinogenic mode of action of benzene, the NTP (2007) assessed benzene's carcinogenic effects in the haploinsufficient p16Ink4a/p19Arf mouse model. Groups of 15 male and 15 female p16Ink4a/p19Arf mice were administered 0, 25, 50, 100, or 200 mg benzene/kg bw per day in corn oil by gavage 5 days per week for 27 weeks. All animals except one male administered 200 mg/kg bw per day survived until the end of the study. The incidence of malignant lymphoma was significantly increased in males exposed to 200 mg benzene/kg bw per day compared with the vehicle controls and exceeded the incidence seen in the historical controls. Malignant lymphomas were not seen in female p16Ink4a/p19Arf mice, which suggests that benzene may be more clastogenic in males than in females. This theory was supported by the micronucleus results, which showed that males exposed to the carcinogenic dose of 200 mg/kg bw per day for 27 weeks had approximately four times the number of micronuclei compared with females.

9.2.6 Immunotoxicity

Concentrations of benzene as low as 10 ppm in air have been reported to cause immunological effects (depression of the response of B cells and T cells) in rats (Rozen et al., 1984). Mice exposed to 300 ppm benzene for 6 hours per day, 5 days per week, for 115 days showed reduced numbers of B cells in the spleen and bone marrow and T cells in the thymus and spleen (Rozen and Snyder, 1985).

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