207
DANGEROUS AND CANCER-CAUSING PROPERTIES OF PRODUCTS AND CHEMICALS IN THE OIL REFINING AND PETROCHEMICAL INDUSTRY - PART II:
CARCINOGENICITY, MUTAGENICITY, AND DEVELOPMENTAL TOXICITY OF 1,3-BUTADIENE M.A. MEHLMAN UMDNJ-Robert Wood Johnson Medical School Department of Environmental and Community Medicine 675 Hoes Lane, Piscataway, New Jersey 08854
M.S. LEGATOR Division
of
Environmental
Toxicology
University of Texas Medical Branch Galveston, Texas 77550
1,3-butadiene (BD) is present in synthetic rubber and motor fuels (gasoline). BD is shown to cause lymphocytic lymphomas, heart hemangiosarcomas, lung alveolar bronchiolar cancers, forestomach-squamous cell cancers, harderian gland neoplasms, preputial gland adenoma or carcinoma, liver-hepatocellular cancers, mammary gland acinar cell carcinomas, ovary-glanulosa cell carcinoma, brain cancers, pancreas adenoma and carcinoma, testis-Leydig cell tumors, thyroid follicular adenoma and carcinoma, and zymbal gland carcinoma in rodents and to date no exposure level has been established at which this chemical does not cause cancers.
In humans BD
increase in lymphomas, leukemias, and other cancers of hematopoietic systems and organs. BD is also a potent alkylating agent, directly toxic to developing embryos and damages progeny after parental exposure. causes
INTRODUCTION
1,3-butadiene (BD) is produced by the oil refining industry as a co-product in the manufacture
ethylene and is used in styrene-butadiene rubber (SBR) latex and polybutadiene rubber (PBR). It is estimated that 12 billion pounds of BD is produced worldwide and 3 billion pounds are produced in the United States each year (Morrow, 1990). In addition to being a primary ingredient of synthetic rubber, butadiene is also a component of plastics, gasoline (in the U.S. alone 140 billion gallons of gasoline are consumed annually), and motor vehicle of
Industrial Health, 7:3, pp. 207-220 Princeton Scientific Publishing Co., Inc. ISSN: 0748-2337
Toxicology and Copyright 1991
Downloaded from tih.sagepub.com at UCSF LIBRARY & CKM on April 21, 2015
208
exhausts (Miller, 1978). It has been shown that exposure to butadiene causes an increase in the incidence of malignant lymphomas, leukemias, cardiac hemangiosarcomas, alveolar neoplasms, tumors of the forestomach, carcinomas of the mammary gland, hepatocellular neoplasms, pancreatic adenomas, uterine sarcomas, zymbal gland carcinomas, thyroid tumors, and testicular cancers in animals (Environ. Health Persp., 1990). This paper we will review (a) the metabolism and genotoxic effects of butadiene, (b) the animal and human data which characterize this chemical as a multiorgan, multispecies carcinogen, and (c) the limited studies which indicate that butadiene is a developmental toxin. METABOLISM AND GENOTOXICITY OF 1,3-BUTADIENE
1,3-Butadiene is Metabolized to Reactive Compounds that Form Adducts with DNA In 1955 Kotin and Falk suggested that epoxides might be responsible for carcinogenic activity from polluted air in Los Angeles. In 1957 McCammon, Kotin, and Falk produced tumors in animals which were exposed to the butadiene-derived diepoxy compound.
Later studies indicated that in the presence of microsomal activating systems, 1,3-butadiene is metabolized to the epoxide 1,2-epoxybutene-3, which in turn can be metabolized further by microsomal activating system to dioxybutane and, in the presence of epoxide hydrolase, to the 3,4-epoxy-1,2-butane-diol (Malvoisin et al., 1985). In vitro studies indicate DNA adducts [7,-(2-hydroxy-buten-1-yl) guanine and 7,-(I-hydroxy-buten-1-yl) guanine], are formed with 1,2-epoxybutene-3 (Citti et al., 1984). Using C 14 labeled butadiene, Laib et al. (1990) found that radioactive compounds were covalently bound to nucleoprotein fractions and to the total liver DNA of rats and mice treated with 1,3-butadiene. Thus, from metabolic pathways supported by both in vitro and in vivo data, there is evidence of interaction with and alteration of DNA by metabolites of 1,3-butadiene. In Vivo Somatic Cell :B1vtCtgen In mutagenicity studies in mice, in vivu cxpusujTjs tu a,3~~br.~:~ciiene of 2 weeks and 13 weeks duration produced dose-related increases in three different endpoints: 1) chromosomal aberrations, 2) sister chromatid exchanges (SCE) in bone marrow cells, and 3) micronuclei in peripheral erythrocytes. In the 2 week exposure, the SCE were significantly increased at exposure rates of 6.25 ppm. In the 13 week exposure, where only micronuclei in peripheral
1,3-Butadiene:
an
blood were evaluated, a statistical increase over control was noted at the 6.25 ppm level. Induction of chromosomal effects were concentration-dependent over a dosage range of two orders of magnitude (Shelby, 1990). Studies in the rat do not indicate similar effects (de Meester, 1988). These species’ differences, however, may reflect the more extensive studies carried out with the mouse, and differences in the durations of exposure. For example in the rat, an exposure of only two days were evaluated. At the present time there are no in vivo data available on gene mutations following sub-chronic exposure. Cancer Caused by 1,3-Butadiene iri Animals The National Toxicology Program (NTP) conducted an inhalation study with butadiene at Battele Pacific Northwest Laboratories with B6C3F1 mice of both sexes. The mice were
Downloaded from tih.sagepub.com at UCSF LIBRARY & CKM on April 21, 2015
209
exposed to 627 and 1236 ppm of butadiene. The study was run for 60 weeks for males and 611 weeks for females. The animals were exposed for a shorter period than planned, due to high mortality from malignant tumors in the exposed animals. The major cause of death in both male and female mice from exposure to butadiene were lymphomas, which may have originated in the thymus, and hemangiosarcoma of the heart. These lymphomas occurred as early as 20 weeks after exposure, and that these lymphomas caused the majority of deaths between 40 and 45 weeks of exposure.
Hemangiosarcomas of the heart are extremely uncommon cancers in rodents, and especially in B6C3Fl mice. The background incidence of hemangiosarcomas in B6C3Fl mice is about 1 in every 2500 control animals. Exposure to butadiene of mice resulted in a high frequency of the rare hemangiosarcomas. In addition to the tumors described above, an increase of tumors in the following organs was observed: lung and forestomach of males and females, and liver, mammary gland, and ovaries of females. There was approximately a 29.7 fold increase in total tumors of mice exposed to
1250 ppm (Table 1). TABLE 1
Summary of
Tumors in B6C3Fl Mice Induced
by Inhalation of
1,3-Butadiene
Second NTP Study by Melnick, Huff, Chou, and Miller In the study by Melnick et al. (1990) using B6C3F1 mice, carcinogenicity was observed at the lowest concentration of 6.25 ppm. A &dquo;no effect&dquo; level was not established. In addition, at higher concentrations, 625 ppm, there was a high frequency of fatal lymphocytic lymphomas, masking the development of tumors at other sites at this exposure level. Response for hemangiosarcomas of the heart and neoplasms of the lung and other tumor sites were demonstrated (Figures 1 and 2).
Survival and Incidence of Tunwrs in Both Male and Female B6C3Fl Mice Melnick et al. (1990) show that exposure of mice to butadiene at 20 ppm or higher results in a very significant decrease in survival from that of control mice. The percent of animals with
Downloaded from tih.sagepub.com at UCSF LIBRARY & CKM on April 21, 2015
210
malignant tumors increased three-fold compared with controls (Table 2). In the highest dose, approximately 90% of the animals had tumors. It is important to note the rapidity with which lymphocytic lymphomas of thymic origin appeared only 23 weeks after exposure.
1. Cumulative incidence of lymphocytic lympohomas or hemangiosarcomas of the heart versus weeks on study for male B6C3Fl mice exposed to 200 or 625 ppm of 1,3butadiene. (Source: Melnick et al., 1990. Reproduced by permission of the publisher.).
- FIGURE
FIGURE 2. Cumulative incidences of lymphocytic lymphomas or alveolar-bronchiolar neoplasms versus weeks on study for female B6CX3F mcie exposed to 200 or 625 ppm, of 1,3-butadiene. (Source: Melnick et al., 1990. Reproduced by permission of the publisher.).
Downloaded from tih.sagepub.com at UCSF LIBRARY & CKM on April 21, 2015
211
The cumulative incidence of lymphocytic lymphomas and alveolar-bronchiolar neoplasms is summarized in Figure 1 (males) and Figure 2 (females). These date demonstrate that butadiene causes a high incidence of lymphocytic lymphoma and bronchiolar neoplasms. TABLE 2
Primary Tumors in Male B6C3Fl Mice 1,3-Butadiene for up to 2 Years (a)
Survival and Incidence of
Exposed
to
is given as the number of animals bearing a neoplastic lesion at a specific anatomic site. Overall rates, based on the number of animals at which that site was examined, are given below the incidence values. Kaplan-Meier estimated tumor rates at the end of the study after adjusting for intei-urrent mortality are given in parentheses. Initial number includes animals removed from the study for interim sacrifices at 40 and 65 weeks. Decreased compared to chamber control (0 ppm), P< 0.05. Increased compared to chamber control (0 ppm), P< 0.05, based on logistic regression analyses with adjustment for intercurrent mortality.
(a) Incidence
(b) + *
Source: Melnick
et al.
(1990).
Downloaded from tih.sagepub.com at UCSF LIBRARY & CKM on April 21, 2015
212
The incidence of hemangiosarcomas of the heart was higher for males than for females at lower concentrations of butadiene exposure. Figure 1 shows the incidence of hemangiosarcomas of the heart in mice exposed to 200 ppm and 625 ppm of butadiene. Results summarized in Table 3 show the frequency of multiple-site neoplasia for a variety of tumors in male mice. In control mice, an average of 2.5% of mice developed tumors at two sites, and none developed tumors at three or more sites. In butadiene-exposed mice, up to 12% of the males had tumors at three sites and in females there were 4% of mice with tumors at four or more sites. The rates of tumor induction for all tumors were significantly increased above that in non-exposed animals. The development of cancer, as early as three months after exposure to butadiene, is an unusual finding. TABLE 3
Frequency of Multiple-site Neoplasia in Male B6C3Fl Mice Exposed to 1,3-Butadiene: Lymphocytic Lymphoma, Hemangiosarcoma of the Heart, Squamous Cell Neoplasm of the Forestomach, and Harderian Gland
Neoplasm
_
Source: Melnick et al.
The incidence of
’
( 1990).
lymphocytic lymphoma
-
,¡
was
greater
at
higher concentrations
for short time
compared to exposure to lower concentrations for much longer periods of exposure. In addition to the NTP
studies, the International Institute of Synthetic Rubber Producers (IISRP) sponsored an inhalation study at Hazleton Laboratory in England using SpragueDawley rats exposed to two doses of butadiene, 1000 ppm and 8000 ppm (Hazleton, 1981). This study showed (Table 4) an increase in the incidence of tumors in the zymbal gland, thyroid, lung, skin, mammary gland, brain, uterus, testes, and kidney. Brain tumors are known to be rare in non-exposed rats. The IISRP study provides evidence of carcinogenicity of BD to a second species of animals.
Downloaded from tih.sagepub.com at UCSF LIBRARY & CKM on April 21, 2015
213
TABLE 4 in Charles River Rats of Cancer Incidence Summary Caused by Inhalation of 1,3-Butadiene
Source: Hazelton
Laboratory, IISRP-sponsored study (1981).
Butadiene-Humnn Studies Evidence of the causal relationship between occupational exposure to butadiene and cancer mortality has been reported in styrene-butadiene rubber (SBR) workers by Meinhardt et al. (1982); Matanoski et al. (1987, 1990); McMichael (1976); Lemen et al. (1990); Downs et al. (1987); Andjelkovic et al. (1976); and Divine (1990). A consistent observed increase in the incidence of lymphomas, leukemias, and other hematopoietic cancers among butadiene workers as compared to non-exposed workers was found.
McMichael et al. (1976) reported that mortality in industrial SBR workers is between 60 to 90% that of general workers. Table 5 shows Standard Mortality Ratios (SMRs) among production workers. The SMR of 7 shows an increased cancer risk to workers from exposure to butadiene.
study by Matanoski et al. (1987) showed a significant increase in SMR for &dquo;other [lymphohematopoietic cancers (LHC)],&dquo; i.e., observed a 9 vs. 3.5 expected in production
The
workers with
an
SMR of 260.
The SMR for leukemia was 710, for all LHC, 504. Thus the results from the case control studies of Matanoski et al (1987) clearly establish an association between butadiene and human leukemia in pioduction workers, the group with the greatest exposure to butadiene. a study by McMichael et al. (1976) of SBR workers at an Akron, Ohio plant from 19641972, it was found that there was excess of mortality due to cancer of the stomach ( SMR 187), prostate (SMR 142), and LSC (SMR 226). In reviewing McMichael’s data with revised
In
&dquo;Ratios of Exposed Rates&dquo; (RERs), the EPA found a dose-response relationship between exposure and cancer, thus greatly strengthening the weight of evidence for causality.
Downloaded from tih.sagepub.com at UCSF LIBRARY & CKM on April 21, 2015
214
TABLE 5 Standard Mortality Ratios (SMRs) for Leukemias and Lymphohematopoietic Cancers
(1976) studied mortality of rubber workers from 1964 to 1973 in a plant located in Akron, Ohio. They reported significant increase in monocytic leukemia (SMR 311) and &dquo;other LHC&dquo; (SMR 192). Thus, the Andjelkovic study is consistent with the McMichael
Andjelkovic
et al.
finding in SBR workers showing LHC&dquo; and lymphatic leukemia.
a
dose-response relationship
between butadiene and &dquo;all
Butadiene-Leukemia and Other Cancers in Humans Table 5 shows SMRs from leukemia, lymphosarcoma/reticular cell sarcoma, and cancers of lymphohematopoietic system. This table shows that the SMR for leukemia is 710; for lymphosarcoma/reticular cell sarcoma, 235; and for &dquo;other LHC,&dquo; 260.
Thus it is
possible to conclude that there is a dose-response relation between 1,3-butadiene and exposure lymphohematopoietic cancers and leukemia. These findings are supported by studies from Downs (1987), Matanoski (1990), and McMichael (1976). On the basis of these epidemiological findings and the NTP animal studies, it is concluded that lymphohematopoietic cancer, leukemia, and other cancers are related to occupational exposure to 1,3butadiene..
,
_
-
EXPERIMENTAL EVIDENCE CHARACTERIZES 1,3-BUTADIENE AS A DEVELOPMENTAL TOXIN
The European Economic Community (EEC) is presently considering categories for labeling chemicals as germinal cell mutagens. Figure 3 illustrates the procedures used to classify a Downloaded from tih.sagepub.com at UCSF LIBRARY & CKM on April 21, 2015
215
germinal cell mutagen. Given the formidable obstacles involved in conductinghuman studies in this area, there are no chemicals in category 1 (human mutagens). Compounds in category 2 are regarded as if they were mutagenic in humans, based on animal data that includes demonstration of their ability to reach the gonads. In category 3 are compounds that raise concern about their being germinal cell mutagens because of positive in vivo and in vitro mutagenicity assays. chemical
as
a
EEC
Categories
for
Labeling
Chemical
Mutagens
FIGURE 3 The
following discussion indicates that 1,3-butadiene would be in category 2a.
The classification of 1,3-butadiene as a developmental toxin would be consistent with its metabolism, its know mechanism of action and distribution, even in the absence of confirmatory experimental evidence. Two studies can be identified which specifically address the developmental effects of this chemical. A teratology study in Sprague-Dawley rats (exposed 6 hrs a day to 0, 200, 1000, or 8000 ppm from day 5 through 15 of gestation) produced the following outcomes (Hazelton Laboratories,
1981): 1. 2.
Concentration-related suppression of maternal weight Increase in morality of postimplantation embryos
Downloaded from tih.sagepub.com at UCSF LIBRARY & CKM on April 21, 2015
gains during exposure
216
.
3. 4. 5.
6. 7.
body weight and crown-rump length of fetuses increase in minor fetal defects in all groups Significant Significant increase in major anomalies (at 1000 and 8000 ppm) Increase in lens opacity and irregularities of ossification at 8000 ppm Increase in wavy ribs in a dose-related manner. Decrease in
’
.
This study indicated developmental toxicity over a range of concentrations, and furthermore, indicates multiple non-specific effects. If either intrauterine growth retardation or structural anomalies are present, this is indicative of developmental toxicity (see above). In the Hazelton study, both of these adverse health effects were found. Since minor anomalies were detected at the lowest concentration studied (200 ppm), other developmental toxicity may be manifest at concentrations below this level. For example, functional outcomes have yet to be investigated, and, since they usually occur at concentrations that are below the level used in this study, would have an adverse effect.
Adverse
developmental outcomes
were
also found in Swiss (CD-1) mice
by Morrissey
et al.
(1990). In this study, rodent were exposed to 1,3-butadiene at levels of 0, 40, 200, and 1000 ppm for 6 hrs a day from day 6 to day 15 of gestation. Fetal body weight was reduced at 200 and 1000 ppm for the female and at all levels for the male; placental weight was reduced at the 200 and 1000 ppm level for males and at the 1000 ppm level for females; maternal toxicity was observed at the 200 and 1000 ppm level; fetal variations (supernumary ribs and reduced ossification of the sterenebrae) were observed at exposure levels of 200 and 1000 ppm. In this study, developmental toxicity in the form of reduced male fetal weights (intrauterine growth retardation) was observed at the lowest level tested, 40 ppm. The effect on male fetuses was seen in the absence of maternal toxicity. This study shows significant effects from exposure to 1,3-butadiene in the mouse, but Morrissey did not find statistically significant differences in
butadiene-exposed rats similar type of exposure. Studies in two species, the mouse (Morrissey) and that rat (the Hazelton study) offer convincing evidence that 1,3-butadiene induces developmental toxicity form in utero exposure. As is often the case with developmental toxins, the form in which the adverse health outcome is manifested will vary with species and experimental conditions. Although the few studies already conducted indicate that 1,3-butadiene is a developmental toxin, it should be noted that critical studies to evaluate functional deficits have yet to be performed. Since functional deficits frequently are encountered at concentrations substantially below the gross effects measured in the present studies, standards set for 1,3-butadiene, on the basis of currently available information, may significantly underestimate the true hazards of this chemical to humans. The distribution of 1,3-butadiene into germinal cells, and the susceptibility of these cell types to DNA alterations by this chemical is evident from the germinal cell cancers that are induced. Leydig Cell tumors of the testes, and uterine/vaginal stromal tumor were increased in the rat (Hazelton Laboratories, 1981); and granuloma cell tumors of the ovary and testicular and ovarian atrophy were seen in the 65 week mouse study (Huff et al., 1985). The distribution to germinal cells, and the effect of 1,3-butadiene on germinal cells is also confinned by the
Downloaded from tih.sagepub.com at UCSF LIBRARY & CKM on April 21, 2015
217
dominant lethal study as described below. The metabolic-somatic mutation model is satisfied by this chemical as indicated by its previously described clastogenic effects in vivo studies. The dominant lethal test establishes a chemical as a germinal cell mutagen. In a dominant lethal study, male mice were exposed to 1,3-butadiene at concentrations of 0, 200, 1000, and 5000 ppm for 5 consecutive days, 6 hrs a day (Morrissey et al., 1990). Females were mated to treated males for one week over an eight week period. Twelve days after the last day of cohabitation with the exposed males, the females were sacrificed. There was no mortality in the fetal CD-1 mice at the highest concentration used (5000 ppm for 5 days). At the 1000 ppm level there was a statistically significant increase in dead implants. At the 200 and 5000 ppm levels there was an increase but it was not significant. In week 2 there was a significant increase in dead implants per pregnancy at the 200 and 1000 ppm levels but not at the 5000 ppm level. These results are consistent with an effect on mature germ cells (spermatozoa and
spermatid). 1,3-butadiene is, therefore, positive in the dominant lethal test at concentrations substantially below a toxic effect. In fact, no toxicity in the treated male was detected, even at the highest concentration used in this investigation. Dominant lethal effects were noted between 200 and 5000 ppm (Morrissey et al., 1990). As previously stated, the most important attribute of the germinal cell mutation procedure is to identify an adverse health outcome in the progeny following exposure to the parent (male). This can truly be considered the &dquo;tip of the iceberg.&dquo; Transmissible genetic damage, displaying a spectrum of abnormal outcomes, can be anticipated at concentrations below those identified in this dominant lethal procedure.
Available information, therefore, establishes 1,3-butadiene as a germinal cell mutagen based on: 1) chemical classification, 2) the known reactivity of its metabolites resulting in the formation of DNA adducts, 3) its distribution in germinal cells, 4) the metabolic-somatic mutation model (where in addition to reactive metabolites reaching germinal cells the chemical has been show to induce chromosomal aberration in animals), and 5) the positive dominant lethal test.
Using the EEC guidelines currently under consideration, 1,3-butadiene would be placed in Category 2A. Category 2A is the highest category in terms of germinal cell mutagens based on
animal studies. CONCLUSIONS AND SUMMARY
1. 1 ,3-butadiene is a potent classical alkylating agent. Sufficient information is available to indicate that the metabolites of 1,3-butadiene alkylate DNA, and that this alteration leads to mutation. Positive in vivo cytogenetic data, detectable after a comparatively short exposure, provide evidence that this chemical causes genetic damage. Cancer bioassay studies extend the data base to indicate that the metabolites of 1,3butadiene alkylate DNA and produce mutations that are fixed and lead to adverse health outcomes. This chemical induces cancer at multiple sites, including germinal cells, and this response occurs at an unusually low concentration. Alkylation of DNA, irreparable genetic
Downloaded from tih.sagepub.com at UCSF LIBRARY & CKM on April 21, 2015
218
damage, and finally, the development of adverse health outcomes are established consequences of exposure to 1,3-butadiene. 2. 1,3-butadiene is directly toxic to developing embryos. The known mechanisms of action of 1,3-butadiene and the demonstrated carcinogenicity would indicate that this chemical is a developmental toxin capable of causing effects such as intrauterine growth retardation, early fetal death, malformations, or functional abnormalities. In the case of 1,3-butadiene, not only can this conclusion be drawn indirectly, but evidence is available from studies in both rats and mice. Intrauterine growth retardation, increased mortality of post-implantation embryos, and malformations have all been demonstrated. in rats produced multiple developmental abnormalities (Hazelton Laboratories, 1981). The rat is allegedly less sensitive to the toxicity of 1,3butadiene than the mouse (Laib, 1990). If this is the correct conclusion from the studies carried out to date, then we have evidence for the developmental toxicity of 1,3-butadiene in an allegedly resistant species. However, the length of exposure in these studies, and the comparatively few experiments that have been carried out would make any conclusions as to the differences between the rat and mouse premature. As to extrapolating experimental animal data to humans, it should be borne in mind that we are an extremely heterogeneous species. This heterogeneity can be translated into unusually high variation in the way we metabolize chemicals. Because of this variability there could well be subsets of our population which are more sensitive to the genotoxic effects of 1,3-butadiene than either rats or mice. It is
interesting
to note that the
study
In terms of the quantitative risk assessment of developmental toxins that affect the embryo, it should be emphasized that humans in general have proven to be a far more sensitive species than non-human animals. Schardein and Keller (1989) have pointed out that the human is remarkably sensitive to those agents characterized as developmental toxicants. Of the 21 agents considered to be direct human developmental toxins, in 19 of the 21 agents the human has been more sensitive than non-human animal models. Ratios of threshold does in the most sensitive animals to those in humans, ranged from 1.2 to 200.
3. 1,3-butadiene is a developmental toxin affecting progeny after parental exposure. If this chemical reaches germinal cells, purely on the basis of theoretical considerations its classification as a germinal cell mutant would be warranted, even in the absence of further experimental evidence. The adverse health outcomes in the progeny following parental exposure would be the same as the somatic mutation effects seen in the developing embryo. Experimental evidence is available, however, to show that this chemical induces dominant lethality following male exposure. We therefore have direct evidence that 1,3-butadiene produces adverse health outcomes in the progeny as a consequence of parental exposure. It should be understood that the dominant lethal test is a highly insensitive procedure, and adverse genetic outcomes other than lethality would be anticipated at concentrations below the recorded dominant lethal effect.
Downloaded from tih.sagepub.com at UCSF LIBRARY & CKM on April 21, 2015
219
4.
Animal cancer studies demonstrate that 1,3-butadiene is a carcinogen in B6C3F1 mice and in Sprague-Dawley rats. Epidemiological studies in workers showed significant increase in lyphomas and leukemias as a result of exposure to 1,3-butadiene.
REFERENCES
ANDJELKOVICH, D., TAULBEE, J., and SYMONS, M. (1976). "Mortality experience of a cohort of rubber workers." J. Occup. Med. 18 (6):387-394. CITTI, L., GERVASI, P.G., TURCHI, G., BELLUCI, G., and BIANCHINI, R. (1984. "The reaction of 3,4-epoxy-1-butene with deoxyguanosine and DNA in vitro: synthesis and characterization of the main adducts." Carcinogenesis 5: 47-52. DE MEESTER, C. (1988). "Genotoxic properties of 1,3-butadiene." Mutation Research 195: 273-281. DIVINE, B.J. (1987). "An update on mortality among workers at a butadiene facility." Am. J. Industri. Med. 12: 311-329. DOWNS, T.D, CRANE, M.M., and KWANGE, K.W. (1987). "Mortality among workers at butadiene facility." Am. J. Industr. Med. 311-329. 12: ENVIRONMENTAL HEALTH PERSPECTIVES (1990). Symposium on Toxicology, Carcinogenesis, and Human Health Aspects of 1,3-Butadiene. Env. Health Persp. 86. HAZELTON LABORATORIES, EUROPE, LTD. (1981). "1,3-Butadiene: Inhalation Teratogenicity Study in the Rat." International Institute of Synthetic Rubber Producers (IISRP) Report No. 2788-522/3. HUFF, J.E, MELNICK, R.L., SOLLEVELD, H.A., HASESMAN, J.K., POWERS, M., and MILLER, R.A. (1985). "Multiple organ carcinogenicity of 1,3-butadiene in B6C3F1 mice after 60 weeks of inhalation exposure." Science 227: 548-549.
HUFF, J.E., HASEMAN, J.K, DEMARINI, D.M., EUSTES, S., MARONPOT, R.R., PETERS, A.C., PERSHING, R.L., CHRISP, C.C., and JACOBS, A.C. (1989).
"Multiple site carcinogenicity of benzene Health Perspec. 32. KOTIN, P. and FALK, H.L.
in Fisher 344 rats and B6C3F1 mice." Env.
30. (1955). Proc. Am. Assoc. Cancer Research 2:
LAIB, R.J., FILSER, J.G, KREILING,R., VANGALA, R.R., and BOLT, H.M. (1990). "Inhalation pharmacokinetics of 1,3-butadiene and 1,2-epoxybutane-3 in rats and mice." Environ. Health
57-63. 86: Perspec.
LEMEN, R.A., MEINHARDT, T.J., CRANDALL, M.S., FAJEN, J.M., and BROWN, D.P.
(1990). "Environmental Epidemiological Investigations in the Styrene-Butadiene Rubber Production Industry." Environ. Health Perspec. 86: 103-106. LOW, J.A., and GALBRAITH, R.S. (1974). "Pregnancy characteristics of intrauterine growth retardation." Obstet. Gynecol. 44: 122. MALVOISIN, E., MERCIER, M., and ROBERFROID, M. (1982). "Enzymic hydration of butadiene monoxide and its importance in the metabolism of butadiene." Adv. Expo. Med. Biol. 38A: 437-444. MATANOSKI, G.M. and SCHWARTZ, L. (1987). "Mortality of workers in styrenebutadiene polymer production" J. Occup. Med. 29 (8):675-680.
Downloaded from tih.sagepub.com at UCSF LIBRARY & CKM on April 21, 2015
220
MATANOSKI, I., SANTOS-BURGOA, C., and SCHWARTZ, L. (1990). "Mortality of
a
Styrene-Butadiene Polymer Manufacturing Industry (1943107-117. Health Perspec. 88:
Cohort of Workers in the
1982). Environ.
MCCAMMON, P. KOTIN, P., and FALK, H.L. (1957). Proc. Am. Assoc. Cancer Research 229. 2:
MCMICHAEL, A.J., SPRITAS, R., GAMBLE, J.F., and TOUSEY, P.M. (1976).
"Mortality among rubber workers: relationship to specific jobs." J. Occup. Med.
18 178:
185.
MEINHARDT, T.J., LEMEN, R.A., CRANDALL, M.S., and YOUNG, R.J. (1982).
epidemiologic investigations of the styrene-butadiene rubber industry: mortality patterns with discussion of the hematopoietic and lymphatic malignancies."
"Environmental
Scand. J. Work Environ. Health 8: 250-259.
MELNICK, R.L., HUFF, J., CHOU, B.J., and MILLER, R.A. (1990). "Carcinogenicity of 1,3-Butadiene in C57BL/6 x C3H F1 Mice at Low Exposure Concentrations." Cancer 6592-6599. Research 50:
MILLER, H.C. (1981). "Intrauterine growth retardation. An
unmet
challenge."
Am. J. Dis.
Child. 135: 944. L.M. MILLER, (1978). "Investigation of Selected Potential Environmental Contaminants: Butadiene and its Oligomers." EPA-560/2-78-008. Washington, D.C., U.S. EPA.
MORRISSEY, R.E., SCHWETZ, B.A., HACKETT, P.L., SIKOV, M.R., HARDIN, B.D., MCCLANAHAN, B.J., DECKER, J.R., and MAST, T.J. (1990). "Overview of reproductive and developmental toxicity studies of 1,3-butadiene in rodents." Environ. Health
79-84. Perspec. 86:
MORROW, N.L. (1990). "The Industrial Production and Use of 1,3-butadiene." Env. Health 7-8. Persp. 86:
NELSON, T., OAKLEY, G.P., and SHEPARD, T.H. (1971). "Collection of human embryos and fetuses. II. Clarification and tabulation of conceptual wastage with observations on type of malformation, sex ratio, and chromosome studies." In: Monitoring Birth Defects & Environment (E.B. Hook, D.T. Janerich, and I.H. Porter, eds.). Academic Press, New York. OSHA (OCCUPATIONAL SAFETY AND HEALTH ADMINISTRATION) (1990). Fed. 155. Regist. 55: SCHARDEIN, J.L. and KELLER, K.A. (1989). "Potential Human Developmental Toxicants and the Role of Animal Testing in Their Identification and Characterization." CRC Critical Reviews in Toxicology 19 (3):251-338. SHELBY, M.D. (1990). "Results of NTP-sponsored mouse cytogenetic studies on 1,371-73. butadiene, isoprene, and chloroprene." Environ. Health Perspec. 86: U.S. ENVIRONMENTAL PROTECTION AGENCY (EPA). (1986). "2,4,5-T and Silvex." Fed. Regist. 44: 874.
Downloaded from tih.sagepub.com at UCSF LIBRARY & CKM on April 21, 2015
221
INFORMATION FOR AUTHORS
Toxicology and Industrial Health welcomes manuscripts dealing with basic and applied research in the fields of toxicology, biochemical toxicology, genetic and cellular toxicology, and risk assessment associated with hazardous wastes and ground water. The journal will accept and publish rapidly brief communications reporting on important research. It will publish several types of articles including papers describing original research results; reviews on subjects of contemporary importance to toxicologists and biochemical scientists; reports or proceedings, which will be published as supplements to issues of the journal at the expense of the organization that submits the proceedings; and brief announcements of scientific meetings or courses of interest to readers. -
Submission of Manuscripts. Prior to submission, manuscripts should have received any required institutional clearance and approval. The original and one copy should be sent to:
_
The decision
Managing Editor Princeton Scientific Publishing Co., Inc. P.O. Box 2155 Princeton, NJ 08543
.
regarding acceptability for publication will come from the Editor-in-Chief.
Manuscript Preparation. The entire manuscript should be double-spaced on standard 8-1/2 x 11 inch paper, typed on one side only with pages numbered consecutively. A cover sheet should contain the following items: 1) title of manuscript; 2) full first names, middle initials, and last names of authors; 3) institutional affiliation of authors; 4) corresponding author’s name, mailing address, and telephone number; 5) abbreviations used in the manuscript, listed alphabetically; 6) 3-6 key words found in the manuscript, listed alphabetically; 7) running title of approximately 45 characters. Papers are to be organized into Abstract, Introduction, Methods, Results, Discussion, Acknowledgments, and References sections. All figures (originals only) must accompany the manuscript at the time of submission. They should be properly identified with authors’ names, figure number, place of insertion, and top of figure. Tables should be numbered sequentially in Arabic numerals and headed with a short title. Each table should be on a separate page and the preferred point of insertion should be indicated in the margin of the manuscript text. Whenever possible, tables should be submitted in camera-ready form. If the manuscript and/or figures and tables are available on Apple Macintosh computer diskettes, please submit a diskette along with the two hard copies of the material and indicate
which software application
was
used.
Downloaded from tih.sagepub.com at UCSF LIBRARY & CKM on April 21, 2015
222
Footnotes and References. Keep footnotes to a minimum. Refer to them numerically as superscripts within the text. Unpublished material and personal communications should be footnoted. Critical statements of fact must be documented by referenced whenever applicable. Citations in the text are by the name of the first author and year of publication. In the case of a multiple citation, the order is chronological.
References should be organized in per the samples below:
alphabetical order, providing complete bibliographical data,
MAIN, A.R. and BRAID, P.E. (1962). &dquo;Hydrolysis of malathion by
Journal:
aliesterase in vitro and in vivo.&dquo; Biochem. J. 84: 255-263.
NICKLESS, G. (1968). Inorganic Sulfur Chemistry, pp. 509-531. Elsevier, New York.
Book:
Book
chapter:
TALCOTT, R.E., KETTERMAN, A., HARGER, W., DENK, H., and ECKERSTORFER, R. (1980). &dquo;Microsomal lipid peroxidation: Catalysis, effects, and inhibition by cytosolic protein.&dquo; In: Microsomes, Drug Oxidations, and Chemical Carcinogenesis (M.J. Coon, A.H. Cooney, R.W. Estabrook, H.V. Gelboin, J.R. Gillette, and P.J. O’Brien, eds.). Vol. 2, pp. 753-761. Academic Press, New York.
Page Charges. A page charge of $40.00 per printed page will be assessed to cover part the cost of publication. Acceptance of the papers is not based on the ability to pay charges.
Reprints.
Forms for
ordering reprints will be sent to authors with their galley proofs.
Downloaded from tih.sagepub.com at UCSF LIBRARY & CKM on April 21, 2015
__
of
DANGEROUS AND CANCER-CAUSING PROPERTIES OF PRODUCTS AND CHEMICALS IN THE OIL REFINING AND PETROCHEMICAL INDUSTRY - PART II:
CARCINOGENICITY, MUTAGENICITY, AND DEVELOPMENTAL TOXICITY OF 1,3-BUTADIENE M.A. MEHLMAN UMDNJ-Robert Wood Johnson Medical School Department of Environmental and Community Medicine 675 Hoes Lane, Piscataway, New Jersey 08854
M.S. LEGATOR Division
of
Environmental
Toxicology
University of Texas Medical Branch Galveston, Texas 77550
1,3-butadiene (BD) is present in synthetic rubber and motor fuels (gasoline). BD is shown to cause lymphocytic lymphomas, heart hemangiosarcomas, lung alveolar bronchiolar cancers, forestomach-squamous cell cancers, harderian gland neoplasms, preputial gland adenoma or carcinoma, liver-hepatocellular cancers, mammary gland acinar cell carcinomas, ovary-glanulosa cell carcinoma, brain cancers, pancreas adenoma and carcinoma, testis-Leydig cell tumors, thyroid follicular adenoma and carcinoma, and zymbal gland carcinoma in rodents and to date no exposure level has been established at which this chemical does not cause cancers.
In humans BD
increase in lymphomas, leukemias, and other cancers of hematopoietic systems and organs. BD is also a potent alkylating agent, directly toxic to developing embryos and damages progeny after parental exposure. causes
INTRODUCTION
1,3-butadiene (BD) is produced by the oil refining industry as a co-product in the manufacture
ethylene and is used in styrene-butadiene rubber (SBR) latex and polybutadiene rubber (PBR). It is estimated that 12 billion pounds of BD is produced worldwide and 3 billion pounds are produced in the United States each year (Morrow, 1990). In addition to being a primary ingredient of synthetic rubber, butadiene is also a component of plastics, gasoline (in the U.S. alone 140 billion gallons of gasoline are consumed annually), and motor vehicle of
Industrial Health, 7:3, pp. 207-220 Princeton Scientific Publishing Co., Inc. ISSN: 0748-2337
Toxicology and Copyright 1991
Downloaded from tih.sagepub.com at UCSF LIBRARY & CKM on April 21, 2015
208
exhausts (Miller, 1978). It has been shown that exposure to butadiene causes an increase in the incidence of malignant lymphomas, leukemias, cardiac hemangiosarcomas, alveolar neoplasms, tumors of the forestomach, carcinomas of the mammary gland, hepatocellular neoplasms, pancreatic adenomas, uterine sarcomas, zymbal gland carcinomas, thyroid tumors, and testicular cancers in animals (Environ. Health Persp., 1990). This paper we will review (a) the metabolism and genotoxic effects of butadiene, (b) the animal and human data which characterize this chemical as a multiorgan, multispecies carcinogen, and (c) the limited studies which indicate that butadiene is a developmental toxin. METABOLISM AND GENOTOXICITY OF 1,3-BUTADIENE
1,3-Butadiene is Metabolized to Reactive Compounds that Form Adducts with DNA In 1955 Kotin and Falk suggested that epoxides might be responsible for carcinogenic activity from polluted air in Los Angeles. In 1957 McCammon, Kotin, and Falk produced tumors in animals which were exposed to the butadiene-derived diepoxy compound.
Later studies indicated that in the presence of microsomal activating systems, 1,3-butadiene is metabolized to the epoxide 1,2-epoxybutene-3, which in turn can be metabolized further by microsomal activating system to dioxybutane and, in the presence of epoxide hydrolase, to the 3,4-epoxy-1,2-butane-diol (Malvoisin et al., 1985). In vitro studies indicate DNA adducts [7,-(2-hydroxy-buten-1-yl) guanine and 7,-(I-hydroxy-buten-1-yl) guanine], are formed with 1,2-epoxybutene-3 (Citti et al., 1984). Using C 14 labeled butadiene, Laib et al. (1990) found that radioactive compounds were covalently bound to nucleoprotein fractions and to the total liver DNA of rats and mice treated with 1,3-butadiene. Thus, from metabolic pathways supported by both in vitro and in vivo data, there is evidence of interaction with and alteration of DNA by metabolites of 1,3-butadiene. In Vivo Somatic Cell :B1vtCtgen In mutagenicity studies in mice, in vivu cxpusujTjs tu a,3~~br.~:~ciiene of 2 weeks and 13 weeks duration produced dose-related increases in three different endpoints: 1) chromosomal aberrations, 2) sister chromatid exchanges (SCE) in bone marrow cells, and 3) micronuclei in peripheral erythrocytes. In the 2 week exposure, the SCE were significantly increased at exposure rates of 6.25 ppm. In the 13 week exposure, where only micronuclei in peripheral
1,3-Butadiene:
an
blood were evaluated, a statistical increase over control was noted at the 6.25 ppm level. Induction of chromosomal effects were concentration-dependent over a dosage range of two orders of magnitude (Shelby, 1990). Studies in the rat do not indicate similar effects (de Meester, 1988). These species’ differences, however, may reflect the more extensive studies carried out with the mouse, and differences in the durations of exposure. For example in the rat, an exposure of only two days were evaluated. At the present time there are no in vivo data available on gene mutations following sub-chronic exposure. Cancer Caused by 1,3-Butadiene iri Animals The National Toxicology Program (NTP) conducted an inhalation study with butadiene at Battele Pacific Northwest Laboratories with B6C3F1 mice of both sexes. The mice were
Downloaded from tih.sagepub.com at UCSF LIBRARY & CKM on April 21, 2015
209
exposed to 627 and 1236 ppm of butadiene. The study was run for 60 weeks for males and 611 weeks for females. The animals were exposed for a shorter period than planned, due to high mortality from malignant tumors in the exposed animals. The major cause of death in both male and female mice from exposure to butadiene were lymphomas, which may have originated in the thymus, and hemangiosarcoma of the heart. These lymphomas occurred as early as 20 weeks after exposure, and that these lymphomas caused the majority of deaths between 40 and 45 weeks of exposure.
Hemangiosarcomas of the heart are extremely uncommon cancers in rodents, and especially in B6C3Fl mice. The background incidence of hemangiosarcomas in B6C3Fl mice is about 1 in every 2500 control animals. Exposure to butadiene of mice resulted in a high frequency of the rare hemangiosarcomas. In addition to the tumors described above, an increase of tumors in the following organs was observed: lung and forestomach of males and females, and liver, mammary gland, and ovaries of females. There was approximately a 29.7 fold increase in total tumors of mice exposed to
1250 ppm (Table 1). TABLE 1
Summary of
Tumors in B6C3Fl Mice Induced
by Inhalation of
1,3-Butadiene
Second NTP Study by Melnick, Huff, Chou, and Miller In the study by Melnick et al. (1990) using B6C3F1 mice, carcinogenicity was observed at the lowest concentration of 6.25 ppm. A &dquo;no effect&dquo; level was not established. In addition, at higher concentrations, 625 ppm, there was a high frequency of fatal lymphocytic lymphomas, masking the development of tumors at other sites at this exposure level. Response for hemangiosarcomas of the heart and neoplasms of the lung and other tumor sites were demonstrated (Figures 1 and 2).
Survival and Incidence of Tunwrs in Both Male and Female B6C3Fl Mice Melnick et al. (1990) show that exposure of mice to butadiene at 20 ppm or higher results in a very significant decrease in survival from that of control mice. The percent of animals with
Downloaded from tih.sagepub.com at UCSF LIBRARY & CKM on April 21, 2015
210
malignant tumors increased three-fold compared with controls (Table 2). In the highest dose, approximately 90% of the animals had tumors. It is important to note the rapidity with which lymphocytic lymphomas of thymic origin appeared only 23 weeks after exposure.
1. Cumulative incidence of lymphocytic lympohomas or hemangiosarcomas of the heart versus weeks on study for male B6C3Fl mice exposed to 200 or 625 ppm of 1,3butadiene. (Source: Melnick et al., 1990. Reproduced by permission of the publisher.).
- FIGURE
FIGURE 2. Cumulative incidences of lymphocytic lymphomas or alveolar-bronchiolar neoplasms versus weeks on study for female B6CX3F mcie exposed to 200 or 625 ppm, of 1,3-butadiene. (Source: Melnick et al., 1990. Reproduced by permission of the publisher.).
Downloaded from tih.sagepub.com at UCSF LIBRARY & CKM on April 21, 2015
211
The cumulative incidence of lymphocytic lymphomas and alveolar-bronchiolar neoplasms is summarized in Figure 1 (males) and Figure 2 (females). These date demonstrate that butadiene causes a high incidence of lymphocytic lymphoma and bronchiolar neoplasms. TABLE 2
Primary Tumors in Male B6C3Fl Mice 1,3-Butadiene for up to 2 Years (a)
Survival and Incidence of
Exposed
to
is given as the number of animals bearing a neoplastic lesion at a specific anatomic site. Overall rates, based on the number of animals at which that site was examined, are given below the incidence values. Kaplan-Meier estimated tumor rates at the end of the study after adjusting for intei-urrent mortality are given in parentheses. Initial number includes animals removed from the study for interim sacrifices at 40 and 65 weeks. Decreased compared to chamber control (0 ppm), P< 0.05. Increased compared to chamber control (0 ppm), P< 0.05, based on logistic regression analyses with adjustment for intercurrent mortality.
(a) Incidence
(b) + *
Source: Melnick
et al.
(1990).
Downloaded from tih.sagepub.com at UCSF LIBRARY & CKM on April 21, 2015
212
The incidence of hemangiosarcomas of the heart was higher for males than for females at lower concentrations of butadiene exposure. Figure 1 shows the incidence of hemangiosarcomas of the heart in mice exposed to 200 ppm and 625 ppm of butadiene. Results summarized in Table 3 show the frequency of multiple-site neoplasia for a variety of tumors in male mice. In control mice, an average of 2.5% of mice developed tumors at two sites, and none developed tumors at three or more sites. In butadiene-exposed mice, up to 12% of the males had tumors at three sites and in females there were 4% of mice with tumors at four or more sites. The rates of tumor induction for all tumors were significantly increased above that in non-exposed animals. The development of cancer, as early as three months after exposure to butadiene, is an unusual finding. TABLE 3
Frequency of Multiple-site Neoplasia in Male B6C3Fl Mice Exposed to 1,3-Butadiene: Lymphocytic Lymphoma, Hemangiosarcoma of the Heart, Squamous Cell Neoplasm of the Forestomach, and Harderian Gland
Neoplasm
_
Source: Melnick et al.
The incidence of
’
( 1990).
lymphocytic lymphoma
-
,¡
was
greater
at
higher concentrations
for short time
compared to exposure to lower concentrations for much longer periods of exposure. In addition to the NTP
studies, the International Institute of Synthetic Rubber Producers (IISRP) sponsored an inhalation study at Hazleton Laboratory in England using SpragueDawley rats exposed to two doses of butadiene, 1000 ppm and 8000 ppm (Hazleton, 1981). This study showed (Table 4) an increase in the incidence of tumors in the zymbal gland, thyroid, lung, skin, mammary gland, brain, uterus, testes, and kidney. Brain tumors are known to be rare in non-exposed rats. The IISRP study provides evidence of carcinogenicity of BD to a second species of animals.
Downloaded from tih.sagepub.com at UCSF LIBRARY & CKM on April 21, 2015
213
TABLE 4 in Charles River Rats of Cancer Incidence Summary Caused by Inhalation of 1,3-Butadiene
Source: Hazelton
Laboratory, IISRP-sponsored study (1981).
Butadiene-Humnn Studies Evidence of the causal relationship between occupational exposure to butadiene and cancer mortality has been reported in styrene-butadiene rubber (SBR) workers by Meinhardt et al. (1982); Matanoski et al. (1987, 1990); McMichael (1976); Lemen et al. (1990); Downs et al. (1987); Andjelkovic et al. (1976); and Divine (1990). A consistent observed increase in the incidence of lymphomas, leukemias, and other hematopoietic cancers among butadiene workers as compared to non-exposed workers was found.
McMichael et al. (1976) reported that mortality in industrial SBR workers is between 60 to 90% that of general workers. Table 5 shows Standard Mortality Ratios (SMRs) among production workers. The SMR of 7 shows an increased cancer risk to workers from exposure to butadiene.
study by Matanoski et al. (1987) showed a significant increase in SMR for &dquo;other [lymphohematopoietic cancers (LHC)],&dquo; i.e., observed a 9 vs. 3.5 expected in production
The
workers with
an
SMR of 260.
The SMR for leukemia was 710, for all LHC, 504. Thus the results from the case control studies of Matanoski et al (1987) clearly establish an association between butadiene and human leukemia in pioduction workers, the group with the greatest exposure to butadiene. a study by McMichael et al. (1976) of SBR workers at an Akron, Ohio plant from 19641972, it was found that there was excess of mortality due to cancer of the stomach ( SMR 187), prostate (SMR 142), and LSC (SMR 226). In reviewing McMichael’s data with revised
In
&dquo;Ratios of Exposed Rates&dquo; (RERs), the EPA found a dose-response relationship between exposure and cancer, thus greatly strengthening the weight of evidence for causality.
Downloaded from tih.sagepub.com at UCSF LIBRARY & CKM on April 21, 2015
214
TABLE 5 Standard Mortality Ratios (SMRs) for Leukemias and Lymphohematopoietic Cancers
(1976) studied mortality of rubber workers from 1964 to 1973 in a plant located in Akron, Ohio. They reported significant increase in monocytic leukemia (SMR 311) and &dquo;other LHC&dquo; (SMR 192). Thus, the Andjelkovic study is consistent with the McMichael
Andjelkovic
et al.
finding in SBR workers showing LHC&dquo; and lymphatic leukemia.
a
dose-response relationship
between butadiene and &dquo;all
Butadiene-Leukemia and Other Cancers in Humans Table 5 shows SMRs from leukemia, lymphosarcoma/reticular cell sarcoma, and cancers of lymphohematopoietic system. This table shows that the SMR for leukemia is 710; for lymphosarcoma/reticular cell sarcoma, 235; and for &dquo;other LHC,&dquo; 260.
Thus it is
possible to conclude that there is a dose-response relation between 1,3-butadiene and exposure lymphohematopoietic cancers and leukemia. These findings are supported by studies from Downs (1987), Matanoski (1990), and McMichael (1976). On the basis of these epidemiological findings and the NTP animal studies, it is concluded that lymphohematopoietic cancer, leukemia, and other cancers are related to occupational exposure to 1,3butadiene..
,
_
-
EXPERIMENTAL EVIDENCE CHARACTERIZES 1,3-BUTADIENE AS A DEVELOPMENTAL TOXIN
The European Economic Community (EEC) is presently considering categories for labeling chemicals as germinal cell mutagens. Figure 3 illustrates the procedures used to classify a Downloaded from tih.sagepub.com at UCSF LIBRARY & CKM on April 21, 2015
215
germinal cell mutagen. Given the formidable obstacles involved in conductinghuman studies in this area, there are no chemicals in category 1 (human mutagens). Compounds in category 2 are regarded as if they were mutagenic in humans, based on animal data that includes demonstration of their ability to reach the gonads. In category 3 are compounds that raise concern about their being germinal cell mutagens because of positive in vivo and in vitro mutagenicity assays. chemical
as
a
EEC
Categories
for
Labeling
Chemical
Mutagens
FIGURE 3 The
following discussion indicates that 1,3-butadiene would be in category 2a.
The classification of 1,3-butadiene as a developmental toxin would be consistent with its metabolism, its know mechanism of action and distribution, even in the absence of confirmatory experimental evidence. Two studies can be identified which specifically address the developmental effects of this chemical. A teratology study in Sprague-Dawley rats (exposed 6 hrs a day to 0, 200, 1000, or 8000 ppm from day 5 through 15 of gestation) produced the following outcomes (Hazelton Laboratories,
1981): 1. 2.
Concentration-related suppression of maternal weight Increase in morality of postimplantation embryos
Downloaded from tih.sagepub.com at UCSF LIBRARY & CKM on April 21, 2015
gains during exposure
216
.
3. 4. 5.
6. 7.
body weight and crown-rump length of fetuses increase in minor fetal defects in all groups Significant Significant increase in major anomalies (at 1000 and 8000 ppm) Increase in lens opacity and irregularities of ossification at 8000 ppm Increase in wavy ribs in a dose-related manner. Decrease in
’
.
This study indicated developmental toxicity over a range of concentrations, and furthermore, indicates multiple non-specific effects. If either intrauterine growth retardation or structural anomalies are present, this is indicative of developmental toxicity (see above). In the Hazelton study, both of these adverse health effects were found. Since minor anomalies were detected at the lowest concentration studied (200 ppm), other developmental toxicity may be manifest at concentrations below this level. For example, functional outcomes have yet to be investigated, and, since they usually occur at concentrations that are below the level used in this study, would have an adverse effect.
Adverse
developmental outcomes
were
also found in Swiss (CD-1) mice
by Morrissey
et al.
(1990). In this study, rodent were exposed to 1,3-butadiene at levels of 0, 40, 200, and 1000 ppm for 6 hrs a day from day 6 to day 15 of gestation. Fetal body weight was reduced at 200 and 1000 ppm for the female and at all levels for the male; placental weight was reduced at the 200 and 1000 ppm level for males and at the 1000 ppm level for females; maternal toxicity was observed at the 200 and 1000 ppm level; fetal variations (supernumary ribs and reduced ossification of the sterenebrae) were observed at exposure levels of 200 and 1000 ppm. In this study, developmental toxicity in the form of reduced male fetal weights (intrauterine growth retardation) was observed at the lowest level tested, 40 ppm. The effect on male fetuses was seen in the absence of maternal toxicity. This study shows significant effects from exposure to 1,3-butadiene in the mouse, but Morrissey did not find statistically significant differences in
butadiene-exposed rats similar type of exposure. Studies in two species, the mouse (Morrissey) and that rat (the Hazelton study) offer convincing evidence that 1,3-butadiene induces developmental toxicity form in utero exposure. As is often the case with developmental toxins, the form in which the adverse health outcome is manifested will vary with species and experimental conditions. Although the few studies already conducted indicate that 1,3-butadiene is a developmental toxin, it should be noted that critical studies to evaluate functional deficits have yet to be performed. Since functional deficits frequently are encountered at concentrations substantially below the gross effects measured in the present studies, standards set for 1,3-butadiene, on the basis of currently available information, may significantly underestimate the true hazards of this chemical to humans. The distribution of 1,3-butadiene into germinal cells, and the susceptibility of these cell types to DNA alterations by this chemical is evident from the germinal cell cancers that are induced. Leydig Cell tumors of the testes, and uterine/vaginal stromal tumor were increased in the rat (Hazelton Laboratories, 1981); and granuloma cell tumors of the ovary and testicular and ovarian atrophy were seen in the 65 week mouse study (Huff et al., 1985). The distribution to germinal cells, and the effect of 1,3-butadiene on germinal cells is also confinned by the
Downloaded from tih.sagepub.com at UCSF LIBRARY & CKM on April 21, 2015
217
dominant lethal study as described below. The metabolic-somatic mutation model is satisfied by this chemical as indicated by its previously described clastogenic effects in vivo studies. The dominant lethal test establishes a chemical as a germinal cell mutagen. In a dominant lethal study, male mice were exposed to 1,3-butadiene at concentrations of 0, 200, 1000, and 5000 ppm for 5 consecutive days, 6 hrs a day (Morrissey et al., 1990). Females were mated to treated males for one week over an eight week period. Twelve days after the last day of cohabitation with the exposed males, the females were sacrificed. There was no mortality in the fetal CD-1 mice at the highest concentration used (5000 ppm for 5 days). At the 1000 ppm level there was a statistically significant increase in dead implants. At the 200 and 5000 ppm levels there was an increase but it was not significant. In week 2 there was a significant increase in dead implants per pregnancy at the 200 and 1000 ppm levels but not at the 5000 ppm level. These results are consistent with an effect on mature germ cells (spermatozoa and
spermatid). 1,3-butadiene is, therefore, positive in the dominant lethal test at concentrations substantially below a toxic effect. In fact, no toxicity in the treated male was detected, even at the highest concentration used in this investigation. Dominant lethal effects were noted between 200 and 5000 ppm (Morrissey et al., 1990). As previously stated, the most important attribute of the germinal cell mutation procedure is to identify an adverse health outcome in the progeny following exposure to the parent (male). This can truly be considered the &dquo;tip of the iceberg.&dquo; Transmissible genetic damage, displaying a spectrum of abnormal outcomes, can be anticipated at concentrations below those identified in this dominant lethal procedure.
Available information, therefore, establishes 1,3-butadiene as a germinal cell mutagen based on: 1) chemical classification, 2) the known reactivity of its metabolites resulting in the formation of DNA adducts, 3) its distribution in germinal cells, 4) the metabolic-somatic mutation model (where in addition to reactive metabolites reaching germinal cells the chemical has been show to induce chromosomal aberration in animals), and 5) the positive dominant lethal test.
Using the EEC guidelines currently under consideration, 1,3-butadiene would be placed in Category 2A. Category 2A is the highest category in terms of germinal cell mutagens based on
animal studies. CONCLUSIONS AND SUMMARY
1. 1 ,3-butadiene is a potent classical alkylating agent. Sufficient information is available to indicate that the metabolites of 1,3-butadiene alkylate DNA, and that this alteration leads to mutation. Positive in vivo cytogenetic data, detectable after a comparatively short exposure, provide evidence that this chemical causes genetic damage. Cancer bioassay studies extend the data base to indicate that the metabolites of 1,3butadiene alkylate DNA and produce mutations that are fixed and lead to adverse health outcomes. This chemical induces cancer at multiple sites, including germinal cells, and this response occurs at an unusually low concentration. Alkylation of DNA, irreparable genetic
Downloaded from tih.sagepub.com at UCSF LIBRARY & CKM on April 21, 2015
218
damage, and finally, the development of adverse health outcomes are established consequences of exposure to 1,3-butadiene. 2. 1,3-butadiene is directly toxic to developing embryos. The known mechanisms of action of 1,3-butadiene and the demonstrated carcinogenicity would indicate that this chemical is a developmental toxin capable of causing effects such as intrauterine growth retardation, early fetal death, malformations, or functional abnormalities. In the case of 1,3-butadiene, not only can this conclusion be drawn indirectly, but evidence is available from studies in both rats and mice. Intrauterine growth retardation, increased mortality of post-implantation embryos, and malformations have all been demonstrated. in rats produced multiple developmental abnormalities (Hazelton Laboratories, 1981). The rat is allegedly less sensitive to the toxicity of 1,3butadiene than the mouse (Laib, 1990). If this is the correct conclusion from the studies carried out to date, then we have evidence for the developmental toxicity of 1,3-butadiene in an allegedly resistant species. However, the length of exposure in these studies, and the comparatively few experiments that have been carried out would make any conclusions as to the differences between the rat and mouse premature. As to extrapolating experimental animal data to humans, it should be borne in mind that we are an extremely heterogeneous species. This heterogeneity can be translated into unusually high variation in the way we metabolize chemicals. Because of this variability there could well be subsets of our population which are more sensitive to the genotoxic effects of 1,3-butadiene than either rats or mice. It is
interesting
to note that the
study
In terms of the quantitative risk assessment of developmental toxins that affect the embryo, it should be emphasized that humans in general have proven to be a far more sensitive species than non-human animals. Schardein and Keller (1989) have pointed out that the human is remarkably sensitive to those agents characterized as developmental toxicants. Of the 21 agents considered to be direct human developmental toxins, in 19 of the 21 agents the human has been more sensitive than non-human animal models. Ratios of threshold does in the most sensitive animals to those in humans, ranged from 1.2 to 200.
3. 1,3-butadiene is a developmental toxin affecting progeny after parental exposure. If this chemical reaches germinal cells, purely on the basis of theoretical considerations its classification as a germinal cell mutant would be warranted, even in the absence of further experimental evidence. The adverse health outcomes in the progeny following parental exposure would be the same as the somatic mutation effects seen in the developing embryo. Experimental evidence is available, however, to show that this chemical induces dominant lethality following male exposure. We therefore have direct evidence that 1,3-butadiene produces adverse health outcomes in the progeny as a consequence of parental exposure. It should be understood that the dominant lethal test is a highly insensitive procedure, and adverse genetic outcomes other than lethality would be anticipated at concentrations below the recorded dominant lethal effect.
Downloaded from tih.sagepub.com at UCSF LIBRARY & CKM on April 21, 2015
219
4.
Animal cancer studies demonstrate that 1,3-butadiene is a carcinogen in B6C3F1 mice and in Sprague-Dawley rats. Epidemiological studies in workers showed significant increase in lyphomas and leukemias as a result of exposure to 1,3-butadiene.
REFERENCES
ANDJELKOVICH, D., TAULBEE, J., and SYMONS, M. (1976). "Mortality experience of a cohort of rubber workers." J. Occup. Med. 18 (6):387-394. CITTI, L., GERVASI, P.G., TURCHI, G., BELLUCI, G., and BIANCHINI, R. (1984. "The reaction of 3,4-epoxy-1-butene with deoxyguanosine and DNA in vitro: synthesis and characterization of the main adducts." Carcinogenesis 5: 47-52. DE MEESTER, C. (1988). "Genotoxic properties of 1,3-butadiene." Mutation Research 195: 273-281. DIVINE, B.J. (1987). "An update on mortality among workers at a butadiene facility." Am. J. Industri. Med. 12: 311-329. DOWNS, T.D, CRANE, M.M., and KWANGE, K.W. (1987). "Mortality among workers at butadiene facility." Am. J. Industr. Med. 311-329. 12: ENVIRONMENTAL HEALTH PERSPECTIVES (1990). Symposium on Toxicology, Carcinogenesis, and Human Health Aspects of 1,3-Butadiene. Env. Health Persp. 86. HAZELTON LABORATORIES, EUROPE, LTD. (1981). "1,3-Butadiene: Inhalation Teratogenicity Study in the Rat." International Institute of Synthetic Rubber Producers (IISRP) Report No. 2788-522/3. HUFF, J.E, MELNICK, R.L., SOLLEVELD, H.A., HASESMAN, J.K., POWERS, M., and MILLER, R.A. (1985). "Multiple organ carcinogenicity of 1,3-butadiene in B6C3F1 mice after 60 weeks of inhalation exposure." Science 227: 548-549.
HUFF, J.E., HASEMAN, J.K, DEMARINI, D.M., EUSTES, S., MARONPOT, R.R., PETERS, A.C., PERSHING, R.L., CHRISP, C.C., and JACOBS, A.C. (1989).
"Multiple site carcinogenicity of benzene Health Perspec. 32. KOTIN, P. and FALK, H.L.
in Fisher 344 rats and B6C3F1 mice." Env.
30. (1955). Proc. Am. Assoc. Cancer Research 2:
LAIB, R.J., FILSER, J.G, KREILING,R., VANGALA, R.R., and BOLT, H.M. (1990). "Inhalation pharmacokinetics of 1,3-butadiene and 1,2-epoxybutane-3 in rats and mice." Environ. Health
57-63. 86: Perspec.
LEMEN, R.A., MEINHARDT, T.J., CRANDALL, M.S., FAJEN, J.M., and BROWN, D.P.
(1990). "Environmental Epidemiological Investigations in the Styrene-Butadiene Rubber Production Industry." Environ. Health Perspec. 86: 103-106. LOW, J.A., and GALBRAITH, R.S. (1974). "Pregnancy characteristics of intrauterine growth retardation." Obstet. Gynecol. 44: 122. MALVOISIN, E., MERCIER, M., and ROBERFROID, M. (1982). "Enzymic hydration of butadiene monoxide and its importance in the metabolism of butadiene." Adv. Expo. Med. Biol. 38A: 437-444. MATANOSKI, G.M. and SCHWARTZ, L. (1987). "Mortality of workers in styrenebutadiene polymer production" J. Occup. Med. 29 (8):675-680.
Downloaded from tih.sagepub.com at UCSF LIBRARY & CKM on April 21, 2015
220
MATANOSKI, I., SANTOS-BURGOA, C., and SCHWARTZ, L. (1990). "Mortality of
a
Styrene-Butadiene Polymer Manufacturing Industry (1943107-117. Health Perspec. 88:
Cohort of Workers in the
1982). Environ.
MCCAMMON, P. KOTIN, P., and FALK, H.L. (1957). Proc. Am. Assoc. Cancer Research 229. 2:
MCMICHAEL, A.J., SPRITAS, R., GAMBLE, J.F., and TOUSEY, P.M. (1976).
"Mortality among rubber workers: relationship to specific jobs." J. Occup. Med.
18 178:
185.
MEINHARDT, T.J., LEMEN, R.A., CRANDALL, M.S., and YOUNG, R.J. (1982).
epidemiologic investigations of the styrene-butadiene rubber industry: mortality patterns with discussion of the hematopoietic and lymphatic malignancies."
"Environmental
Scand. J. Work Environ. Health 8: 250-259.
MELNICK, R.L., HUFF, J., CHOU, B.J., and MILLER, R.A. (1990). "Carcinogenicity of 1,3-Butadiene in C57BL/6 x C3H F1 Mice at Low Exposure Concentrations." Cancer 6592-6599. Research 50:
MILLER, H.C. (1981). "Intrauterine growth retardation. An
unmet
challenge."
Am. J. Dis.
Child. 135: 944. L.M. MILLER, (1978). "Investigation of Selected Potential Environmental Contaminants: Butadiene and its Oligomers." EPA-560/2-78-008. Washington, D.C., U.S. EPA.
MORRISSEY, R.E., SCHWETZ, B.A., HACKETT, P.L., SIKOV, M.R., HARDIN, B.D., MCCLANAHAN, B.J., DECKER, J.R., and MAST, T.J. (1990). "Overview of reproductive and developmental toxicity studies of 1,3-butadiene in rodents." Environ. Health
79-84. Perspec. 86:
MORROW, N.L. (1990). "The Industrial Production and Use of 1,3-butadiene." Env. Health 7-8. Persp. 86:
NELSON, T., OAKLEY, G.P., and SHEPARD, T.H. (1971). "Collection of human embryos and fetuses. II. Clarification and tabulation of conceptual wastage with observations on type of malformation, sex ratio, and chromosome studies." In: Monitoring Birth Defects & Environment (E.B. Hook, D.T. Janerich, and I.H. Porter, eds.). Academic Press, New York. OSHA (OCCUPATIONAL SAFETY AND HEALTH ADMINISTRATION) (1990). Fed. 155. Regist. 55: SCHARDEIN, J.L. and KELLER, K.A. (1989). "Potential Human Developmental Toxicants and the Role of Animal Testing in Their Identification and Characterization." CRC Critical Reviews in Toxicology 19 (3):251-338. SHELBY, M.D. (1990). "Results of NTP-sponsored mouse cytogenetic studies on 1,371-73. butadiene, isoprene, and chloroprene." Environ. Health Perspec. 86: U.S. ENVIRONMENTAL PROTECTION AGENCY (EPA). (1986). "2,4,5-T and Silvex." Fed. Regist. 44: 874.
Downloaded from tih.sagepub.com at UCSF LIBRARY & CKM on April 21, 2015
221
INFORMATION FOR AUTHORS
Toxicology and Industrial Health welcomes manuscripts dealing with basic and applied research in the fields of toxicology, biochemical toxicology, genetic and cellular toxicology, and risk assessment associated with hazardous wastes and ground water. The journal will accept and publish rapidly brief communications reporting on important research. It will publish several types of articles including papers describing original research results; reviews on subjects of contemporary importance to toxicologists and biochemical scientists; reports or proceedings, which will be published as supplements to issues of the journal at the expense of the organization that submits the proceedings; and brief announcements of scientific meetings or courses of interest to readers. -
Submission of Manuscripts. Prior to submission, manuscripts should have received any required institutional clearance and approval. The original and one copy should be sent to:
_
The decision
Managing Editor Princeton Scientific Publishing Co., Inc. P.O. Box 2155 Princeton, NJ 08543
.
regarding acceptability for publication will come from the Editor-in-Chief.
Manuscript Preparation. The entire manuscript should be double-spaced on standard 8-1/2 x 11 inch paper, typed on one side only with pages numbered consecutively. A cover sheet should contain the following items: 1) title of manuscript; 2) full first names, middle initials, and last names of authors; 3) institutional affiliation of authors; 4) corresponding author’s name, mailing address, and telephone number; 5) abbreviations used in the manuscript, listed alphabetically; 6) 3-6 key words found in the manuscript, listed alphabetically; 7) running title of approximately 45 characters. Papers are to be organized into Abstract, Introduction, Methods, Results, Discussion, Acknowledgments, and References sections. All figures (originals only) must accompany the manuscript at the time of submission. They should be properly identified with authors’ names, figure number, place of insertion, and top of figure. Tables should be numbered sequentially in Arabic numerals and headed with a short title. Each table should be on a separate page and the preferred point of insertion should be indicated in the margin of the manuscript text. Whenever possible, tables should be submitted in camera-ready form. If the manuscript and/or figures and tables are available on Apple Macintosh computer diskettes, please submit a diskette along with the two hard copies of the material and indicate
which software application
was
used.
Downloaded from tih.sagepub.com at UCSF LIBRARY & CKM on April 21, 2015
222
Footnotes and References. Keep footnotes to a minimum. Refer to them numerically as superscripts within the text. Unpublished material and personal communications should be footnoted. Critical statements of fact must be documented by referenced whenever applicable. Citations in the text are by the name of the first author and year of publication. In the case of a multiple citation, the order is chronological.
References should be organized in per the samples below:
alphabetical order, providing complete bibliographical data,
MAIN, A.R. and BRAID, P.E. (1962). &dquo;Hydrolysis of malathion by
Journal:
aliesterase in vitro and in vivo.&dquo; Biochem. J. 84: 255-263.
NICKLESS, G. (1968). Inorganic Sulfur Chemistry, pp. 509-531. Elsevier, New York.
Book:
Book
chapter:
TALCOTT, R.E., KETTERMAN, A., HARGER, W., DENK, H., and ECKERSTORFER, R. (1980). &dquo;Microsomal lipid peroxidation: Catalysis, effects, and inhibition by cytosolic protein.&dquo; In: Microsomes, Drug Oxidations, and Chemical Carcinogenesis (M.J. Coon, A.H. Cooney, R.W. Estabrook, H.V. Gelboin, J.R. Gillette, and P.J. O’Brien, eds.). Vol. 2, pp. 753-761. Academic Press, New York.
Page Charges. A page charge of $40.00 per printed page will be assessed to cover part the cost of publication. Acceptance of the papers is not based on the ability to pay charges.
Reprints.
Forms for
ordering reprints will be sent to authors with their galley proofs.
Downloaded from tih.sagepub.com at UCSF LIBRARY & CKM on April 21, 2015
__
of
