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Concise International Chemical Assessment Document 70

First draft prepared by Drs J. Kielhorn, S. Schmidt, and I. Mangelsdorf, Fraunhofer Institute of Toxicology and Experimental Medicine, Hanover, Germany; and Dr P. Howe, Centre for Ecology & Hydrology, Monks Wood, United Kingdom

Published under the joint sponsorship of the United Nations Environment Programme, the International Labour Organization, and the World Health Organization, and produced within the framework of the Inter-Organization Programme for the Sound Management of Chemicals.

The International Programme on Chemical Safety (IPCS), established in 1980, is a joint venture of the United Nations Environment Programme (UNEP), the International Labour Organization (ILO), and the World Health Organization (WHO). The overall objectives of the IPCS are to establish the scientific basis for assessment of the risk to human health and the environment from exposure to chemicals, through international peer review processes, as a prerequisite for the promotion of chemical safety, and to provide technical assistance in strengthening national capacities for the sound management of chemicals.

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WHO Library Cataloguing-in-Publication Data

Heptachlor.

(Concise international chemical assessment document ; 70)

First draft prepared by J. Kielhorn, S. Schmidt and I. Mangelsdorf.

1. Heptachlor - adverse effects. 2. Heptachlor - toxicity. 3. Environmental exposure.
4. Risk assessment. I. Kielhorn, Janet. II. Schmidt, S. III. Mangelsdorf, Inge. IV. World
Health Organization. V. International Programme on Chemical Safety, VI. Series.

ISBN 92 4 153070 7          (NLM Classification: WA 240)
ISBN 978 92 4 153070 5

©World Health Organization 2006

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Advice from Risk Assessment Steering Group Criteria of priority: there is the probability of exposure; and/or there is significant toxicity/ ecotoxicity. Thus, it is typical of a priority chemical that it is of transboundary concern; it is of concern to a range of countries (developed, developing, and those with economies in transition) for possible risk management; there is significant international trade; the production volume is high; the use is dispersive. Special emphasis is placed on avoiding duplication of effort by WHO and other international organizations. A prerequisite of the production of a CICAD is the availability of a recent high-quality national/regional risk assessment document = source document. The source document and the CICAD may be produced in parallel. If the source document does not contain an environmental section, this may be produced de novo, provided it is not controversial. If no source document is available, IPCS may produce a de novo risk assessment document if the cost is justified. Depending on the complexity and extent of controversy of the issues involved, the steering group may advise on different levels of peer review: standard IPCS Contact Points above + specialized experts above + consultative group

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This CICAD² on heptachlor was prepared by the Fraunhofer Institute of Toxicology and Experimental Medicine, Hanover, Germany. It is an update of the Environmental Health Criteria document on heptachlor (IPCS, 1984) and includes data from the IARC (2001) and JMPR (1992) reports. A comprehensive literature search of relevant databases was conducted from 2000 up to February 2004 to identify any relevant references published subsequent to those incorporated in these reports. Information on the source documents is presented in Appendix 2. Information on the peer review of this CICAD is presented in Appendix 3. This CICAD was considered and approved as an international assessment at a meeting of the Final Review Board, held in Hanoi, Viet Nam, on 28 September – 1 October 2004. Participants at the Final Review Board meeting are presented in Appendix 4. The International Chemical Safety Card on heptachlor (ICSC 0743), produced by the International Programme on Chemical Safety (IPCS, 2003), has also been reproduced in this document.

Heptachlor (CAS No. 76-44-8) is a chlorinated dicyclopentadiene insecticide that is persistent in the environment and accumulates in the food-chain. Although its use has been banned or severely restricted in many countries since the 1980s, it is still detected as a contaminant in some food commodities. This is due to its persistence, but it also suggests the illegal use of this pesticide in the recent past or present (or maybe its permitted use in some countries). Heptachlor is one of several organochlorine pesticides that are persistent in the environment. Concentrations in body tissues and in the environment of all these compounds together are several times greater than those of heptachlor and/or heptachlor epoxide (a persistent heptachlor metabolite) alone.

Heptachlor released into the environment can be transformed by abiotic processes, such as the transformation by photochemically produced hydroxyl radicals, and it is transformed in the presence of water to compounds such as 1-hydroxychlordene or heptachlor epoxide (such as, for example, in moist soils). In addition, it can be removed to some extent from aquatic systems by evaporation and has a limited potential to leach from soil into groundwater due to its elevated soil sorption coefficient. It is not readily biodegraded, but it is transformed biologically (i.e. by bacteria, fungi, plants, animals), mainly to the stable heptachlor epoxide. The data available on the bioconcentration potential of this lipophilic chlorinated hydrocarbon indicate that it and its stable epoxide will bioaccumulate, which can be shown from the extent of heptachlor/heptachlor epoxide still detected in environmental samples.

The main exposure routes for heptachlor are probably via application-related inhalation or skin penetration, from extended exposure to dusts containing heptachlor in, for example, homes treated with this compound to control termites, and indirectly by uptake from food contaminated with heptachlor from crops or from other foods via the food-chain. However, heptachlor is a component of technical chlordane as well as a metabolite of chlordane, and thus identification of heptachlor or heptachlor epoxide does not always signify unequivocally that the primary exposure was to heptachlor (or heptachlor epoxide) per se.

A survey of recent studies shows that heptachlor and/or heptachlor epoxide are found in all environmental compartments — air, water, soil, and sediment — as well as in plants (vegetables), fish and other aquatic organisms, amphibians and reptiles, birds and bird eggs, and aquatic and terrestrial mammals. They are found particularly in adipose tissues, where they accumulate. They pass up the food-chain. They are detected in human serum, adipose tissue, including breast tissue, and human breast milk.

Heptachlor is readily absorbed via all routes of exposure and is readily metabolized. The major faecal metabolites include heptachlor epoxide, 1-hydroxychlordene, and 1-hydroxy-2,3-epoxychlordene. In liver microsomes incubated with heptachlor, 85.8% was metabolized to heptachlor epoxide in rats, but only 20.4% in humans. Other metabolites identified in the human liver microsome system were 1-hydroxy-2,3-epoxychlordene (5%), 1-hydroxychlordene (4.8%), and 1,2-dihydroxydihydrochlordene (0.1%). Heptachlor epoxide is metabolized slowly and is the most persistent metabolite; it is stored mainly in adipose tissue, but also in liver, kidney, and muscle. Females appear to store more heptachlor epoxide than males. A period of 12 weeks was required for complete disappearance from the fat after discontinuing heptachlor feeding in rats.

In humans and laboratory animals, placental transfer of heptachlor and/or heptachlor epoxide has been shown.

Acute oral LD₅₀s for heptachlor for the rat and mouse are 40–162 and 68–90 mg/kg body weight, respectively. The acute toxicity of heptachlor in animals is associated with central nervous system disturbances, such as hyperexcitability, tremors, convulsions, and paralysis. The acute toxicity of heptachlor epoxide is greater than that of heptachlor, whereas that of the other metabolites is much less.

In animals fed heptachlor/heptachlor epoxide by diet, gavage, or subcutaneous injection, there is a sharp dose–response curve for mortality. Usually, no marked differences were seen between the treated animals and the controls with respect to body weights and food consumption. However, liver enlargement has been described, associated with accentuated lobulation, and histopathological findings showed enlargement of centrilobular and midzonal hepatocytes.

Fertility studies in rats injected with heptachlor subcutaneously resulted in LOAELs of 5 mg/kg body weight per day for suppression of reproductive hormone levels, disruptions in female cyclicity, and delays in mating behaviour.

In developmental toxicity studies, there were usually no clinical signs of maternal toxicity (dose-related alterations in weight gain) until mortality occurred [NOAEL for maternal toxicity = 3 mg/kg body weight per day]. In one study, reduced litter sizes were noted, but postnatal mortality of the pups was the most obvious finding [NOAEL for pre- or postnatal survival of pups = 6 mg/kg body weight per day]. No teratological effects were observed.

There is accumulating evidence that the nervous system and its development are influenced by cyclodiene pesticides. The profile of effects produced by repeated heptachlor administration to female rats consisted of altered activity, hyperexcitability, and autonomic effects [NOAEL = 2 mg/kg body weight per day]. Neurotoxicological studies on perinatal heptachlor exposure in the rat (0.03, 0.3, or 3 mg/kg body weight per day) suggested developmental delays, alterations in GABAergic neurotransmission, and neurobehavioural changes, including cognitive deficits at all doses.

Immunological studies in rats indicate the suppression of the primary IgM and secondary IgG anti-sheep red blood cell responses following perinatal exposure to all tested doses (0.03, 0.3, or 3 mg/kg body weight per day) of heptachlor.

Heptachlor, technical-grade heptachlor, heptachlor epoxide, and a mixture of heptachlor and heptachlor epoxide have been tested for carcinogenicity by oral administration in several strains of mice and rats. Heptachlor/heptachlor epoxide and technical-grade heptachlor were shown to be carcinogenic in male and female mice but not in rats. In an initiation–promotion assay, heptachlor was active as a promoter after initiation by .-nitrosodiethylamine.

Heptachlor shows mostly negative responses in in vitro and in vivo genotoxicity testing. Heptachlor causes in vitro inhibition of gap junctional intercellular communication, also suggesting a non-genotoxic carcinogenic mechanism.

Available epidemiological data do not show a clear relationship between adverse health effects and exposure to heptachlor. A tolerable intake was therefore developed from experimental studies. As hepatic tumours induced by heptachlor in mice are likely to be induced by a non-genotoxic mechanism and as non-neoplastic effects were observed at doses 1/20th of those inducing tumours, non-neoplastic effects (i.e. histopathological effects in the liver, neurotoxicological effects, and immunotoxicological effects) were used to derive the tolerable intake. The NOAEL for hepatic effects observed in dogs was 25 µg/kg body weight per day, and that for neurotoxicity and immunotoxicity observed in studies in rats was 30 µg/kg body weight per day. Applying an uncertainty factor of 10 for each of inter- and intraspecies variation and an additional factor of 2 for inadequacy of the database to the NOAEL in dogs gives a tolerable intake of 0.1 µg/kg body weight per day for the non-neoplastic effects.

Daily dietary intakes of heptachlor and heptachlor epoxide in Poland were estimated at 0.51–0.58 µg per person (about 0.01 µg/kg body weight, assuming a mean weight of 64 kg). This value is 10-fold less than the tolerable intake of 0.1 µg/kg body weight. However, if food is contaminated with heptachlor, such as fish from contaminated rivers (e.g. concentrations in fish in the 0.1–1 mg/kg range reported recently in some areas), vegetables from fields contaminated with heptachlor (up to 16 mg/kg), or contaminated milk (e.g. in the microgram per kilogram to milligram per kilogram range in some regions), then the dietary intake of this chemical would be much higher, and there would be a likely health risk if the contaminated food is ingested for a long period of time. For breast-fed children, taking the highest reported values for heptachlor epoxide in human breast milk and assuming a daily milk consumption of 150 g/kg body weight and an average milk fat content of 3.1%, a mean intake of 1.5 µg/kg body weight can be calculated. This value is more than 10-fold higher than the tolerable intake of 0.1 µg/kg body weight per day and, if the concentrations reported are correct, should be a cause of concern.

The acute toxicity of heptachlor was tested using a variety of aquatic species from different trophic levels. Heptachlor was shown to be toxic to fish and other aquatic species. However, there is a great deal of variability in the levels of toxicity reported, possibly due to evaporation of heptachlor, thereby reducing the actual concentration of the test compound from the nominal test concentration over time.

For the freshwater environment, 23 toxicity values were chosen to derive a guidance value. A guidance value for heptachlor, based on the species sensitivity distribution, for the protection of 99% of species with 50% confidence was derived at 10 ng/l. In many locations, freshwater heptachlor concentrations exceed the guidance value; the highest reported heptachlor concentration measured in fresh surface water, 62 000 ng/l, exceeds it more than 1000-fold.

For the marine environment, 18 toxicity values were chosen to derive a guidance value. A guidance value for heptachlor, based on the species sensitivity distribution, for the protection of 99% of species with 50% confidence was derived at 5 ng/l. For seawater, the highest reliable value for heptachlor present is about 0.15 ng/l, so the guidance value is not exceeded, suggesting a low risk for the marine environment.

From the few data available, heptachlor appears to exhibit moderate toxic effects upon terrestrial vertebrates. None of the studies appears reliable enough to serve as a basis for a quantitative risk characterization. It should be remembered that heptachlor is used as a termiticide.

In studies on rats, heptachlor has been shown to be neurotoxic and immunotoxic at 0.03 mg/kg body weight per day. The Göksu Delta, Turkey, which is one of the most important breeding and wintering areas for birds in the world, is contaminated by organochlorine pesticides from soils from agricultural areas that have been transported to the delta by the Göksu River. From this region, levels of heptachlor/heptachlor epoxide have been detected in birds and bird eggs in the lower milligram per kilogram range. The effect of such concentrations of heptachlor on the bird populations can only be speculated at present due to lack of data; however, there is a potential risk for the terrestrial environment in this location.

Heptachlor (1,4,5,6,7,8,8-heptachloro-3a,4,7,7a-tetrahydro-1.-4,7-methanoindene; CAS No. 76-44-8; C₁₀H₅Cl₇) is a chlorinated dicyclopentadiene belonging to the so-called persistent organic pollutants, which were restricted by the Stockholm treaty in 2001 (http:// www.chem.unep.ch/pops/). In its pure form at room temperature, it appears as a white or light tan, crystalline solid with a mild camphor- or cedar-like odour, exhibiting a melting point of about 95–96 °C. However, the technical product is a soft wax and has a melting point of about 46–74 °C, depending on its composition.

The technical product usually contains about 72% heptachlor and about 28% related compounds, such as trans-chlordane (20–22%) and trans-nonachlor (4–8%) (NCI, 1977).

The environmentally relevant physicochemical properties of heptachlor and its stable and recalcitrant transformation product heptachlor epoxide (2,3-epoxy-1,4,5,6,7,8,8-heptachloro-2,3,3a,4,7,7a-hexahydro-4,7-endomethanoindane; CAS No. 1024-57-3; C₁₀H₅Cl₇O) are summarized in Table 1. Additional physical and chemical properties are presented in the International Chemical Safety Card (ICSC 0743) reproduced in this document. Their structures are shown in Figure 1.

Fig. 1: Structure of heptachlor and heptachlor epoxide.

Table 1: Physicochemical properties of heptachlor and its epoxide.

A few selected examples of the analytical methods for the detection and quantification of heptachlor and its epoxide in different matrices are presented below. Further detailed information is available in IARC (2001).

Residues of heptachlor and its epoxide in solid materials such as animal tissues or sediments can be determined by GC using various detection methods, usually following appropriate cleanup and/or extraction procedures. For example, Lu & Wang (2002) employed Soxhlet extraction of ground rainbow trout tissue followed by column purification and GC/ECD. Diop et al. (1999) used solvent extraction followed by column purification and HRGC/ECD for plant material, whereas lyophilized sediment has been analysed by GC/ECD following Soxhlet extraction and column purification (González-Farias et al., 2002). Analysis of heptachlor in air usually involves sorption of the organochlorine insecticide onto a solid matrix followed by thermal or solvent desorption prior to HRGC/MS (e.g. Buser & Müller, 1993).

There are no known natural sources of heptachlor. However, its epoxide is not produced commercially but rather is formed by abiotic or biotic transformation of heptachlor in the environment.

The Stockholm treaty of 2001 restricted or banned the use of heptachlor, as this compound was recognized as a so-called persistent organic pollutant (http:// www.chem.unep.ch/pops/).

Heptachlor is a pesticide, and the technical grade contains structurally related compounds (see section 2). Heptachlor also occurs as a component of technical chlordane, which is also used as a pesticide (IARC, 2001). Chlordane consists of four major components — heptachlor (22%), 1,2-dichlorochlordane (13.2%), trans-chlordane (27.5%), and cis-chlordane (11.9%) — and seven minor components (Tsushimoto et al., 1983). Schmitt et al. (1999) give <10% heptachlor in chlordane.

The main use of heptachlor by farmers is to kill termites, ants, and soil insects in seed grains and on crops and by exterminators and home owners to kill termites in wooden structures. Agricultural consumption of heptachlor in the USA was about 550 tonnes per year in the early 1970s (Fendick et al., 1990). However, most applications of this insecticide were banned or at least severely restricted in the early 1980s in many countries. The only permitted commercial use of heptachlor products in the USA since 1987 is for fire ant control in power transformers and in underground cable television and telephone cable boxes (ATSDR, 1993). However, the pesticides heptachlor and chlordane were still produced for export. In 1997, Velsicol ceased production in the USA. United States customs data show that at least 2028 tonnes of technical chlordane (containing about 446 tonnes heptachlor) and 2584 tonnes of heptachlor were exported from the USA between 1991 and 1994 (therefore about 760 tonnes per year), and these figures are thought to greatly underestimate the actual figures (PANNA, 1997). In the Republic of Korea, about 560 tonnes of heptachlor were imported and employed in agriculture from 1961 until its use came to an end in 1980 (Kim & Smith, 2001). According to Bouwman (2003), South Africa currently still imports about 180–270 tonnes of heptachlor per year but will probably phase out its registration. However, use of chlordane (containing heptachlor) is still permitted for protecting buildings from termites. As in many other countries, stocks of obsolete pesticides have been accumulating for various reasons, and their disposal is a worldwide problem (FAO, 1999).

According to a survey on sources of persistent organic pollutants, performed by the United Nations Environment Programme, the global import figures of heptachlor in 1993 and 1994, based on the response of 61 countries, were 389 and 435 tonnes, respectively, with South America and Oceania being the largest consumers (UNEP, 1996). In those countries where use is restricted, but may continue, the applications are restricted to seed treatment, structural termite control, or wood treatment. In tropical and subtropical countries that have resumed use of heptachlor for seed treatment or preplanting agricultural use, heptachlor is restricted to crops that form edible portions above ground and, in particular, to crops with long growing seasons that undergo processing before consumption (FAO/UNEP, 1996; IARC, 2001).

Buildings constructed in temperate regions may contain added pesticides in materials. Therefore, their destruction or demolition may induce local environmental contamination (Offenberg et al., 2004).

Heptachlor has also been used as a component in plywood glues — e.g. two Finnish products contained 17–25% heptachlor and 6–9% chlordane (Mussalo-Rauhamaa et al., 1991).

Several recent monitoring studies have shown evidence of recent heptachlor usage (see section 6 and Appendix 5).

Heptachlor and heptachlor epoxide are subject to long-range transport and removal from the atmosphere by wet and dry deposition. Heptachlor epoxide was present in 20 of 21 snowpack samples collected in the Northwest Territories, Canada (1985–1986), at a mean concentration of 0.18 ng/l. No known local sources for heptachlor in Canadian Arctic snow were identified (Gregor & Gummer, 1989).

The environmental transport and distribution of heptachlor are reflected in the data shown in the tables in Appendix 5; heptachlor has been found in samples in various matrices all over the world where no local sources are to be found (e.g. in mammal tissue in the Arctic, etc.; Table A5-9).

The predominant target compartments for heptachlor in the environment are soil and sediment (about 43% and 55%) and, to a lesser extent, water (about 2%; Level III fugacity calculation, using EPI suite 3.10). Due to its experimentally determined water solubility of about 180 µg/l at 25 °C, only a limited proportion of this compound is expected to be removed from the atmosphere by wet deposition (dissolution in clouds, rainfall, etc.). According to Thomas (1990), the measured Henry’s law constant of 29.75 Pa·m³/mol indicates a moderate volatility of heptachlor from the aqueous phase.

In general, agricultural drains are a means of transporting the pesticides far from their original place of use (González-Farias et al., 2002).

Under environmental conditions, heptachlor will not be prone to wash out from soil, as its ._oc value of about 16 000 (Johnson, 1991) indicates a low mobility in soil; together with its limited water solubility, this would restrict its potential for leaching to groundwater. Therefore, heptachlor was characterized as a non-leacher pesticide (Johnson, 1991). In principle, however, a long residence time may nevertheless result in an appreciable movement of heptachlor and its stable metabolite heptachlor epoxide, posing a potential risk for groundwater.

Heptachlor is moderately persistent in soil, where it is mainly transformed into its epoxide. It may undergo significant photolysis, oxidation, and volatilization (ATSDR, 1993).

Application-related release of heptachlor to soil surfaces can result in volatilization from the surface, but volatilization of heptachlor incorporated into soil will be slower (Fendick et al., 1990). In water and in moist soils, the reaction of heptachlor with water can give rise to the formation of 1-hydroxychlordene (Bowman et al., 1965) and, to a lesser extent, heptachlor epoxide (Miles et al., 1969). Chapman & Cole (1982) reported half-lives of heptachlor dissolved in phosphate-buffered sterile water–ethanol (99:1) of 0.77 (pH 4.5), 0.62 (pH 5), 0.62 (pH 6), 0.64 (pH 7), and 0.43 (pH 8) weeks, whereas Johnson (1991) reported a half-life of 180 days.

Assuming a hydroxyl radical concentration of 1.5 × 10⁶ molecules per cubic centimetre and a 12-h day, half-lives of about 2.1 h for heptachlor and of about 24 h for the stable epoxide in the atmosphere (applying the USEPA program AOPWIN v. 1.9) can be calculated for indirect photochemical transformation by hydroxyl radicals in the atmosphere. Further, although heptachlor is apparently stable in the light (Fendick et al., 1990), Mansour & Parlar (1978) reported a phototransformation of heptachlor and its epoxide to photoisomerization products. The caged photoisomer derived from heptachlor by intramolecular cycloaddition upon UV irradiation (Buser & Müller, 1993) is known to be even more toxic and persistent than heptachlor and heptachlor epoxide (Zhu et al., 1995). Podowski et al. (1979) reported that heptachlor is converted into its photoisomer on exposure to low-intensity (longwave) UV light. This photolysis can also occur on plant leaves in the presence of sunlight or UV light. Photoheptachlor is about 20 times more toxic to rats than heptachlor.

In aerobic biodegradation tests performed according to OECD guideline 301C, heptachlor was not readily biodegradable (BOD 0% of theoretical oxygen demand, incubation for 28 days) (MITI, 1992). In an earlier report, Leigh (1969) demonstrated that there was no significant difference between abiotic and biological removal (as BOD₅) of heptachlor. However, in contrast to the desired aerobic biodegradation (i.e. mineralization), which does not take place to any considerable extent, the simple and rapid biotransformation of heptachlor to transformation products such as the heptachlor epoxide was reported for typical soil bacteria (Miles et al., 1969; Bourquin et al., 1972; Beeman & Matsumura, 1981), soil fungi (Miles et al., 1969; Iyengar & Rao, 1973), higher fungi, such as the white rot fungus (Phanerochaete chrysosporium) (Kennedy et al., 1990; Arisoy, 1998), mixed microbial cultures from soil (Miles et al., 1971; Bourquin et al., 1972), plants (i.e. cabbage and wheat; Weisgerber et al., 1972), and animals such as water flea (Daphnia magna) (Feroz et al., 1990) and goldfish (Carassius auratus) (Feroz & Khan, 1979). Under anaerobic conditions, both heptachlor and its epoxide showed only limited conversion (Hill & McCarty, 1967). Johnson (1991) cited an aerobic biotransformation half-life for heptachlor of 2000 days and an anaerobic biotransformation half-life of 39 days. It is interesting to note that the presence of heptachlor epoxide in ambient air samples is due to the emission of this stable metabolite from soil where it is formed from heptachlor by microbial activity rather than to the photooxidation of heptachlor in the atmosphere (Bidleman et al., 1998a,b).

The experimentally determined bioconcentration factors (measured corresponding to OECD guideline 305C) in the range of 2000–17 000 (MITI, 1992) strongly indicate a high bioaccumulation potential. Bioconcentration may nevertheless be limited in the aquatic environment by the rapid transformation of heptachlor in water to hydroxychlordene and the sorption of heptachlor to sediment or particulate matter. However, as heptachlor epoxide is more stable in water, it is bioconcentrated extensively (Lu & Wang, 2002), with a measured concentration of about 5000 µg/kg (on a wet weight basis) after 20 days’ exposure of rainbow trout to heptachlor epoxide present in water at a concentration of about 1.5 µg/l.

Log ._ow values for heptachlor and heptachlor epoxide of 6.1 and 5.1, respectively, suggest a high potential for bioaccumulation and biomagnification. An example illustrating the way in which heptachlor might enter the food-chain to end up as heptachlor epoxide in dairy milk for human consumption is provided by the report describing an application to control ants on pineapple plantations in Hawaii, USA (Baker et al., 1991). In this instance, chopped leaves (from the pineapple plants) were fed to dairy cattle, with subsequent excretion of metabolically formed epoxide into the milk.

Organochlorinated pesticides such as heptachlor, owing to their chemical stability, low aqueous solubility, and high lipophilicity, become concentrated along the food-chain, reaching higher concentrations at higher trophic levels. They reach the human body in the daily diet and are deposited and accumulated in adipose tissues.

Several recent monitoring studies have shown evidence of recent heptachlor usage, including, for example, elevated levels of heptachlor in air in Africa and the USA (Jantunen et al., 2000; Karlsson et al., 2000a), its presence in polluted rivers (e.g. Ayas et al., 1997), and some high residue levels found in vegetables (Bakore et al., 2002). Further, heptachlor and/or its epoxide are still present in sediment and microflora and are bioavailable to plants/crops (CU, 1999; Miglioranza et al., 2003) and thereafter biomagnify further up the food-chain. In an extensive data collection, Osibanjo (2003) showed the presence of heptachlor in the Nigerian environment (water, soil, sediment, etc.) and the food-chain (vegetables, meat, drinking-water).

Data from recent studies on levels of heptachlor and heptachlor epoxide in the environment are given in Appendix 5, Tables A5-1–A5-14.

6.1.1 Atmosphere

Table A5-1 gives an overview of some recent studies on concentrations of heptachlor and heptachlor epoxide in ambient and indoor air. Ambient air concentrations are generally in the low picogram per cubic metre range. Higher values are to be found in agricultural areas where heptachlor was previously used as a pesticide and where the pesticide is still being released from the soil; for example, Meijer et al. (2003) detected heptachlor epoxide at 550 pg/m³ 3 cm above the soil at a soya bean site and at 25 pg/m³ at 150 cm above the soil.

In houses where heptachlor was used against termites, indoor concentrations in the nanogram per cubic metre range have been measured; for example, Anderson & Hites (1989) reported heptachlor concentrations ranging from about 4 to 110 ng/m³ for basement levels and from about 3 to 66 ng/m³ for first-floor levels. More recently, Leone et al. (2000) found heptachlor concentrations in homes in the corn belt region in the USA ranging from not detectable to 79 ng/m³.

6.1.2 Hydrosphere

Heptachlor might enter the hydrosphere due to its use and application, mainly by drift or surface runoff. However, it can evaporate from the aqueous phase to some extent, and it can be converted in the presence of water (e.g. to yield hydroxychlordene); thus, the concentration of heptachlor in the hydrosphere may decrease.

Table A5-2 gives an overview of some recent studies on heptachlor and heptachlor epoxide concentrations in water samples.

6.1.2.1 Rainwater

Although the use of heptachlor has been banned in Europe for several years, there are reports that this pesticide is still present in rainwater. Heptachlor epoxide was detected in rainwater (wet and dry precipitation) in Poland (10 different sampling stations in the Gdansk region) at average concentrations ranging from 0.09 ng/l up to 0.58 ng/l (Grynkiewicz et al., 2001). A follow-up study (Polkowska et al., 2002) demonstrated the presence of heptachlor epoxide in the runoff water collected from roofs in an urban region (Gdansk, Poland) at concentrations up to 1.49 ng/l. In the Netherlands, while both heptachlor and its epoxide were detected in rainwater collected in typical agricultural locations (intense greenhouse horticulture and flower bulb production) at maximum concentrations of 3 ng/l (heptachlor) and 7 ng/l (heptachlor epoxide), only heptachlor was detected (maximum concentration 3 ng/l) in corresponding background locations (Hamers et al., 2003). Similar results were found by van Maanen et al. (2001).

6.1.2.2 Drinking-water

In an intense monitoring programme in 21 counties in California, USA, 332 wells were sampled for heptachlor and 335 wells were sampled for heptachlor epoxide. Neither compound was detected (CEPA, 2000). However, in a state-wide survey (December 1985 to February 1986), heptachlor was detected in 1% of about 100 wells tested in Kansas, USA, at an average concentration of about 0.025 µg/l (Steichen et al., 1988). In New York City, USA, from 1989 to 1993, heptachlor was detected in influent and effluent of municipal treatment facilities at concentrations ranging from 0.021 to 0.35 µg/l and from 0.02 to 0.45 µg/l, respectively (Stubin et al., 1996).

6.1.2.3 Surface and river water

The concentrations of heptachlor/heptachlor epoxide vary greatly from, for example, the lower picogram per litre range in surface water from Lake Malawi, southern Africa, to a mean value of 19 000 ng/l for heptachlor epoxide in water from around Göksu Delta, Turkey (Ayas et al., 1997) and a maximum value of 27 800 ng/l for heptachlor epoxide in water samples from canals and drains, El-Haram Giza, Arab Republic of Egypt (El-Kabbany et al., 2000). Göksu Delta is one of the most important breeding and wintering areas for birds in the world, so it is of concern that the environment is contaminated by organochlorine pesticides from soils from agricultural areas that have been transported by the Göksu River to the delta (González-Farias et al., 2002).

6.1.2.4 Ocean water

Jantunen & Bidleman (1998) reported the presence of the stable heptachlor epoxide in water samples from the western Arctic Ocean at an average concentration of 14.8 pg/l. However, the origin of the stable epoxide in polar water is not known. Water samples from the Bering and Chukchi seas contained heptachlor epoxide at concentrations up to 223 pg/l (Yao et al., 2002). Seawater surface samples from the Straits of Jahore (Singapore/Malaysia) apparently contained heptachlor at concentrations up to 233 ng/l (Basheer et al., 2002).

6.1.2.5 Wastewater

Heptachlor epoxide was detected in all samples of raw wastewater (82–1100 ng/l; median 200 ng/l) and secondary sedimentation effluent (6–120 ng/l; median 13 ng/l) at the municipal wastewater treatment plant in Thessaloniki, northern Greece (Katsoyiannis & Samara, 2004).

6.1.3 Soil and sediment

Several studies on sediment show concentrations of heptachlor/heptachlor epoxide in the lower microgram per kilogram (nanogram per gram) range (see Table A5-3). González-Farias et al. (2002) found heptachlor in single agricultural drains at high concentrations (49 and 65 ng/g dry weight for heptachlor and its epoxide, respectively), although officially the pesticide was not being used. In other studies, much higher concentrations were found; for example, means of 1377 and 244 ng/g for heptachlor and heptachlor epoxide, respectively, were measured in the Göksu Delta (Ayas et al., 1997). It should be noted that in all these studies, other organochlorine pesticides are also present at a 10-fold or higher concentration than heptachlor and heptachlor epoxide, and other pollutants are present as well. The study of Zhang et al. (2003) (Tables A5-3 and A5-4) shows how heptachlor accumulates in sediment, leaches out of sediment into the sediment porewater, and then transfers to surface water by processes such as diffusion.

Recent measurements of heptachlor and heptachlor epoxide in soil samples are given in Table A5-4. Kim & Smith (2001) reported soil levels of heptachlor up to 2.8 ng/g and of heptachlor epoxide up to 48 ng/g for top soil samples (mainly rice and vegetable farming) analysed in the Republic of Korea. Heptachlor was found not only in agricultural soil in Argentina but also in the surface of natural soil on highland hills where heptachlor was never directly applied but was probably blown by winds. The heptachlor was then metabolized in the soil to heptachlor epoxide. The majority of the heptachlor/heptachlor epoxide is found in the top layers of soil and does not penetrate to any great extent to lower layers (Miglioranza et al., 2003). In the study on soil from the Göksu Delta, heptachlor concentrations ranged from a maximum of 9616 ng/g (mean 4777 ng/g) in the agricultural area to a mean of 32 ng/g in the dune area (Ayas et al., 1997).

6.1.4 Fish and aquatic organisms

Tables A5-5 and A5-6 give an overview of concentrations of heptachlor and heptachlor epoxide in fish and other aquatic organisms, respectively.

Fish from several areas had heptachlor concentrations below 1 ng/g wet weight or in the low ng/g lipid weight range. Much higher values (in the 100–1000 ng/g range) were reported from Ganges Estuary, Bangladesh, from Bay of Bengal, and from Göksu Delta, Turkey (see Table A5-5 for details).

Concentrations of heptachlor/heptachlor epoxide in crabs, mussels, and whelks were below 1 ng/g lipid (from White Sea; Muir et al., 2003) or just above 1 ng/g lipid (Gulf of Gdansk: Falandysz et al., 2001) or 1 ng/g dry weight (marine [coastal] Pacific and Caribbean coasts: Castillo et al., 1997). Higher values were reported in estuarine Nicoya Gulf (Castillo et al., 1997), New Bedford Harbor, Massachusetts, USA (Hofelt & Shea, 1997), in the Karachi Coast area (Munshi et al., 2001), and in Australia (Connell et al., 2002). Concentrations in zooplankton were 0.02–0.55 ng/g lipid weight in the White Sea (Muir et al., 2003).

6.1.5 Birds and bird eggs

A study was conducted (see Table A5-7) during the period of October 1991 to October 1993 to determine organochlorine pesticide residues (including heptachlor) in waterbirds — Eurasian coot (Fulica atra), mallard (Anas platyrhynchos), and little egret (Egretta garzetta) — at Göksu Delta–Tasucu, which is an internationally important wetland for the waterbirds (Ayas et al., 1997). The results showed that environs and organisms were contaminated by 13 different organochlorine pesticides. Heptachlor and heptachlor epoxide generally accumulated in adipose tissue (e.g. mean heptachlor epoxide concentration of 2744 ng/g in mallards) and in the eggs of waterbirds (e.g. mean heptachlor concentration of 980 ng/g in eggs of the little egret). In comparison, heptachlor epoxide was detected in eggs of great black-backed gulls (Larus marinus) in Lake Ontario in North America at 90–140 ng/g wet weight (Weseloh et al., 2002), in an egg pool of black-footed albatross (Phoebastria nigripes) from Midway Atoll, Pacific, at 3.4 ng/g wet weight (Muir et al., 2002), in great blue heron (Ardea herodias) eggs, Upper Mississippi River, USA, at 20–100 ng/g wet weight, mean of 10 colonies (Custer et al., 1997a), and in eggs of Eurasian buzzard (Buteo buteo), La Segarra, north-eastern Spain, at 1–233 ng/g wet weight (Manosa et al., 2003).

6.1.6 Amphibians and reptiles

Table A5-8 summarizes concentrations of heptachlor and heptachlor epoxide in amphibians and reptiles.

A survey performed in a Costa Rican tropical conservation area showed the presence of heptachlor in amphibians. The highest detected mean level of about 32 ng/g wet weight was observed in Mexican giant leopard frog (Rana forreri). Heptachlor was detected in turtles (Rhinoclemmys pulcherrima) at the highest detected mean level of about 17 ng/g wet weight (Klemens et al., 2003).

6.1.7 Mammals

Levels of heptachlor in mammals are given in Table A5-9. Northwest Atlantic pilot whales (Globicephala melas) stranded in Massachusetts, USA, between 1990 and 1996 showed mean concentrations of heptachlor and heptachlor epoxide of 39 and 56 ng/g lipid weight, respectively (Weisbrod et al., 2000a). Higher concentrations (up to a mean of 1287 ng/g lipid weight in winter) were found in right whales (Eubalaena glacialis) that migrate in early spring from southern waters to the Bay of Fundy in Canada following warming water temperatures and plankton blooms and migrate back south in winter. Biopsies collected during winter had lower concentrations of lipid than biopsies collected during summer (Weisbrod et al., 2000b).

Several recent studies have measured the concentrations of heptachlor/heptachlor epoxide in seal blubber and polar bear fat in the Arctic (see Table A5-9). With a few exceptions, mean concentrations were up to 50 ng/g lipid weight in seal blubber and up to 475 ng/g lipid weight in polar bears.

6.1.8 Food

Table A5-10 gives an overview of concentrations of heptachlor and heptachlor epoxide in food.

Fruits and vegetables sampled from different markets in Dakar, Senegal, showed the presence of heptachlor at concentrations ranging from 9 µg/kg (mandarins) up to 42 µg/kg (tomatoes) (Diop et al., 1999). Similarly, medicinal plants examined by these authors contained heptachlor, with concentrations ranging from 2.25 up to 17 µg/kg. However, heptachlor and its epoxide were not detected in different herbal formulations used as dietary supplements from South-East Asia (detection limit 0.03 ng/g; Hwang & Lee, 2000). Nakamura et al. (1994) failed to detect heptachlor or its epoxide in a range of agricultural products, such as rice, cabbage, and cucumber, in Japan (detection limit 1 ng/g).

Vegetables from Jaipur City, Rajasthan, India, analysed at the end of the season, contained much higher levels of heptachlor plus heptachlor epoxide: up to about 15.9 (tomatoes), 16 (cabbage), 9.3 (okra), 9.4 (spinach), and 1.5 (cauliflower) mg of heptachlor plus heptachlor epoxide per kilogram (Bakore et al., 2002). Dairy milk as well as buffalo milk sampled from 1993 to 1996 in Jaipur City were shown to contain heptachlor and its epoxide in milligram per litre amounts (John et al., 2001). Furthermore, concentrations of heptachlor in blood of breast cancer patients and controls from Jaipur City were also very high (Mathur et al., 2002). It is not known whether these extremely high values in vegetables, cow milk, and human samples are correct (which would indicate extreme pollution with organic pesticides) or whether this is a measurement or calculation error.

Heptachlor epoxide was reported in dairy milk for human consumption as a result of the feeding of chopped leaves from the pineapple plants to dairy cattle, with subsequent excretion of metabolically formed epoxide into the milk (Baker et al., 1991). The concentrations of heptachlor epoxide were 0.12 µg/g fat basis (October 1980 – April 1981); 1.20–5.00 µg/g (April 1981 – April 1982), and <0.30 µg/g (April 1982 – December 1982) (Le Marchand et al., 1986).

Heptachlor and/or heptachlor epoxide were not detected in samples of dairy milk retailed in China (detection limit 0.002 µg/g; Zhong et al., 2003).

Heptachlor has been reported in infant food from Nigeria and Italy at concentrations of 0.09 (ND–0.87) ng/g and 9.80 (ND–72) ng/g, respectively (Osibanjo, 2003).

6.2.1 Levels of heptachlor and heptachlor epoxide in human fluids and tissues

6.2.1.1 Occupational exposure

Approximately 200 plywood workers in Finland were reported to be exposed to heptachlor, either through working with sizings that contain heptachlor or through stacking plywood for heat pressure treatment. Residue levels of heptachlor, heptachlor epoxide, and other chlordane compounds were determined in sera from 74 Finnish plywood workers and 52 controls. Concentrations of heptachlor epoxide in plywood workers varied from below the detection limit of 0.1 ng/g to 19.2 ng/g serum (1 ng/g = 0.98 µg/l); the mean and standard deviation were 3.2 and 3.9 ng/g, respectively. Heptachlor epoxide values in controls varied from below the detection limit to 1.2 ng/g serum. The exposure time (e.g. the number of years spent working with plywood sizings that contained heptachlor) correlated with the residue levels of heptachlor epoxide that were measured in serum samples obtained from employees at two companies (. = 0.03) (Mussalo-Rauhamaa et al., 1991).

6.2.1.2 General population exposure

Consumption of foods containing pesticides such as heptachlor leads to an accumulation of these compounds in human tissues. Concentrations of heptachlor and heptachlor epoxide in various human tissues and fluids from recent studies are given in Appendix 5: Table A5-11, serum and blood samples; Table A5-12, human breast milk; Table A5-13, adipose tissue; and Table A5-14, breast adipose tissue.

The most significant source of exposure of infants to heptachlor and its metabolites appears to be breast milk, in which the concentrations can be much higher than those found in dairy milk. A large international survey performed in the 1970s found that the mean concentrations of heptachlor and heptachlor epoxide in human breast milk ranged from 2 to 720 ng/g of fat (IPCS, 1984). Concentrations of heptachlor epoxide in breast milk from women in a number of countries are given in Table A5-12. Although heptachlor has not been found in all samples, it has been found in at least some samples in most surveys, showing that this organochlorine compound is a ubiquitous contaminant. Mean concentrations from recent reports are much below 100 ng/g on a milk fat basis; however, studies in some countries give noticeably higher values — e.g. Jordan (mean of 600 ng of heptachlor epoxide per gram of milk fat; Alawi & Khalil 2002) and Thailand (360 ng of heptachlor epoxide per gram of milk fat; Stuetz et al., 2001).

In a breast milk study in Victoria, Australia, a correlation was found between the levels of heptachlor epoxide in breast milk and the use of heptachlor as a termiticide (Sim et al., 1998).

Heptachlor that had been formerly sprayed on pineapple plants to control ants responsible for the spread of mealy bugs remained on the leaves that were chopped and fed to dairy cows on Oahu, Hawaii, USA. Heptachlor-contaminated "green chop" led to contamination of the commercial milk supply on Oahu for as long as 15 months during 1981–1982, at levels as high as 1.2 µg/g fat basis (Baker et al., 1991; Allen et al., 1997). Lactating women who consumed dairy products and beef from local sources had average levels of 123 ng of heptachlor epoxide per gram of fat, with a maximum concentration of >250 ng/g of fat (Baker et al., 1991). Heptachlor is usually only one of the organochlorine pesticides present in breast milk.

A compilation of data from the 1960s and 1970s indicated that the mean concentration of heptachlor epoxide in adipose tissue from the general population ranged from 10 to 460 ng/g of fat (IARC, 1991; Kutz et al., 1991). Data from more recent years still fall in this range. However, one report from Jordan gave values above 1000 ng/g of fat (Alawi et al., 1999; see Table A5-13).

6.2.2 Estimates of human exposure

The main exposure routes for heptachlor are probably via application-related inhalation or skin penetration, from extended exposure to dusts containing heptachlor in, for example, homes treated with this compound to control termites, and indirectly by uptake from food contaminated with heptachlor from crops or from other foods via the food-chain.

A study of the exposure of applicators and residents to heptachlor when used for subterranean termite control in homes indicated that applicator exposure by the dermal route to hands, forearms, and lower legs was 71.3, 29.3, and 17.1 µg/region per hour, respectively, and by the respiratory route in the breathing zone, 33.4 (range 2–176) µg/m³. Exposure of residents to heptachlor in ambient air during termiticide treatments was a maximum of 5 µg/m³, which decreased to 2.86 µg/m³ after 24 h (Kamble et al., 1992).

The most likely route of exposure of the general population to heptachlor is via food-related uptake. This is due to the fact that heptachlor will bioaccumulate in lipid phases. In particular, foods such as milk, fish, and meats, which have a higher fat content, and vegetables directly contaminated with the pesticide are likely to contain higher concentrations of heptachlor.

The estimated dietary intake of heptachlor epoxide in the 1960s and 1970s in the USA was about 0.3–2 µg/day (Duggan & Corneliussen, 1972; Peirano, 1980; IPCS, 1984). The average daily intake of heptachlor found in the United States Food and Drug Administration Total Diet Study (1986–1998) analysis was <0.0001 µg/kg body weight per day for all age/sex groups (i.e. 6–11 months; 2 years; 14–16 years, females, and 14–16 years, males; 25–30 years, females, and 25–30 years, males; 60–65 years, females, and 60–65 years, males) (Gunderson, 1995). Based on a total diet study conducted in the period April 1982 – April 1984, the estimated daily intakes of heptachlor and heptachlor epoxide for men aged 25–30 were 0.007 µg and 0.184 µg, respectively (Gunderson, 1988).

The intake of heptachlor epoxide in a Basque population in Spain in 1990–1991 was estimated to be <0.1 µg/day on average (Urieta et al., 1996).

For several countries, Kannan et al. (1997) summarized the average daily uptake values (in micrograms per person) for heptachlor and its epoxide from the available data as follows: 0.07 (India, 1989, heptachlor only), 0.08 (Thailand, 1980), 0.06 (Japan, 1992–1993), 1.1 (Australia, 1990–1992), 8.4 (Italy, 1971–1972), 0.18 (Italy, 1978–1984), 0.49 (Finland, 1983), 0.1 (USA, 1987), and 0.04 (USA, 1990).

Estimated heptachlor and heptachlor epoxide exposure from food items in Poland in 1970–1996 was calculated by multiplying annualized mean consumption rates by residue concentrations in the food. The daily dietary intakes of heptachlor and heptachlor epoxide were from 0.51 to 0.58 µg per person (Falandysz, 2003) (about 0.01 µg/kg body weight, assuming a mean weight of 64 kg). In this study, the main sources of heptachlor and heptachlor epoxide were thought to be meat, meat products, and animal fats.

However, if fish from contaminated rivers (e.g. concentrations in fish in the 0.1–1 mg/kg range reported from Ganges Estuary, Bangladesh, Bay of Bengal, and Göksu Delta, Turkey) were ingested, a much higher daily intake would be likely. This would also be the case for vegetables ingested from fields contaminated with heptachlor or contaminated milk (e.g. in the microgram per kilogram range in Dakar, Senegal; in the milligram per kilogram range in Jaipur City, Rajasthan, India).

The daily intake of heptachlor by breast-fed babies in Jordan calculated from the data of Alawi & Khalil (2002), assuming a daily milk consumption of 150 g/kg body weight and an average milk fat content of 3.1%, was a mean of 0.67 µg/kg body weight. The corresponding daily intake of heptachlor epoxide by babies in 2000 was a mean of 1.5 µg/kg body weight (Alawi & Khalil, 2002). In contrast, Rogan & Ragan (1994) calculated, on the basis of the 90th percentile of 100 ng/g fat (Table A5-12; Rogan et al., 1991), an average daily heptachlor epoxide dose of 0.003 µg/kg body weight.

Heptachlor is readily absorbed via all routes of exposure and is readily metabolized. The results in excretion studies on orally administered [¹⁴C]heptachlor in rats show that about 72% of the radioactivity was eliminated by the faeces in the form of metabolites and 26.2% as the parent compound by day 10. The major faecal metabolites include heptachlor epoxide, 1-hydroxychlordene, and 1-hydroxy-2,3-epoxychlordene (Tashiro & Matsumura, 1978). The metabolite 1-exo-hydroxychlordene epoxide was detected only in the urine of treated rats (Klein et al., 1968). In liver microsomes incubated with heptachlor, 85.8% was metabolized to heptachlor epoxide in rats, but only 20.4% in humans. Other metabolites identified in the human liver microsome system were 1-hydroxy-2,3-epoxychlordene (5%), 1-hydroxychlordene (4.8%), and 1,2-dihydroxydihydrochlordene (0.1%) (Tashiro & Matsumura, 1978). A scheme for the metabolism in rats is given in Figure 2.

Fig. 2: Metabolic scheme for heptachlor in rats [1-hydroxychlordene
is also designated as 1-exohydroxychlordene; 1-hydroxy-2,3-exo-epoxychlordene
is also designated as 1-exohydroxy-2,3-exo-epoxychlordene]
(from Tashiro & Matsumura, 1978).

Heptachlor epoxide is metabolized slowly and is the most persistent metabolite; it is stored mainly in adipose tissue, but also in liver, kidney, and muscle (Radomski & Davidow, 1953). The concentrations of heptachlor epoxide in serum, adipose tissue, bile, and gallstone of a pesticide worker were 3, 400, 1, and 1 ng/g, respectively (Paschal et al., 1974). The mean ratio between concentrations of heptachlor epoxide in fat and blood was 880 for 10 workers at a technical heptachlor plant (Nisbet, 1986). The bioaccumulation factors (mg/g in fat / mg/g in diet) in the tissues of rats were 1.0 for males and 5.0 for females (Adams et al., 1974). Studies in male dogs indicated an average bioaccumulation factor for heptachlor epoxide of 22 (Radomski & Davidow, 1953). After prolonged exposure to heptachlor epoxide, cattle had bioaccumulation factors of 5 or more in males and 10–25 in females (Nisbet, 1986).

Heptachlor epoxide accumulated rapidly in the fat of rats fed heptachlor at a level of 30 mg/kg in the diet for 12 weeks. The maximum concentration in the fat was reached in 2–4 weeks. A period of 12 weeks was required for complete disappearance from the fat after discontinuing heptachlor feeding. The concentration of heptachlor epoxide in the fat bears a relationship to the concentration of heptachlor in the diet (Radomski & Davidow, 1953). In female rats, the amount of heptachlor and heptachlor epoxide found in milk, blood, fat, and tissues was proportional to the dose of heptachlor administered (Smialowicz et al., 2001). Heptachlor was not detected in rat pup tissues, but heptachlor epoxide was detected in fat, brain, liver, and plasma at concentrations proportional to the dose administered (Moser et al., 2001).

The presence of heptachlor epoxide in the adipose tissue of stillborn infants (Wassermann et al., 1974) and in the cord blood of newborns (D’Ercole et al., 1976) demonstrates placental transfer of heptachlor and/or heptachlor epoxide.

Acute toxicity studies of heptachlor and heptachlor epoxide in several animal species are reviewed in IPCS (1984) and FAO/WHO (1967) and summarized in JMPR (1992). Acute oral LD₅₀s for heptachlor for the rat and mouse are 40–162 and 68–90 mg/kg body weight, respectively. Dermal LD₅₀s for heptachlor for the rat are 119–250 mg/kg body weight (IPCS, 1984). The symptoms associated with acute heptachlor toxicity include hyperexcitability, tremors, convulsions, and paralysis.

The acute toxicity of heptachlor epoxide is greater than that of heptachlor; for example, the intravenous lethal doses for heptachlor and heptachlor epoxide are 40 and 10 mg/kg body weight, respectively (IPCS, 1984). However, four other heptachlor metabolites (chlordane, 3-chlordene, 1-hydroxychlordene, and chlordane epoxide) were found to have a much lower toxicity, with acute oral LD₅₀ values of greater than 4600 mg/kg body weight (Mastri et al., 1969

Groups of 10 Charles River CD-1 mice per sex per dose were fed a mixture of heptachlor/heptachlor epoxide (1:3) at dietary concentrations of 1, 5, 10, 25, and 50 mg/kg for 30 days (Wazeter et al., 1971a). Nine males and eight females of the highest dose group and one female in the 25 mg/kg group died. No marked differences were seen between the treated animals and the controls with respect to body weights and food consumption. Liver enlargement in the 10, 25, and 50 mg/kg groups in both sexes was associated with accentuated lobulation. Histopathological findings showed enlargement of centrilobular and midzonal hepatocytes in these groups and in the 5 mg/kg female dose group. The NOAEL was given as 1 mg of heptachlor per kilogram diet [NOAEL = 0.13 mg/kg body weight per day].

In a study to investigate the effect of heptachlor on the development of the reproductive system (see section 8.5), pregnant Sprague-Dawley rats were administered heptachlor by oral gavage at doses of 0, 0.5, and 5.0 mg/kg body weight per day from GD 8 through PND 21, which was the day of weaning (. = 7–8 per group). Two of the dams in the 5.0 mg/kg body weight per day group died. Pups in the highest dose group weighed significantly less than those in the other two groups on PND 0. All but one litter of the 5.0 mg/kg body weight per day group died within the first 4 postnatal days (Lawson & Luderer, 2004).

Groups of eight female Fischer 344 rats were dosed daily (0, 2, or 7 mg/kg body weight per day) by oral gavage for 14 consecutive days. Hepatocytomegaly was seen at 2 mg/kg body weight per day in two out of eight rats and all seven surviving rats in the 7 mg/kg body weight per day group. These doses also increased liver weight and decreased thymus weight (Berman et al., 1995; see also studies on reproductive effects [Narotsky & Kavlock, 1995] in section 8.5 and on neurobehavioural effects [Moser et al., 1995] in section 8.6) [LOAEL = 2 mg/kg body weight per day].

Two dogs given heptachlor dissolved in corn oil orally at 5 mg/kg body weight per day died within 21 days, whereas three of four dogs given heptachlor dissolved in corn oil orally at 1 mg/kg body weight per day died within 424 days (Lehman, 1952).

Three dogs given heptachlor epoxide orally in dosages of 2, 4, and 8 mg/kg body weight per day for 5 days per week died after 22, 10, and 3 weeks, respectively. Daily oral doses of 0.25 and 0.5 mg/kg body weight did not cause illness during 52 weeks, but 0.25 mg/kg body weight, estimated to be equivalent to 6 mg/kg in the diet, was reported as the minimal dose producing a pathological effect (Velsicol Corporation, 1959).

8.3.1 Neoplastic effects

Technical-grade heptachlor (72 ± 3% heptachlor, 18% trans-chlordane, 2% cis-chlordane, 2% nonachlor, 1% chlordene, 0.2% hexachlorobutadiene, and 10–15 other compounds) was tested for carcinogenicity using both sexes of Osborne-Mendel rats and B6C3F1 mice (NCI, 1977). Heptachlor was administered in the diet for 80 weeks using two separate dose levels for each sex and species. Owing to the observable toxic effects, the dose levels were changed during the course of the study. The time-weighted average doses³ were 39 and 78 mg/kg for male rats and 26 and 51 mg/kg for female rats. The average body weights of rats treated with high doses were consistently lower than those of untreated controls, while body weights of low-dose rats were unaffected. Mortality was dose related for female rats but not for male rats. No hepatic tumours were observed in the rats.

For mice, the time-weighted average doses were 6 and 14 mg/kg for the males and 9 and 18 mg/kg for the females. Mortality was dose related for female mice but not for male mice. A review of the liver samples from this study by the panel of the United States National Academy of Sciences (NAS, 1977) indicated a significant increase in the combined incidence of hepatocellular carcinoma and "nodular changes" in males and females receiving the higher concentration (see Table 2).

Table 2: Tumour incidence in B6C3F1 mice treated with technical-grade heptachlor.^a

An oral carcinogenicity study on heptachlor and its epoxide was carried out by the United States Food and Drug Administration in 1965 and was later published in a summarized form (Epstein, 1976). Three groups of 100 male and 100 female C3H mice were fed diets containing heptachlor at 0 or 10 mg/kg or heptachlor epoxide at 10 mg/kg for 24 months. A review of the histopathology of liver samples by the United States National Academy of Sciences (NAS, 1977) indicated a significant increase in the incidence of hepatocellular carcinomas in females but not in males given heptachlor and in both males and females given heptachlor epoxide. There was also a significant increase in the combined incidence of carcinomas and nodules in both males and females given heptachlor or heptachlor epoxide (IARC, 2001; see Table 3).

Table 3: Tumour incidence in C3H mice treated with heptachlor or heptachlor epoxide.^a

^a From Epstein (1976); NAS (1977); IARC (2001).

In a further study on heptachlor and its epoxide conducted by the International Research and Development Corporation in 1973 and later published in a summarized form (Epstein, 1976), groups of 100 male and 100 female CD-1 mice were fed diets containing a mixture of 75% heptachlor epoxide and 25% heptachlor at a concentration of 0, 1, 5, or 10 mg/kg for 18 months. After exclusion of 10 animals from each group that were killed for interim study at 6 months, the mortality rate at 18 months was 34–49%, with the exception of the group receiving the highest dose, for which the rate was about 70%. A review of the histopathology of liver samples from this study by the United States National Academy of Sciences (NAS, 1977) indicated a significant increase in the combined incidence of hepatocellular carcinomas and nodules in the groups at the highest concentration (Table 4).

Table 4: Tumour incidence in CD-1 mice treated with a mixture of heptachlor and heptachlor epoxide (25%:75%).^a

^a From NAS (1977); IARC (2001).

8.3.2 Administration with known carcinogens

Groups of male B6C3F1 mice, 8 weeks of age, were given drinking-water containing the tumour initiator .-nitrosodiethylamine at 0 or 20 mg/l for 14 weeks. After 4 weeks with no treatment, mice received diets containing technical heptachlor at 0, 5, or 10 mg/kg for 25 weeks. All surviving animals were killed at 43 weeks; five mice from each group were sacrificed after 8 and 16 weeks’ administration of heptachlor. The effect of .-nitrosodiethylamine was assessed by the presence of altered foci displaying abnormalities in glucose-6-phosphatase. The incidence of hepatocellular adenomas and carcinomas was significantly increased with heptachlor compared with .-nitrosodiethylamine alone (Williams & Numoto, 1984; see Table 5). Heptachlor showed tumour promoter activity.

Table 5: Preneoplastic and neoplastic liver lesions in B6C3F1 mice treated with heptachlor in the diet after initiation with .-nitrosodiethylamine (NDEA).

8.3.3 Non-neoplastic effects

Groups of beagle dogs (four per sex per dose) were fed heptachlor epoxide at concentrations of 0, 1, 3, 5, 7, and 10 mg/kg in the diet for 2 years (Wazeter et al., 1971b). After this time, two dogs per sex per dose were sacrificed and necropsied, while the other two dogs per sex per dose were maintained on the control diet for an additional 6 months. In addition, the test animals were also mated during the study and employed as the P1 parental animals for a two-generation reproduction and teratology study. Neither deaths nor compound-related behavioural changes were seen during the study. No marked differences were seen between the treated animals and the controls with respect to body weights and food consumption. There were increases in alkaline phosphatase activities in males and females at the 3 mg/kg dose and above; these increases, in some dogs, were more marked towards the end of the treatment period and tended to persist through the recovery period. The serum albumin and total protein levels were slightly decreased in 10 mg/kg dose male and female dogs during the treatment, extending into the recovery period. After 1 year of treatment, the animals in the 7 mg/kg group also showed an increase in the alanine aminotransferase level, which lasted into the recovery period.

There was an increase in liver weights in the 10 mg/kg male and female dogs relative to those of the controls, and this increase persisted with a slight attenuation during the recovery period.

Histopathological examination of the dogs (two dogs per sex per dose) sacrificed at the end of the treatment period showed an increase in the incidence of liver changes (e.g. enlargement and vacuolation of groups of centrilobular hepatocytes) in animals at 3 mg/kg or above. These changes were also noted in the dogs at 3 mg/kg and above after 6 months of recovery. No compound-related histopathological changes were seen in the 1 mg/kg dogs. Based upon changes in biochemical parameters and the histological changes in the liver, the NOAEL was given as 1 mg/kg diet [NOAEL = 0.025 mg/kg body weight per day] (Wazeter et al., 1971b).

The genotoxicity of heptachlor and heptachlor epoxide has been reviewed by IARC (1991, 2001), where further details of the studies are to be found (see also Appendix 6).

Heptachlor did not induce DNA damage or point mutations in bacteria (Simmon et al., 1977; Griffin & Hill, 1978; Probst et al., 1981; Gentile et al., 1982; Moriya et al., 1983; Rashid & Mumma, 1986; Zeiger et al., 1987; Mersch-Sundermann et al., 1988; Matsui et al., 1989), gene conversion in Saccharomyces cerevisiae (Gentile et al., 1982), or sex-linked recessive lethal mutations in Drosophila melanogaster (Benes & Šram, 1969). It did not induce unscheduled DNA synthesis in cultured rat, mouse, or hamster hepatocytes in the absence of metabolic activation (Maslansky & Williams, 1981; Probst et al., 1981; Williams et al., 1989); however, there was a statistically significant increase in unscheduled DNA synthesis in human fibroblasts in the presence of metabolic activation (Ahmed et al., 1977). Heptachlor induced gene mutations at the Tk (McGregor et al., 1988) but not at the Hprt locus in rodent cells (Telang et al., 1982). It inhibited gap junctional intercellular communication in cultured rodent and human cells (Telang et al., 1982; Ruch et al., 1990; Nomata et al., 1996).

IARC (2001) noted that it is aware of unpublished studies by the United States National Toxicology Program on the effect of heptachlor on sister chromatid exchange (weak positive with rat liver S9) and chromosomal aberrations (negative).

Heptachlor did not induce dominant lethal mutations in mice in vivo (Epstein et al., 1972; Arnold et al., 1977). It showed negative results for lacI mutations in liver DNA in C57BL/6 (Big Blue^®) transgenic mice (Gunz et al., 1993).

Heptachlor epoxide did not induce forward mutation, mitotic crossing-over, or aneuploidy in Aspergillus (Crebelli et al., 1986) or reverse mutation in Salmonella typhimurium (Marshall et al., 1976). However, there was a statistically significant increase in unscheduled DNA synthesis in human fibroblasts in vitro in the presence of metabolic activation (Ahmed et al., 1977), and heptachlor epoxide inhibited gap junctional intercellular communication in rat liver (Matesic et al., 1994) and human breast epithelial cells in vitro (Nomata et al., 1996), without metabolic activation.

8.5.1 Effects on fertility

Male Sprague-Dawley rats (five per group) were injected subcutaneously every other day with heptachlor in corn oil at 5, 10, 15, 20, or 25 mg/kg body weight per day for 2 weeks. The rats did not show any adverse clinical signs throughout the treatment period. There was no clear dose-related effect of heptachlor on body weight. Plasma testosterone levels were suppressed (. < 0.05) and the plasma luteinizing hormone level was elevated (. < 0.01) at all doses, but the changes were not dose related. Cortisol levels were significantly elevated (. < 0.02) compared with control rats. The testes in the group treated with 25 mg/kg body weight per day showed some pathological changes (Wango et al., 1997).

Adult female Sprague-Dawley rats (10 in each dose group, 20 controls) were injected subcutaneously with corn oil or heptachlor solution at 5 or 20 mg/kg body weight every other day for up to 18 days (Oduma et al., 1995a). Animals in the lower dose group showed some disruptions in cyclicity, but the effects were more pronounced with 20 mg/kg body weight. Disruptions became more marked with continued treatment. Heptachlor affected body weight gain in a dose-related manner. In the second part of the study, female rats (10 per dose group) were injected with saline, corn oil, or heptachlor at 5 or 20 mg/kg body weight as above. Heptachlor caused a delay in mating behaviour in a dose-related manner. In summary, effects including disruptions in cyclicity and delayed mating behaviour were seen at all tested doses >5 mg/kg body weight.

Adult female Sprague-Dawley rats (10 per group) were injected subcutaneously with corn oil or heptachlor solution at 5, 20, 25, or 30 mg/kg body weight every other day for up to 18 days (total of nine injections). The stage of the estrous cycle was determined by vaginal smears a day following the last injection, and the rats were sacrificed. Blood samples were taken by cardiac puncture and assayed for progesterone and estrogen. A suppression of blood progesterone and estradiol levels was observed, depending on the dose and stage of the estrous cycle. Ovarian cells from rats treated with low doses of heptachlor (5 mg/kg body weight) showed an increased production of progesterone, whereas high doses (>20 mg/kg body weight) suppressed production (Oduma et al., 1995b). In summary, effects were seen at all tested doses >5 mg/kg body weight.

Prenatal toxicity studies were also carried out in albino CFT Wistar rats dosed by gavage at 0, 45.3, or 90.5 mg/kg body weight for 70 days prior to mating for males and at 25 or 50 mg/kg body weight for 14 days prior to mating for females (Amita Rani et al., 1992, 1993; Amita-Rani & Krishnakumari, 1995). However, the doses chosen resulted in high morbidity, and the studies are not described further here.

Groups of 6- to 9-month-old beagle dogs (four per sex per dose) were fed heptachlor epoxide at dietary concentrations of 0, 1, 3, 5, 7, and 10 mg/kg for 2 years (Wazeter et al., 1971c; JMPR, 1992; see also Wazeter et al., 1971b, in section 8.3.3). When the female dogs reached the age of 14 months, they were mated twice with male dogs from the same dose group. The females were allowed to deliver and to nurse their pups. For the F2 generation, four female and two male pups of the F1 generation were selected from each dose level to be the parental animals (P2) of the F2 generation. At an approximate age of 14 months, the animals were mated, and the pregnant females were allowed to deliver and to nurse their pups to 6 weeks of age, when the females and their pups were sacrificed. Neither deaths nor compound-related behavioural changes were seen in treated P1 or P2 animals or their offspring during the study. No marked differences were seen between the treated animals and the controls with respect to body weights and food consumption. Owing to the small number of animals and the limited data, it could not be determined whether heptachlor epoxide produced any reproductive effects.

8.5.2 Developmental toxicity

In the beagle dog study described in the previous section (Wazeter et al., 1971c), there was a significant increase in the mortality rate of F1 pups in the 10 mg/kg group. Only one male pup survived to scheduled sacrifice, and no female was available to serve as a P2 parental animal. There were slight increases in death rates of F2 pups at 3 and 7 mg/kg relative to the controls. The 5 mg/kg group had no pups. The limited necropsy data showed that in F2 pups, 1/4 females at 7 mg/kg and 3/10 males and 3/7 females at 10 mg/kg had pale or greasy liver. This observation was compound related. Based on mortality rate, the NOAEL was given as 1 mg/kg diet (Wazeter et al., 1971c; JMPR, 1992).

Three reproduction studies in rats were evaluated by JMPR in 1966 and 1970 (FAO/WHO, 1967, 1971). In a three-generation reproduction study, a group of 80 rats was given heptachlor at 6 mg/kg in the diet daily for 3 months prior to mating. The only effect on reproduction was a decrease in the litter size (Mestitzova, 1966, 1967). In two other reproduction studies in rats, dietary levels of heptachlor ranging from 0.3 to 10 mg/kg were tested. No adverse effects on fertility or reproduction were seen at 10 mg/kg diet. A slight increase in postnatal mortality of pups was seen at 10 mg/kg (FAO/WHO, 1967).

Pregnant female Sprague-Dawley rats were dosed by gavage with heptachlor (0, 0.03, 0.3, or 3 mg/kg body weight per day) from GD 12 to PND 7, followed by direct dosing of the pups with heptachlor through PND 42. The doses were set so that the low dose, 0.03 mg/kg body weight per day, produced heptachlor and heptachlor epoxide levels in rat dam milk that matched the 95th percentile of human milk values on Oahu, Hawaii, USA, in 1981 (Siegel, 1988). Evaluation of the effects on the reproductive system included the following: monitoring the development of the reproductive system in males and females (i.e. anogenital distance at birth, age at vaginal opening, age at preputial separation); vaginal cytology monitoring over a 2-week period to assess cyclicity; two mating trials with untreated mates; and a necropsy, including organ weights and histology, measures of epididymal sperm motility and count, and testicular spermatid counts. Necropsies were also performed at the end of dosing (PND 46), and again in the adults, to assess organ weight and histology (liver, kidneys, adrenals, thymus, spleen, ovaries, uterus/vagina, testes, epididymides, seminal vesicles/coagulating glands, and ventral and dorsolateral prostate (Smialowicz et al., 2001).

There were no effects on the number or survival of pups born to heptachlor-exposed dams or to pups exposed postnatally. There were no effects on the number of treated dams delivering litters or on litter size, nor were there any effects on any of the reproductive end-points examined in the F0 or F1 rats. There was no detectable histopathology in any tissue examined. There were no changes in the fertility of adult males or females when mated to untreated partners. Body weights and somatic and reproductive organ weights of males and females exposed to heptachlor were unchanged at terminal necropsy (Smialowicz et al., 2001) [NOAEL = 3 mg/kg body weight per day, the highest dose tested].

Pregnant Fischer-344 rats were treated by gavage with vehicle or heptachlor (0, 4.5, or 6 mg/kg body weight per day) on GD 6–19 (Narotsky & Kavlock, 1995). The dams were allowed to deliver, and their litters were examined on PND 1, 3, 6, and 21. Litter weights were determined on PND 1, 6, and 21. Implants were also counted to determine prenatal loss. Heptachlor caused reduced maternal weight gains (reduced extrauterine weight gains) at both dose levels. Heptachlor at these doses had no effect on either pre- or postnatal survival. Pup weights were significantly reduced at both dose levels on PND 6 only but were comparable among treatment groups at PND 21 [maternal LOAEL for weight gain = 4.5 mg/kg body weight per day; NOAEL for pre- or postnatal survival of pups = 6 mg/kg body weight per day].

In another study in Fischer-344 rats (Narotsky et al., 1995), heptachlor was tested at doses of 0, 5.1, 6.8, 9.0, and 12.0 mg/kg body weight per day by gavage on GD 6–15. There were no clinical signs of maternal toxicity (dose-related alterations in weight gain) up to 9.0 mg/kg body weight per day (at 12.0 mg/kg body weight per day, 5 of 13 treated died), only a slight potential for developmental toxicity (reduced postnatal growth at 6.8 mg/kg body weight per day and above), and dramatically increased postnatal mortality (at 9.0 and 12.0 mg/kg body weight per day) [maternal NOAEL for weight gain = 6.8 mg/kg body weight per day for 9 days; NOAEL for postnatal growth = 5.1 mg/kg body weight per day (note postnatal mortality at 9.0 and 12.0 mg/kg body weight per day)].

Sprague-Dawley rats received oral doses of heptachlor during different developmental periods: 0, 4.2, and 8.4 mg/kg body weight per day for perinatal and gestational studies, and 0, 0.3, 1.0, and 3.0 mg/kg body weight per day for perinatal/adolescent studies (Purkerson-Parker et al., 2001). There were dose-related decreases in maternal weight gain and pup survival as well as delayed righting reflex at heptachlor doses >3.0 mg/kg body weight per day (see also section 8.6).

In a study to investigate the effect of heptachlor on the development of the reproductive system, pregnant Sprague-Dawley rats were administered heptachlor by oral gavage at doses of 0, 0.5, and 5.0 mg/kg body weight per day from GD 8 through PND 21, which was the day of weaning (. = 7–8 per group). Litters were standardized to four males and four females on the day of birth. Two of the dams in the 5.0 mg/kg body weight per day group died. Pups in the highest dose group weighed significantly less than those in the other two groups on PND 0. All but one litter of the 5.0 mg/kg body weight per day group died within the first 4 postnatal days. Age at eye opening was delayed with increasing heptachlor dose. There was no effect of treatment on pup weight gain in the surviving litters. There was no effect of treatment on anogenital distance, age at puberty, nipple retention past the infantile period in males, estrous cycling, serum sex steroid concentrations, reproductive organ weights, or testicular or ovarian histology, suggesting that heptachlor exposure during gestation and lactation does not disrupt development of the reproductive system in rats (Lawson & Luderer, 2004).

In the study by Oduma et al. (1995a) described above (section 8.5.1) in which female rats were injected subcutaneously every other day with heptachlor at 5 or 20 mg/kg body weight before mating, the weights of all pregnant animals increased with age of gestation but were not significantly different from the controls. Animals treated with 20 mg/kg body weight had longer mean gestational lengths of 25 ± 2.9 days, which were significantly different from those of the corn oil–treated controls (. < 0.05) of 22.7 ± 0.5 days, but not significantly different from rats treated with heptachlor at 5 mg/kg body weight. All pups were born alive, but the proportion of pups still alive by weaning time was lower for rats treated with 20 mg/kg body weight than for the other groups [NOAEL for litter size and gestational length = 5 mg/kg body weight (note postnatal mortality)].

Similar results were found in a dietary study on the reproductive function in mink (Mustela vison) (Crum et al., 1993). Maternal mortality was 0, 8, 67, and 100% for doses of 0, 6.25, 12.5, and 25 mg/kg in diet. Survival of kits was adversely affected in the two upper dose groups. Even at the lowest dose (6.25 mg/kg in diet), kit body weights were significantly less than for the control kits.

There is accumulating evidence that nervous system development is influenced by cyclodiene pesticides. Cyclodiene pesticides have been shown to act on the GABA_A receptor, by binding at the chloride channel portion of the receptor and thereby blocking the inhibitory actions of the transmitter GABA. Heptachlor epoxide was more potent than heptachlor in the inhibition of the GABA-induced ³⁶Cl⁻ influx in rat brain (Gant et al., 1987). Acute actions of cyclodiene pesticides (e.g. heptachlor) include excitation, hyperstimulation, and convulsions (see section 8.1).

In a study on the neurobehavioural effects of heptachlor, female Fischer 344 rats (eight per dose level) were orally administered doses of 7, 23, 69, and 129 mg/kg body weight (Moser et al., 1995). No lethality occurred with single acute dosing. With repeated dosing, however (doses of 2, 7, 23, and 69 mg/kg body weight per day for 14 days), all rats receiving the two highest doses died during dosing, and one rat in the next lowest dose (7 mg/kg body weight per day) died shortly after the last dose. A neurobehavioural screening battery consisting of a functional observational battery and automated motor activity measurements was used to evaluate the neurotoxic potential of these chemicals. The magnitude of acute heptachlor effects on activity and excitability was greatest 4 h after dosing, and excitability changes were also observed at 24 h. The profile of effects produced by repeated heptachlor administration consisted of altered activity, hyperexcitability, and autonomic effects [NOAEL = 2 mg/kg body weight per day for a 14-day exposure].

In a study to investigate the neurological effects of perinatal heptachlor exposure, doses were set so that the low dose (0.03 mg/kg body weight per day) produced heptachlor and heptachlor epoxide levels in rat dam milk that matched the 95th percentile of human milk values on Oahu, Hawaii, USA, in 1981 (Siegel, 1988). Pregnant Sprague-Dawley dams were dosed by gavage from GD 12 to PND 7, whereupon the rat pups were dosed directly by gavage until PND 21 (group A) or PND 42 (group B). Dose levels were 0, 0.03, 0.3, or 3 mg/kg body weight per day (Smialowicz et al., 2001; see section 8.5). For the neurotoxicological evaluations, one male and one female from each of 10 litters were used for each dose group. The studies included screening evaluations (functional observational battery, motor activity, development of righting reflex), cognitive tests (associative and non-associative learning, spatial learning and memory, and working memory), and measures of GABA_A receptor function and expression (Moser et al., 2001). The neurotoxicological outcomes of perinatal heptachlor exposure in the rat suggested developmental delays, alterations in GABAergic neurotransmission, and neurobehavioural changes, including cognitive deficits. Females were somewhat more affected, as were rats dosed until PND 42. Heptachlor had the most profound effects on cognitive function (slowed acquisition of the spatial task and impaired recall during probe trials), and these effects on probe trials were significant even at the lowest dose level [LOAEL = 0.03 mg/kg body weight per day].

There is increasing concern that environmental factors such as exposure to pesticides, and in particular to cyclodienes, may play a role in the onset of Parkinson’s disease (Priyadarshi et al., 2000). Changes in biochemical status of nerve terminals in the corpus striatum, one of the primary brain regions affected in Parkinson’s disease, were studied in groups of retired breeder male C57BL/6 mice treated by intraperitoneal injection of heptachlor 3 times over a 2-week period at doses of 3, 6, 12, 25, 50, or 100 mg/kg body weight (Kirby et al., 2001). No outward signs of intoxication or death were observed in mice given heptachlor at <25 mg/kg body weight. However, some mice treated at heptachlor doses of 50 or 100 mg/kg body weight showed hyperexcitability and became convulsive, which resulted in the death of some animals. The dopamine transporter normally functions to allow rapid uptake of dopamine and is therefore important in regulating dopamine actions. Dopamine transporter binding decreases with age and is additionally decreased due to neuronal loss in Parkinson’s disease (e.g. Fischman et al., 1998). On average, the maximal rate of striatal dopamine uptake increased >2-fold in mice treated at heptachlor doses of 6 mg/kg body weight and 1.7-fold at 12 mg/kg body weight, which was attributed to the induction of dopamine transporter. At higher dose levels, no increase in maximal dopamine intake was seen, which was suggested to be due to the toxic effects of heptachlor epoxide (Kirby et al., 2001). The dopaminergic system seems to be specifically sensitive to heptachlor, because no effects on serotonergic pathways were seen.

In an earlier study, retired breeder male C57BL/6 mice (8–10 months old) were treated intraperitoneally with heptachlor (0, 3, 6, 9, or 12 mg/kg body weight) administered 3 times over a 2-week period (Miller et al., 1999). An increase in both plasma membrane dopamine transporter as well as the vesicular monoamine transporter was reported in the striatum of the mice.

The dopamine transporter may also play an important role in neuronal development, appearing first at GD 14 (Fauchey et al., 2000). Therefore, changes in dopamine transporter number and dopamine levels may affect neuronal development early on and may play a role in the etiology of Parkinson’s disease later in life. To investigate this, Sprague-Dawley rats received oral doses of heptachlor during different developmental periods: 0, 4.2, or 8.4 mg/kg body weight per day for perinatal and gestational studies, and 0, 0.3, 1.0, or 3.0 mg/kg body weight per day for perinatal/adolescent studies (Purkerson-Parker et al., 2001). There were dose-related decreases in maternal weight gain and pup survival as well as delayed righting reflex at heptachlor doses >3.0 mg/kg body weight per day. Gestational, perinatal, and/or adolescent exposure to heptachlor produced an increase in dopamine transporter binding in the striatum as early as PND 10, and this change persisted into adulthood.

In a study on the effects of perinatal/juvenile heptachlor exposure (0, 0.03, 0.3, or 3 mg/kg body weight per day) in rats (see section 8.5; Smialowicz et al., 2001), heptachlor-exposed offspring were evaluated for a variety of innate and specific immune function end-points at 8 weeks of age (i.e. 2 weeks after cessation of dosing) and older. Perinatal/juvenile exposure of male and female rats to heptachlor did not alter spleen weight or cellularity or thymus weight, nor did it affect ex vivo immune function tests (i.e. splenic lymphoproliferative responses to mitogens of allogeneic cells and splenic natural killer cell activity) in a mixed lymphocyte response assay at 8 weeks of age. Further, in vivo delayed-type hypersensitivity and contact hypersensitivity were not affected by heptachlor exposure at 10 or 17 weeks of age, respectively. However, the primary IgM response to anti-sheep red blood cells was suppressed in male, but not in female, rats in a dose-related manner at 8 weeks of age. The percentage of B lymphocytes (OX12⁺OX19⁻) in spleen was also reduced in high-dose males. This suppression of antibody responses persisted for up to 20 weeks after the last exposure to heptachlor at a total heptachlor exposure of approximately 1.5 mg/kg body weight per rat. At 26 weeks of age, the secondary IgG antibody response to sheep red blood cells was suppressed in all of the heptachlor-exposed males but not females. The T cell–dependent antibody response to sheep red blood cells has been demonstrated to be one of the most commonly affected and most sensitive functional parameters in animals exposed to chemical immunosuppressants, requiring the interaction of three major immune cell types (i.e. macrophage, T-helper cell, and B cell; Luster et al., 1992).

Immunomodulatory effects have been shown by heptachlor in peripheral blood mononuclear cells from blood samples from male rhesus monkeys. Heptachlor at 80 µmol/l completely suppressed the proliferation and IL-2 release of the monkey lymphocytes (Chuang et al., 1992). Heptachlor inhibited the chemokine-induced chemotaxis of monkey neutrophils and monocytes at concentrations as low as 10⁻¹⁴–10⁻⁵ mol/l (Miyagi et al. 1998). This migration of neutrophils and monocytes towards chemokines normally plays a profound role in the body’s innate immune response towards infections (Chuang et al., 1999).

In rodent studies, heptachlor has a sharp dose–response curve for mortality (see section 8.2) without showing outward clinical signs of toxicity (with the exception of neurotoxic effects). At much lower doses, immunological and neurological effects have been shown. Heptachlor is a non-genotoxic carcinogen. Heptachlor shows tumour-promoting characteristics and down-regulates the tumour suppresser gene p53. There is increasing evidence that heptachlor in vitro causes inhibition of gap junctional intercellular communication. The following are recent studies into the mode of action of heptachlor.

Heptachlor triggered significant proliferation in quiescent rat hepatocytes (Okoumassoun et al., 2003). The key kinases that are a part of the signalling pathways known to be involved in cell proliferation were investigated. Exposure to heptachlor led to activation of protein kinase C MAPKs (Okoumassoun et al., 2003). This supports earlier studies that showed that chlordane stimulated protein kinase C activity in the rat brain (Bagchi et al., 1997).

Heptachlor (80 µmol/l) has been shown to decrease ras expression in human myeloblastic leukaemia (ML-1) cells (Chuang & Chuang, 1991). Studies on the signal transduction pathway using cultured human lymphocytes have shown that heptachlor down-regulated the tumour suppressor retinoblastoma (Bb) protein (Rought et al., 1999) and down-regulated p53 gene expression (Rought et al., 1998). Heptachlor exposure reduced the cellular levels of MAPK cascade proteins, which are important intermediates in the signal transduction pathway of immune cells (Chuang & Chuang, 1998). The activation of MAPKs may be one of the pathways used by heptachlor to exert its mitogenic action (resulting, for example, in cancer promotion).

Studies were undertaken to link cell cycle events and signal transduction pathways within heptachlor-treated cells. Heptachlor was found to block the cell cycle by preventing progression into S phase with a concomitant accumulation of cells in G1 phase; this is associated with a decrease (deactivation) in cyclin-dependent kinase cdk2 and dephosphorylation (activation) of cyclin-dependent kinase cdc2. The altered cell cycle progression may trigger the cell’s apoptotic potential, as indicated by the reduced amount of the anti-apoptotic protein Bcl-2 synthesized inside heptachlor-treated cells (Chuang et al., 1999).

Heptachlor strongly inhibited transforming growth factor beta–induced apoptosis and cytochrome c release into the cytosol in quiescent rat hepatocytes. The levels of Bcl-2 were also increased in the presence of heptachlor (Okoumassoun et al., 2003).

Two biochemical entities thought to be associated with promotion of liver cancer are the phosphoinositide signal transduction pathway and AP-1 nuclear transcription factors, of which protein kinase C is a critical enzyme. In vivo exposure of B6C3F1 male mice to heptachlor epoxide at 1, 10, or 20 mg/kg in the diet has been shown to selectively down-regulate particulate novel protein kinase C epsilon in B6C3F1 male mouse liver tissue, while persistently up-regulating AP-1. DNA binding activity (a critical factor in tumour promotion) was substantially increased at 3 and 6 h with 3.7 mg/kg intraperitoneal heptachlor epoxide and at 3 and 10 days with 20 mg/kg dietary heptachlor epoxide (Hansen & Matsumura, 2001a). Studies into the effects of heptachlor epoxide in mouse 1c1c7 hepatoma cells showed that many hepatocellular effects or changes occurred, suggesting a cellular programme shift. The tyrosine kinase growth factor receptor pathway seemed to be the probable critical pathway for heptachlor-induced tumour promotion, with the critical target most likely being upstream of PLC_gamma1 and AP-1 (Hansen & Matsumura, 2001b).

Rought et al. (2000) reported that heptachlor by itself was able to stimulate apoptosis protease CPP32 at relatively high concentrations. When combined with the chemotherapeutic agent doxorubicin, a known CPP32 activator, a dual effect was noted. Low concentrations of heptachlor (5–10 µmol/l) suppressed doxorubicin-induced CPP32 activity, and high concentrations of heptachlor (80–120 µmol/l) augmented it. Rought et al. (2000) demonstrated that heptachlor has tumour promoting–like effects at lower concentrations and at higher concentrations induces apoptosis as a mechanism of toxicity.

Only a few of the studies that were analysed by IARC (2001) on the effects of chlordane and heptachlor mentioned or included exposure to heptachlor or heptachlor epoxide. These studies are summarized below. Further details are given in IARC (2001).

9.1.1 Cross-sectional studies