SAFETY EVALUATION OF CERTAIN
FOOD ADDITIVES AND CONTAMINANTS

Coplanar PCBs may induce their own metabolism. In rodents exposed to relatively high doses of 2,3,4,7,8-TCDF and TCDD, a twofold induction of their metabolism was observed (Brewster & Birnbaum, 1987; Leung et al., 1990b; McKinley et al., 1993). Similarly, clear biphasic elimination of 2,3,4,7,8-PeCDF and 1,2,3,4,7,8-HxCDF was observed in Yu-cheng and Yusho patients, indicating that they had been exposed to concentrations well above background for induction of metabolism (Ryan et al., 1993a). The metabolism of TCDD was found to be substantially induced in two patients with TCDD poisoning, the half-lives being 200 and 230 days (Geusau et al., 1999).

TCDD readily crossed the placenta of pregnant Long-Evans rats given a single oral dose of TCDD at 1.2 µg/kg bw in corn oil on day 8 of gestation. The concentrations of TCDD found in the fetal compartment (fetuses plus their placentae) were 39 pg/g (0.01% of the administered dose) on day 9 of gestation, when the maternal blood concentration was 15 pg/g; 26 pg/g (0.11% of the administered dose) on day 16, with a maternal blood concentration of 18 pg/g; and 21 pg/g (0.7% of the administered dose on day 21, with a maternal blood concentration of 8 pg/g. In individual embryos, the TCDD concentrations were 40, 18 and 22 pg/g on days 9, 16 and 21. The embryo/fetal compatment may therefore be considered a nonsequestering maternal compartment (Hurst et al., 1998).

Pregnant Long Evans rats received a single oral dose of TCDD at 0.05, 0.2, 0.8, or 1 µg/kg bw in corn oil on day 15 of gestation. On day 16, these doses resulted in concentrations of 6.8, 15, 50 and 61 pg/g in the fetal compartment and 5.3, 13, 39 and 56 pg/g in single, whole fetuses, with associated maternal body burdens of 31, 97, 520 and 580 pg/g. On day 21 of gestation, the concentration of TCDD were 4.3, 14, 32 and 37 pg/g in the fetal compatment, 4.3, 15, 32 and 36 pg/g in single, whole fetuses and 27, 76, 330 and 430 pg/g in the maternal body. On day 16 of gestation, there was a good correlation between the fetal and maternal body burden and the fetal body burden and maternal blood concentration, suggesting that, at a critical time, maternal blood concentrations provide an estimate of the concentrations of dioxin in the developing fetus. On day 16, 60% of the administered dose was recovered in the dams (Hurst et al., 2000a).

Long-Evans rats were given TCDD repeatedly before (5 days/week for 13 weeks) and during gestation at a dose of 1, 10, or 30 ng/kg bw in corn oil. On day 16 of gestation, the concentration of TCDD in single fetuses was 1.4, 7.8 and 16 pg/g, and the associated maternal body burdens were 19, 120 and 300 ng/kg bw, respectively (Hurst et al., 2000b).

2.1.2 Physiologically based pharmacokinetics modelling

As described above, the toxicokinetics of dioxin-like compounds involves the complex interaction of absorption, transport via the blood, distribution in the lipid and protein fractions of the blood and organs, Ah receptor-dependent induction of hepatic CYP proteins and elimination from the body by metabolism and/or transfer to faecal lipid. These concomitant processes can be quantified by physiologically based pharmacokinetics modelling, in which the toxicokinetics of chemicals is described mathematically within a physiological context, i.e. organs connected by the bloodstream and organ-specific responses after exposure to a chemical (Figure 2).

The first physiologically based pharmacokinetics models of dioxin-like compounds (2,3,7,8-TCDF, King et al., 1983; TCDD, Leung et al., 1988) allowed for only linear kinetics, i.e. they described the accumulation of dioxin-like compound in the body as a process which depends linearly on the administered dose. However, these models could not describe the hepatic sequestration of dioxin-like compounds, in particular the binding of TCDD to induced hepatic CYP 1A2. This deficiency was overcome by introducing Ah receptor-dependent CYP induction and subsequent binding of TCDD to induced CYP 1A2 in the model. The latter model was found to describe well the non-linear kinetics of TCDD, as observed in rodents exposed to TCDD at doses that induce Ah receptor-dependent CYP proteins in the liver (Leung et al., 1990a,b; Andersen et al, 1993; Kohn et al., 1993, 1994, 1996; Andersen et al, 1997a,b; Wang et al., 1997a,b; Zeilmaker & van Eijkeren, 1997).

Physiologically based pharmacokinetics modelling has also been used to describe the kinetics of dioxin-like compounds in humans. Assuming that the body burden of dioxin-like compounds is composed mainly of the amounts in the liver and adipose tissue, Carrier et al. (1995a,b) modelled the non-linear kinetics of dibenzo-para-dioxins and dibenzofurans resulting from their preferential accumulation in human liver. The model was used to describe the kinetics of 2,3,4,7,8-PeCDF in Yu-Cheng patients (see section 2.3.2). van der Molen (1998) developed a generic physiologically based pharmacokinetics model to describe the accumulation of dibenzo-para-dioxins and dibenzofurans in the blood and maternal milk of persons who had been exposed to background concentrations of these compounds. Kreuzer et al. (1997) and Pollitt (1999) used this type of modelling to evaluate the effect of exposure to these compounds in mother’s milk on the long-term body burden. In both cases, the exposure was found to have only a limited effect on the long-term body burden. Zeilmaker and van Eijkeren (1997; 1998) and Zeilmaker et al. (1999) used a slightly different approach: a physiologically based pharmacokinetics model of TCDD in rodents was scaled to humans. Figures 3, 4 and 5 show typical simulations made with this model of the accumulation of TCDD in the human body as expected after a variety of exposure scenarios.

2.1.3 Biochemical effects

Cells exposed to chemicals may respond by increasing the activity of metabolizing enzymes, in particular phase I and phase II enzymes (enzyme induction). Although this mechanism can lead to the removal of chemicals with deletorious effects, enzyme induction also has clear disadvantages. As the induced enzymes often have broad substrate specificity, increased activity may increase the metabolism of chemicals other than the inducing compound. In particular, exposure to persistent chemicals like PCDDs, PCDFs and PCBs can lead to sustained, unwanted changes in chemical metabolism. Examples of the latter effect are the increased metabolism of thyroid hormones after induction of UDP-glucuronosyl transferase (UGT1) activity (see section on Thyroid hormones) and increased estrogen metabolism in the liver (see section on TGF-alpha/EGF pathway).

The following working model prevails for enzyme induction by TCDD (Whitlock et al., 1997). After TCDD has diffused into the cell, it binds to the intracellular Ah receptor protein, which is maintained in its ligand inactivated state by complexation with heat shock protein (hsp)-90. After binding of TCDD, the TCDD–Ah receptor complex dissociates from the hsp-90 protein. This complex than translocates to the nucleus, where it combines with the Ah receptor nuclear translocator (Arnt) to a transcription factor, which may bind to a specific DNA enhancer site, the so-called xenobiotic responsive element. Concurrently, transcription factors bind to gene-specific promotor sites, thereby increasing gene transcription. In this way, TCDD may modulate the transcription of CYP 1A1 (Kedderis et al., 1991; Tritscher et al., 1992; DeVito et al., 1996), CYP 1A2 (DeVito et al., 1996), CYP 1B1, NAD(P)H: quinone oxidoreductase, gluthathione A-transferase Ya subunit and UGT (Whitlock et al., 1997). Increased concentrations of protein may manifest as increased enzyme activities, the activity of ethoxyresorufin O-deethylase (EROD) relating mainly to CYP 1A1 and that of acetanilide-4-hydroxylase and methoxyresorufin O-demethylase mainly to CYP 1A2 (De Jongh et al., 1995; DeVito et al., 1996).

TCDD efficiently induced CYP 1A1 and CYP 1A in rats, in which constitutive and inducible expression of CYP 1A2 is observed only in the liver. After administration of a single dose of TCDD ranging from 1 to 3000 ng/kg bw per day, < 50-fold induction of hepatic EROD activity was observed. Statistically significant induction was observed even at 3 ng/kg bw per day (Abraham et al., 1988). In concordance with this result, single doses of 0.1–1 ng of TCDD led to significant induction of CYP 1A1 mRNA in rat liver. A good correlation was found between hepatic CYP mRNA and EROD activity (r² = 0.93) (van den Heuvel et al., 1994).

Similarly, CYP 1A1 and CYP 1A2 were induced by 23- and 5-fold, respectively, in rats given repeated doses of 3.5–125 ng of TCDD (Tritscher et al., 1992). As shown in Figure 6, whereas the hepatic TCDD concentration increased linearly as a function of the administered dose, the induced protein concentrations increased non-linearly as a function of the hepatic concentration of TCDD, until a maximum was reached.

TCDD-induced EROD activity is not limited to the liver. When B6C3F₁ mice were given TCDD orally at a dose of 1.5–150 ng/kg bw per day on 5 days per week for 13 weeks, the activities of both enzymes were induced in the lungs and the skin at the lowest dose, being 30 times higher than the basal hepatic EROD activity in the lungs and 140 times higher in the skin. The dose–response characteristics of EROD induction were similar in the two organs. The lowest dose also significantly increased the phosphorylation of Cdc2 cyclin-dependent kinase, a protein associated with the G₂ to M phase transition of cells, in the liver but not in skin (DeVito et al., 1994).

Whereas induction of acetanilide 4-hydroxylase is limited to the liver (DeVito et al., 1994), a single dose of TCDD at 0.1, 1, or 10 µg/kg bw to mice induced EROD activity in a dose-dependent fashion in liver, lungs and skin, the EROD activity in the lungs and skin being 6% and 0.6% of the corresponding hepatic activity (Diliberto et al., 1995). Similar observations were made in rats, in which dose-dependent induction of EROD activity and the amount of CYP 1A1 was observed in the liver, lungs and kidneys of rats given a single dose of 0.1, 1, or 10 µg/kg bw. As in mice, the induced EROD activity in the lungs and kidneys represented only a small fraction of the corresponding hepatic activity (lungs, 0.8%; kidneys, 2.5%). In all three organs, a strong correlation was found between induced EROD activity and the amount of CYP 1A1 protein. Similar observations were made for hepatic methoxyresorufin O-demethylase activity, with a dose-dependent increase in activity in the liver, which correlated well with induced CYP 1A2 protein levels. As expected, hardly any CYP 1A2 protein was observed in the lungs or kidneys (Santastefano et al., 1996).

CYP 1A1 and CYP 1A2 can also be induced by compounds other than TCDD. Administration of 2,3,4,7,8-PeCDF at single a dose of 300 µg/kg bw to C57BL/6N and 129/Sv mice caused marked induction of EROD and acetanilide 4-hydroxylase activity in the liver and of EROD activity in the lungs. Similar effects were not found after administration of 2,2´,4,4´,5,5´-HxCB at a dose of 36 mg/kg bw (Diliberto et al., 1999). Hepatic EROD and acetanilide 4-hydroxylase activities were induced in B6C3F₁ mice treated orally on 5 days per week for 4 weeks with TCDD at 0.15 µg/kg bw per day, with 2,3,7,8-TCDF at 1.5 µg/kg bw per day, with OCDF at 150 µg/kg bw per day, with 3,3´,4,4´-TCB at 15 mg/kg bw per day, with 2,3,4´,4,4´,5-HxCB at 30 µg/kg bw per day or with 3,3´,4,4´,5-PeCB at 1.5 µg/kg bw per day. No induction was found with 1,2,3,7,8-PeCDF at 9 µg/kg bw per day or with the PCBs 2,3,3´,4,4´-PeCB at 3 mg/kg bw per day, 2,3,3´,4,4´,5´-HxCB at 300 µg/kg bw per day, or 3,3´,4,4´,5,5´-HxCB at 3 µg/kg bw per day. Although the absolute activity of EROD in the liver was 15-fold higher than that in the lungs, similar dose–response relationships were found in these organs. In skin, increased EROD activity was found only with TCDD, OCDF and the PCBs 3,3´,4,4´-TCB and 2,3,3´,4,4´,5-HxCB (DeVito et al., 1993). Administration of OCDD on 5 days/week for 13 weeks at a dose of 50 µg/kg led to significant induction of EROD in the livers of Fischer 344 rats (Couture et al., 1988).

Significant EROD induction was found in the liver and skin of B6C3F₁ mice given 2,3,7,8-TCDF at 1500 ng/kg bw for 4 and 13 weeks (DeVito & Birnbaum, 1995).

After administration of a single oral dose of 2,2´,4,4´,5,5´-HxCB at 91 mg/kg bw to C57BL/6J mice, no EROD induction was observed in the liver, but induction was observed with the PCBs 2,3,3´,4,4´,5-HxCB at 17 mg/kg bw and 3,3´,4,4´,5,5´-HxCB at 2.1 mg/kg bw. The induction was potentiated by concomitant administration of 2,3,3´,4,4´,5-HxCB and 2,2´,4,4´,5,5´-HxCB, whereas no such potentiation was found with 3,3´,4,4´,5,5´-HxCB, 1,2,3,7,8-PeCDD, 1,2,3,6,7,8-HxCDD, or 2,3,4,7,8-PeCDF (De Jongh et al., 1993). A single dose of 1000 µmol of 2,2´,4,4´,5,5´-HxCB doubled hepatic EROD and acetanilide 4-hydroxylase activity in C57BL/6J mice (De Jongh et al., 1995). Similarly, B6C3F₁ mice given 2,2´,4,4´,5,5´-HxCB at a single dose of 360 mg/kg bw had a 2.5-fold increase in hepatic EROD and MROD and 17-fold increase in that of pentoxyresorufin-O-depentylase (van Birgelen et al., 1996c).

Dietary administration of TCDD (14–1024 ng/kg bw per day), 3,3´,4,4´,5-PeCB (0.47–10.1 µg/kg bw per day), or 2,3,3´,4,4´,5-HxCB (81–729 µg/kg bw per day) to Sprague-Dawley rats clearly induced EROD activity. No such induction was observed with 2,2´,4,4´,5,5´-HxCB at 0.7–6 mg/kg bw per day (van Birgelen, 1995b). The 95% CIs for the NOELs were 0.35–0.89 for TCDD-induced EROD activity and 0.55–3.8 ng/kg bw per day for that of acetanilide 4-hydroxylase (van Birgelen et al., 1995a).

Oral administration of 3,3´,4,4´,5-PeCB to B6C3F₁ mice at a dose of 0.015–1.5 µg/kg bw per day induced EROD and acetanilide 4-hydroxylase activity in the liver and EROD activity in the skin and lungs (LOEL, 0.045–1.5 µg/kg bw per day). The activities of liver-specific enzymes were induced by 2,3,3´,4,4´,5-HxCB at 450 µg/kg bw per day, and the activity of EROD in skin and lung was induced by doses > 1500 µg/kg bw per day. 3,3´,4,4´,5,5´-HxCB at doses up to 3 µg/kg bw per day did not induce enzyme activity. The LOEL for the induction of hepatic EROD activity was 3900 µg/kg bw per day with the PCB 2,3,3´,4,4´-PeCB and 300 µg/kg bw per day with 2,3´,4,4´,5-PeCB, whereas that with TCDD was 0.0015 µg/kg bw per day (DeVito et al., 2000).

Induction of EROD by PCDDs and PCDFs in primary hepatocytes has been found to be a sensitive end-point. Half-maximum EROD activity was achieved by incubating the cells with 16 pmol/l of TCDD, 90 pmol/l of 1,2,3,7,8-PeCDD, 184 pmol/l of 1,2,3,4,7,8-HeCDD, 329 pmol/l of 1,2,3,7,8,9-HeCDD, 441 pmol/l of 1,2,3,6,7,8-HeCDD, 702 pmol/l of 1,2,3,4,6,7,8-HpCDD, or 3859 pmol/l of OCDD (Schrenk et al., 1991). TCDD also induced EROD activity in primary human hepatocytes, although with very different dose–response characteristics. Half-maximum EROD activity was observed in cells exposed to ~ 100 pmol of TCDD (Schrenk et al., 1995).

Like CYPs 1A1 and 1A2, CYP 1B1 is induced by TCDD. When C57BL/6J and DBA/2J mice were given single intraperitoneal doses of TCDD ranging from 0.001 to 50 µg/kg bw, dose-dependent accumulation of CYP 1B1 mRNA was observed in the liver. C57BL/6J mice showed a significant increase in mRNA at doses as low as 0.1 µg/kg bw, and maximum induction (200 times background) was found at 10 µg/kg bw. A much steeper dose–response curve was observed with CYP 1A1 mRNA than with CYP 1B1 mRNA (increase from 0.01 µg/kg; maximum induction at 1 µg/kg). The estimated effective doses at which half the maximum inducibility of CYP 1A1 and CYP 1B1 was observed (ED₅₀) were 0.08 and 1.3 µg/kg bw. Similarly, in DBA/2J mice, significant induction of CYP 1A1 and 1B1 mRNA was found at doses of 1 and 10 µg/kg bw, respectively. Again, the dose–response curve of CYP 1A1 induction was much steeper than that for CYP 1B1, the ED₅₀ values for CYP 1A1 and CYP 1B1 induction being 1.5 and 3.4 µg/kg bw, respectively. The differences in susceptibility of C57BL/6J and DBA/2J mice are due to several mutations in the Ahr^d allele of the Ah receptor, resulting in lower ligand binding affinity: C57BL/6J mice carry the Ahr^b-1 allele, conferring relative high susceptibility to TCDD, and DBA/2J mice carry the Ahr^d allele, conferring relatively low susceptibility to TCDD (Abel et al., 1996).

In rat liver, the induction of UGT1, PAI2 and transforming growth factor (TGF)-alpha mRNA clearly deviated from that of CYP 1A1 mRNA. Although the dose required to increase UGT1 mRNA (1 µg/kg) was much higher than that required to induce CYP 1A1 mRNA (1 ng/kg), doses of TCDD up to 100 µg/kg did not induce PAI2 or TGF-alpha mRNA (Van den Heuvel et al., 1994).

Epidermal growth factor (EGF) is a plasma membrane receptor which, after binding a specific ligand, functions as a signal tranducer regulating cellular proliferation. This effect is mediated by internalization of the ligand–receptor complex and then phosphorylation of intracellular targets by tyrosine kinase. The effects of TCDD on hepatic EGF and their relationship to hepatic carcinogenesis are as follows. TCDD is a known inducer of liver tumours in female, but not male, rats (Kociba et al., 1978). It also induces proliferation of hepatocytes and preneoplastic foci in intact, but not ovariectomized, female rats (Lucier et al., 1991), indicating an important role of estrogens in hepatocarcinogenesis. Furthermore, treatment with TCDD results in a dose-dependent decrease in the number of EGF binding sites in the liver (Lucier et al., 1991; Kohn et al., 1993, short-term exposure to 3.5–125 ng/kg bw per day), indicating internalization of the receptor after TCDD binding (Kohn et al., 1993) and induction of TGF-alpha, a ligand of the EGF receptor.

The interactions of TCDD, EGF, TGF-alpha and inducible CYP enzymes in the liver are shown schematically in Figure 7. The hepatic TCDD–Ah receptor induces not only CYP 1A1 and 1A2 but also expression of the TGF-alpha gene and/or post-transcriptional or post-translational TGF-alpha modifications. This expression may be enhanced by the estrogen receptor–estrogen complex. As a consequence, more TGF-alpha is secreted into the interstitial space in the liver, where it may combine with EGF on the liver cell membrane. The TGF-alpha–EGF receptor complex may then be internalized to exert its biochemical signalling function. The TCDD–Ah receptor complex inhibits synthesis of the estrogen receptor. Finally, estrogen metabolism (estradiol-2-hydroxylase activity) is catalysed by CYP 1A2.

PCDDs, PCDFs and PCBs may affect plasma thyroid hormone levels in one of three ways:

(1) By induction of hepatic microsomal enzymes, resulting in accelerated metabolism and excretion in the bile (Bastomsky, 1977). The stimulation of thyroid metabolism may be compensated by increased amounts of thyroid-stimulating hormone (TSH) in the blood. When sustained, such compensation may result in chronic stimulation of the thyroid and, ultimatally, thyroid cancer (Kohn et al., 1996; Whitlock et al., 1997).

The main pathway is glucuronidation of thyroxine (3,5,3´,5´-tetra-iodothyronine, T4) by UGT1, which catalyses the formation of T4 glucuronides, which are excreted in the bile. At least two forms of glucuronosyltransferase contribute to the activity of T4 UGT: UGT 1A1 (known to be induced by 3-methylcholantrene) and UGT 1A2 (known to be induced by phenobarbital).

Decreased plasma T4 levels were found after exposure of rats to phenobarbital, 3-methylchlolanthrene or the PCB mixture Aroclor 1254. This decrease correlated well with increased T4 UGT activity (Barter & Klaassen, 1992). Furthermore, PCBs and phenobarbital caused increased biliary clearance of T4 (Bastomsky, 1974; McClain et al., 1989; Beetstra et al., 1991). Short-term dietary intake of TCDD (14–1000 ng/kg bw per day) by rats lowered the concentrations of total T4 and free T4 in plasma at doses > 47 ng/kg bw per day. No effect was found on total triiodothyronine (T3) (van Birgelen et al., 1995b). Furthermore, short-term dietary intake of TCDD (14–1000 ng/kg bw per day), 3,3´,4,4´,5-PeCB (0.47–10 µg/kg bw per day) or 2,3,3´,4,4´,5-HxCB (81–730 µg/kg bw per day) resulted in dose-dependen increases in the activity of hepatic UGT 1A1, T4 UGT and CYP 1A1 and concomitant decreases in free and total T4 in plasma. Total T4 in plasma and hepatic T4 UGT activity were negativily correlated, whereas UGT 1A1 and CYP 1A1 activities were positivily correlated. This suggests involvement of the Ah receptor in inducing hepatic T4 glucuroni-dation and, consequently, in modulating thyroid hormone metabolism (van Birgelen et al., 1994a,b, 1995a).

(2) By binding of hydroxy metabolites of PCBs to transthyretin, thereby affecting the binding of T4 (and retinol) to transthyretin, its major transport protein in blood (Brouwer & van den Berg, 1986)

(3) By a direct effect of PCBs on the functioning of the thyroid gland. For example, Aroclor 1254 inhibits proteolysis of thyroglobulin, the protein responsible for release of thyroid hormones from the gland (Collins & Capen, 1980). Furthermore, exposure to TCDD results in an increase in serum TSH concentration and, after long-term exposure, to an increased volume of thyroid follicular cells, increased thyroid weight and thyroid hyperplasia (Bastomsky, 1977; Andrae & Greim, 1992; Hill et al., 1989).

In rats, short-term dietary intake of TCDD (14–1000 ng/kg bw per day) reduced the hepatic concentrations of retinol and retinylpalmitate at the lowest dose tested. The concentration of plasma retinol was increased concomitantly. The mechanism behind this effect consists of induction of CYP enzymes, which oxidize retinol and reduce the activity of acyl coenzyme A:retinyl acyltransferase and lecithin:retinol acyltransferase. Both enzymes participate in the esterification of retinol. In a short-term study in Sprague-Dawley rats treated in the diet, the NOEL of 2,3,3´,4,4´,5-HxCB for depletion of hepatic retinoids was 81 µg/kg bw per day (van Birgelen et al., 1994a). In a similar study, the LOEL of 3,3´,4,4´,5-PeCB for this effect was 0.47 µg/kg bw per day (van Birgelen et al., 1994b).

Disturbed biosynthesis of haem may lead to porphyria, a condition in which precursors of haem accumulate in blood and are hence excreted in the urine.

Short-term intake by rats of diets containing TCDD (14–1000 ng/kg bw per day), 3,3´,4,4´,5-PeCB (0.47–10 µg/kg bw per day), 2,3,3´,4,4´,5-HxCB (81–730 µg/kg bw per day) or 2,2´,4,4´,5,5´-HxCB (0.7–6 mg/kg bw per day) maximally induced a twofold increase in hepatic porphyrin concentration (lowest effect doses: TCDD, 47 ng/kg bw per day; 3,3´,4,4´,5-PeCB, 3.2 µg/kg bw per day; 2,3,3´,4,4´,5-HxCB, 360 mg/kg bw per day). Concomitant administration of TCDD and 3,3´,4,4´,5-PeCB or 2,3,3´,4,4´,5-HxCB resulted in an additional twofold increase in porphyrin concentration. When these compounds were administered with a non-inducing dose of TCDD (33 ng/kg bw per day), however, an 800-fold increase in accumulation of porphyrins was seen, in particular uroporphyrin III and heptacarboxylic porphyrin, in the liver, indicating a strong synergistic action. Furthermore, induced acetanilide 4-hydroxylase activity correlated well with induction of accumulation of porphyrins in the liver. The mechanism behind these effects consisted of CYP 1A2-mediated oxidation of uroporphyrinogen III to uroporphyrin III and induction of the activity of delta-aminolaevulinic acid synthetase, the rate-limiting enzyme in haem biosynthesis, by 2,2´,4,4´,5,5´-HxCB (van Birgelen et al., 1996a).

Short-term intake of TCDD (0.15–450 ng/kg bw per day), 1,2,3,7,8-PeCDD (90–9000 ng/kg bw per day), 2,3,7,8-tetrabromodibenzo-para-dioxin (30–3000 ng/kg bw per day), 2,3,7,8-TCDF (15–1500 ng/kg bw per day), 1,2,3,7,8-PeCDF, 90–9000 ng/kg bw per day), 2,3,4,7,8-PeCDF, 9–900 ng/kg bw per day), OCDF (1.5–150 µg/kg bw per day), 3,3´,4,4´,5-PeCB (0.3 and 15 µg/kg bw per day), 2,3,3´,4,4´-PCB (390–39 000 µg/kg bw per day), 2,3´,4,4´,5-PeCB (3000–30 000 µg/kg bw per day) or 2,3,3´,4,4´,5-HxCB (45–4500 µg/kg bw per day) by mice resulted in hepatic porphyria in all cases (van Birgelen et al., 1996b). The relative potencies of these compounds to induce hepatic porphyrin accumulation were TCDD > PeCDD = tetrabromodibenzo-para-dioxin = 4-Pe-CDF ^a TCDF > 3,3´,4,4´,5-PeCB ^a 1-PeCDF > OCDF > 2,3,3´,4,4´,5-HxCB > 2,3´,4,4´,5-PeCB ^a 2,3,3´,4,4´-PeCB. Again, hepatic CYP 1A2 enzyme activity and total hepatic porphyrin accumulation correlated well.

The Ah receptor appears to play an important role in dioxin-like porphyria, as hepatic porphyria is associated with the inducibility of Ah hydroxylase after administration of TCDD to Ah-responsive C57Bl/6 and Ah-nonresponsive DBA mice (Jones & Sweeney, 1980). Furthermore, non-coplanar PCBs, such as 2,2´,4,4´,5,5´-HxCB and 2,2´,3´,4,4´,5,5´-HpCB do not induce porphyria (Stonard & Greig, 1976; Van Birgelen et al., 1996b; Koss et al., 1993). Finally, hepatic porphyrin accumulation and Ah-receptor dependent benzo[a]pyrene hydroxylation (a well known Ah-receptor response) are increased by 2,2´,3,3´,4,4´-HxCB and 2,2´,3,4,4´,5´-HxCB (Stonard & Greig, 1976).

2.1.4 Toxic equivalency factors

Exposure to PCDDs, PCDFs and coplanar PCBs actually consists of exposure to a mixture of congeners with toxic effects (dermal toxicity, immunotoxicity, carcinogenicity, reproductive and developmental toxicity, disruption of endocrine functions) similar to those of TCDD, the most toxic congener of this class of compounds. This finding led to the development of the concept of toxic equivalency factors (TEFs). In this concept, the toxic potency of a congener, i.e. its toxicity as described in all studies in vivo and in vitro, is expressed relative to that of a reference compound, in this case TCDD (for which the TEF is arbitrarily set at 1).

Application of the TEF concept rests on the following assumptions (van den Berg et al, 1998), noting that the TEF concept does not apply to toxicity that is not mediated by the Ah receptor and does not take into account modulation of Ah receptor-dependent responses by non-Ah-receptor ligands:

• PCDDs, PCDFs and (some) PCBs have a common mechanism of action. In this case, the mechanism consists of binding to the Ah receptor. This binding is considered to be the first, necessary though not sufficient, step in the toxicity of dioxins.
• Congeners exert their combined (dose or concentration) effect additively; i.e. in a mixture of congeners, each congener exerts its toxicity independently of the other congeners in the mixture.

Derivation of congener-specific TEFs is based on evaluation and interpretation of the results of all available studies of toxicity on the basis of expert judgement. The first consultations on TEF concluded that the concept can be applied to PCDDs and PCDFs (international TEFs; NATO/CCMS, 1988) and non-ortho and mono-ortho PCBs (Ahlborg et al., 1994). The TEF concept was re-evaluated in 1998 (van den Berg et al., 1998, 2000).

In attributing a TEF to a compound, the following criteria were used:

• The compound shows a structural relationship to PCDDs and PCDFs.
• The compound binds to the Ah receptor
• The compound elicits Ah receptor-mediated biochemical and toxic effects.
• The compound is persistent and accumulates in the food chain.

Furthermore, TEFs in mammals were derived mainly from studies conducted in vivo, in preference to data generated in vitro and/or quantitative structure–activity relationships. Studies of toxicity were placed in the following order of priority: long-term > short-term > acute. Similarly, Ah receptor-dependent toxic end-points were given priority over biochemical end-points such as enzyme induction. TEFs for mammals were considered to apply to humans too.

Currently, there is consensus on the TEFs for mammals listed in Table 1 (van den Berg et al., 1998, 2000; Scientific Committee on Food, 2000). When combined with data on residues in matrices such as tissue, soil and water, TEFs allow determination of the toxic equivalents concentration of the residue. For a particular residue containing a mixture of i PCDDs, j PCDFs and k PCBs, the toxic equivalence is calculated according to the following equation:

In using the TEF concept, it should be kept in mind that non-Ah receptor-mediated toxicity (decreased dopamine concentration, effects on retinoid and thyroid hormone concentrations and estrogen receptor binding), shown by some PCBs, is not covered. Similarly, the TEF concept does not apply to halogenated compounds other than PCDDs, PCDFs and PCBs which show Ah receptor-dependent toxicity (brominated and chloro or bromo analogues of PCDDs, PCDFs, naphthalenes, diphenyl ethers, diphenyl toluenes, phenoxyanisoles, biphenyl anisoles, xanthenes, xanthones, anthracenes, fluorenes, dihydroanthracenes, biphenyl methanes, phenylxylyl-ethanes, dibenzothiophenes, quaterphenyls, quaterphenyl ethers and biphenylenes). Furthermore, non-additivity was found with mixtures of PCDDs, PCDFs and PCBs. For example, antagonistic effects of non-coplanar PCBs have been described on Ah receptor-dependent effects like induction of EROD and fetal cleft palate in mice. However, synergistic interactions between PCBs and PCDDs/PCDFs have also been reported for effects such as those on CYP 1A1 and thyroid hormone concentrations (van den Berg et al., 1998). Whereas the currently agreed TEFs are based on toxicological evaluations of dose–response relationships between external exposure, i.e. the levels of intake of congeners, and toxicity in organs, future TEFs will be based on the dose–response relationship between internal exposure, i.e. the actual concentrations of congeners in tissues, and toxicity in organs (van den Berg et al., 1998).

2.2.1 Acute toxicity

The acute toxicity of TCDD and related dioxins and furans substituted in at least the 2, 3, 7 and 8 positions can vary widely between and among species (Table 3). For example, in guinea-pigs, an LD₅₀ of 0.6 µg/kg bw was recorded after oral administration, as compared with an LD₅₀ of > 5000 µg/kg bw in Syrian hamsters. Explanations for this variation include differences in the Ah receptor, such as size, transformation and binding to the dioxin response element, pharmacokinetics (metabolic capacity, tissue distribution) and body fat content (Geyer et al., 1990; van den Berg et al., 1994; Pohjanvirta et al., 1998). While data on acute toxicity were available for various commercial PCB mixtures (LD₅₀ values usually > 100 mg/kg bw), few data were available on the acute toxicity of the individual coplanar PCB congeners in mammals. In Ah-responsive rodent species, it is thought that lethality correlates to Ah receptor binding affinity.

One of the commoner symptoms associated with dioxin-induced death is generalized delayed wasting syndrome, characterized by inhibition of gluconeo-genesis, reduced feed intake and loss of body weight. Although some differences exist between species, other toxic responses observed after single doses of dioxins include haemorrhage in a number of organs, thymic atrophy, reduced bone-marrow cellularity and loss of body fat and lean muscle mass. For example, in groups of five or six golden Syrian hamsters of each sex given a single oral or intraperitoneal dose of TCDD at 0, 5, 25, 100, 250, 500, 750, 1000, 2000 or 3000 µg/kg bw, deaths occurred at doses > 500 µg/kg bw, oral administration generally being more toxic. At the highest dose, 33% of males treated intraperitoneally and 80% of those treated orally died. The consistent pathological findings included gradual loss of adipose and muscle tissue (wasting syndrome), thymic atrophy and gastrointestinal lesions. The estimated LD₅₀ values were > 3000 µg/kg bw for intraperitoneal administration and 1200 µg/kg bw for oral dosing (Olson et al., 1980).

Groups of 11 male C57BL/6J mice that were responsive or sensitive to TCDD (Ahb/b) were given a single oral dose of TCDD at 0, 5, 100, 200, 300 or 400 µg/kg bw, and a congenic non-responsive strain (Ahd/d) was given a single oral dose of 0, 400, 800, 1600, 2400 or 3200 µg/kg bw. All mice were observed for 35 days before necropsy. Significant mortality occurred in groups of the Ahb/b strain given doses > 200 µg/kg bw, 100% of animals at the two higher doses dying by about 21 days after dosing; an LD₅₀ value of 160 µg/kg bw was estimated by probit analysis. Conversely, few deaths were seen in the Ahd/d strain, only 33% of those at the high dose dying; an LD₅₀ value of 3400 µg/kg bw was estimated. Decreased body-weight gain, increased liver weight and thymic and splenic atrophy were observed in both strains of mice (Birnbaum et al., 1990).

Groups of 30–60 female Sprague Dawley rats were given 1,2,3,4,6,7,8-HpCB at a total dose of 0, 2.5, 2.8, 3.1, 3.4, 3.8, 4.1, 5 or 10 mg/kg bw by gavage (four doses over 2 days) and then observed for deaths. Many deaths due to lethal wasting, haemorrhage and/or anaemia were observed at the four higher doses, 83–100% of the animals dying within 25 days after dosing. None of the animals at the lowest dose died, while 8.3%, 31% and 66% of those at the next higher doses, respectively, died by day 44 after dosing. The author noted that 30% (9/30) of animals at the lowest dose died from squamous-cell carcinoma of the lungs (Rozman, 1999).

2.2.2 Short-term studies of toxicity

The toxicity of TCDD and related coplanar chemicals in short-term studies is characterized, depending on the route and species, by similar biological and toxicological effects.

In studies of the porphyrinogenic potential of various chlorinated dioxins, furans and coplanar PCBs, groups of five female B6C3F₁ mice were treated on 5 days per week for 13 weeks by gavage with concentrations related by TEFs to doses of TCDD of 0, 0.15, 0.45, 1.5, 4.5, 15, 45, 150 or 450 ng/kg bw per day: 1,2,3,7,8-PeCDD, TEF, 0.05; 2,3,7,8-tetrabromodibenzo-para-dioxin, TEF, 0.15; 2,3,7,8-TCDF, TEF, 0.3; 1,2,3,7,8-PeCDF, TEF, 0.05; 2,3,4,7,8-PeCDF, TEF, 0.5; OCDF, TEF, 0.003; 2,3,3´,4,4´-PeCB, TEF, 0.00001; 2,3´,4,4´,5-PeCB, TEF, 5 x 10^–6; 2,3,3´,4,4´,5-HxCB, TEF, 0.0001; 3,3´,4,4´,5-PeCB, TEF, 0.03. Analysis of hepatic tissue for highly carboxylated porphyrins and CYP 1A1 and 1A2 induction indicated that the binding affinity of the congeners to the Ah receptor in vivo was related to CYP 1A2 induction, which was in turn correlated to hepatic porphyrin accumulation. The LOELs associated with significant increases in total hepatic porphyrin content were: 15 ng/kg bw per day for TCDF; 45 ng/kg bw per day for TCDD; 300 ng/kg bw per day for PeCDD, 2,3,4,7,8-PeCDF and 3,3´,4,4´,5-PeCB, 900 ng/kg bw per day for tetrabromodibenzo-para-dioxin and 1,2,3,7,8-PeCDF; 45 µg/kg bw per day for OCDF; 1500 µg/kg bw per day for 2,3,3´,4,4´,5-HxCB; 7500 µg/kg bw per day for 2,3´,4,4´,5-PeCB; and 13 000 µg/kg bw per day for 2,3,3´,4,4´-PeCB. Induction of hepatic porphyrins by the mono-ortho-substituted PCBs was greater than that estimated from the TEFs assigned to them, which was considered to be related in part to non-Ah receptor-dependent induction of CYP 2B1 and delta-aminolaevulinic acid synthetase (van Birgelen et al., 1996b).

Groups of 12 Sprague-Dawley rats of each sex given TCDD at 0, 0.001, 0.01, 0.1 or 1 µg/kg bw per day by gavage on 5 days/week for 13 weeks had decreased organ and body weights, haematological effects and deaths at the two higher doses. The deaths occurred only at the highest dose, four females dying during the 13 weeks of treatment and two males and two females dying between 14 and 49 days during the 13-week post-dosing observation period. Minor increases in relative liver weight (5–8%) observed in animals at 0.01 µg/kg bw per day were considered to be an adaptive response, as no corresponding histopathological changes were seen. The NOEL was thus 0.01 µg/kg bw per day (equivalent to 0.007 µg/kg bw per day when averaged over the 13 weeks), which resulted in TCDD concentrations in the liver of 2.6–3.7 µg/kg (Kociba et al., 1976).

Groups of six male and six female rats of the same strain were fed diets containing a variety of penta- and hexachlorinated dioxins and dibenzofurans, separately and in a mixture, for 13 weeks: TCDD at 0.2, 2 or 20 µg/kg bw per day; 2,3,4,7,8-PeCDF, 1,2,3,7,8-PeCDF and 1,2,3,6,7,8-HxCD at 2, 20 or 200 µg/kg bw per day; or a mixture of TCDD at 0.2 or 2 µg/kg bw per day, 1,2,3,7,8-PeCDD at 1 or 10 µg/kg bw per day, 2,3,4,7,8-PeCDF at 2 or 20 µg/kg bw per day and 1,2,3,6,7,8-HxCD at 1 or 10 µg/kg bw per day. On the basis of decreases in body weight, histological changes in the liver and thymus and deaths, the relative toxicity of each congener was seen to be related to its binding affinity to the Ah receptor, while the toxicity of the mixture concurred with an additivity concept (TCDD > 2,3,4,7,8-PeCDF > 1,2,3,6,7,8-HxCDF > 1,2,3,7,8-PeCDF > 1,2,3,4,8-PeCDF) (Poiger et al., 1989b).

Male and female Fischer 344 rats were given oral doses of TCDD designed to generate a liver concentration of 0.03, 30 or 150 ng/g. After an initial loading dose of 0.005, 2.5 or 12 µg/kg bw, three maintenance doses of 0.0009, 0.60 or 3.5 µg/kg bw were given every fourth day, and the animals were killed at various times up to day 14. The terminal body weights of males and females at the highest dose were significantly reduced by approximately 11%, while the relative liver weights were increased on average by 30% and 43% at the two higher doses, respectively. Induction of hepatic CYP 1A1 (all doses) and CYP 1A2 (two higher doses) was determined by northern blot hybridization, and dose-dependent induction of a human TCDD-responsive CYP gene was detected in the livers of rats of each sex. Induction of this gene has not been associated with various treatments designed to induce hepatocellular proliferation (Fox et al., 1993).

Two weeks after receiving a protocol designed to initiate preneoplastic lesions (N-nitrosomorpholine at 80 mg/l of drinking-water for 25 days), female Wistar rats, were given subcutaneous injections of various dioxins every 2 weeks for 13 weeks, at approximate daily doses as follows: TCDD, 0, 2, 20 or 200 ng/kg bw; 1,2,3,4,6,7,8-HpCDD, 0, 0.2, 2 or 20 µg/kg bw; or a defined mixture of 49 dioxin congeners, 0, 0.2, 2 or 20 µg/kg bw. At the end of treatment, the body weights of animals at the highest doses of all three treatments were decreased by 5–7%, and the relative liver weights of animals were increased at the two higher doses of TCDD and the highest doses of the other compounds. Hepatic EROD activity was significantly increased in all treated groups when compared with controls, with similar induction at the low, intermediate and high doses. Linear regression analysis of hepatic EROD activity and measured toxic equivalents (ng/g liver) revealed slight differences in the slope of the regression lines (m = 0.87, 0.76, 0.67 for TCDD, HpCDD and the dioxin mixture, respectively; all r₂ = 0.92). When the tumour promoting ability of the three treatments was assessed (relative focal volume of ATPase-deficient preneoplastic liver tissue), similar results were obtained after modelling the toxic equivalents for liver with TCDD and the dioxin mixture; however, the response to the latter was about twofold lower at the highest dose. The authors concluded that TEFs based on enzyme induction in vitro provide only an approximation of the tumour promoting ability of dioxin congeners and gave an overestimate of the response to the HpCDD (Schrenk et al., 1994).

Groups of six male and six female Sprague-Dawley rats were treated by gavage with total doses of 1,2,3,6,7,8-HxCDF of 0, 18, 220, 1300, 4000 or 6000 µg/kg bw for females and 0, 31, 370, 2200, 6700 or 10 000 µg/kg bw for males over 13 weeks; the total dose was divided into four daily loading doses, each comprising about 13% of the total dose, and was followed by six maintenance doses given every 2 weeks, each equivalent to 7.8% of the total dose. Treatment induced a variety of biochemical and toxic effects similar to those seen with TCDD in a group of rats given a total dose of 42 µg/kg bw for females and 70 µg/kg bw for males by the same dosing regimen. On the basis of the measured end-points (deaths, hepatic EROD induction, decreased plasma T4 concentration, haematological indices), the TEF for this congener was estimated to be 0.007, in close agreement with the WHO-assigned TEF of 0.01 (Viluksela et al., 1997).

In an experiment of a similar design, groups of 20 rats of the same strain were given total toxic equivalents of 0, 0.14, 1.7, 10, 31 or 47 µg/kg bw for females and 0, 0.22, 2.6, 16, 47 or 70 µg/kg bw for males, with contributions of similar toxic equivalents from TCDD (TEF, 1), 1,2,3,7,8-PeCDD (TEF, 0.2), 1,2,3,4,7,8-HeCDD (TEF, 0.05) and 1,2,3,4,6,7,8-HeCDD (TEF, 0.007). The same regimen of four daily loading doses (each comprising about 10% of the total toxic equivalents) followed by six maintenance doses (each comprising about 10% of the total toxic equivalents) every 2 weeks for 13 weeks. Whereas there was a significant, dose-dependent increase in hepatic EROD activity even at the lowest toxic equivalents (0.14 and 0.22 µg/kg bw for females and males, respectively), most of the additional biochemical end-points (decreased hepatic phosphoenolpyruvate carboxykinase activity, serum glucose and serum total T4) were affected only by total toxic equivalents > 10 µg/kg bw. Overall, the effects seen with the toxic equivalent mixture, TCDD or 1,2,3,6,7,8-HxCDD alone were comparable (mortality rate, growth reduction, hepatic enzyme induction, haematological effects), providing support for the TEF and additivity concept for chlorinated dioxins (Viluksela et al., 1998a,b).

In an experiment designed to identify the lowest effective doses and body burdens of TCDD in rats, groups of eight female Sprague-Dawley rats were fed diets formulated to deliver TCDD at a dose of 0, 14, 26, 47, 320 or 1000 ng/kg bw per day for 13 weeks. The most sensitive effects, seen at the lowest dose, included hepatic CYP 1A1 and 1A2 induction and significant decreases in thymus weights and hepatic retinol concentration. At the higher doses, differences in liver, kidney and spleen weights and decreases in plasma T3 and free T4 concentrations were seen. The estimated NOEL (by sigmoidal curve fitting) for hepatic EROD induction was 0.35 ng/kg bw per day, which corresponds to a liver TCDD content of 0.037 ng/g (van Birgelen et al., 1995a,b).

Thyroid hormone status was assessed in rats in a short-term assay for tumour promotion. After initiation with N-nitrosodietheylamine at 70 days of age, female Sprague-Dawley rats were treated with TCDD by gavage every 2 weeks for 30 weeks at doses designed to deliver 0, 0.1, 0.35, 1, 3.5, 11, 36 or 125 ng/kg bw per day. The rats were necropsied 1 week after the last TCDD dose, and serum samples were analysed for T3, T4 and TSH. T4 concentrations were significantly reduced at doses of TCDD > 11 ng/kg bw per day in the initiated rats and > 36 ng/kg bw per day in the uninitiated animals (maximum reduction, 42%). While there was no effect on T3 concentration, that of serum TSH was increased by about 2.5-fold in uninitiated rats at the highest dose (3.3 ng/ml, with 1.3 ng/ml in controls). Histological changes in the thyroid included diffuse follicular hyperplasia; in rats at 3.5 ng/kg bw per day, the ratio of parenchymal to follicular area was significantly increased. Hepatic CYP 1A1 and UGT1 mRNA levels were increased at doses > 0.35 ng/kg bw per day and > 3.5 ng/kg bw per day, respectively (Sewall et al., 1995).

Groups of 10 Hartley guinea-pigs of each sex were given diets containing TCDD at a concentration of 0, 2, 10, 76 or 430 ng/kg of diet for 13 weeks (equal to 0, 0.12, 0.61, 4.9 and 26 ng/kg bw per day for males and 0, 0.14, 0.68, 4.9 and 31 ng/kg bw per day for females). The effects induced were similar to those in rats, including deaths at the highest dose. The NOEL for changes in organ and body weights and clinical effects was 0.61 ng/kg bw per day, confirming the greater sensitivity of this species to TCDD (DeCaprio et al., 1989).

When eight female rhesus monkeys were given a diet containing TCDD at a concentration of 500 pg/g for 9 months, dermatological effects were seen by 3 months and changes in haemoglobin and erythrocyte volume fraction by 6 months, which persisted and increased in severity up to the end of treatment. One animal died after 7 months on the diet, and four further deaths occurred within 2 months after removal from the diet, after total TCDD intakes estimated to be 19 and 12 µg/kg bw per day (Allen et al., 1977). Similar effects were seen when three male rhesus monkeys were fed diets containing 2,3,7,8-TCDF at 5 or 50 µg/g for up to 6 months, except that the animals that survived to the end of treatment tended to recover quickly after being removed from the diets (McNulty et al., 1981).

2.2.3 Long-term studies of toxicity and carcinogenicity

Previous WHO expert groups have evaluated the carcinogenicity of PCDDs, PCDFs and PCBs (IARC 1987, 1997; van Leeuwen & Younes, 2000). Most of the long-term experiments designed to determine the toxicity of dioxin and coplanar chemicals were conducted with various rodent species or non-human primates. The carcinogenic effects assessed in long-term studies are summarized in Table 4.

Groups of 45 male Swiss mice were given TCDD by gavage at a dose of 0, 0.007, 0.7 or 7 µg/kg bw per week, equal to 0.001, 0.1 and 1 µg/kg bw per day, for up to 1 year. Dose-dependent increases in the incidence of both ulcerative skin lesions and amyloidosis were observed at the two lower doses and a decreased lifespan in mice at the highest dose. An increased incidence of liver tumours (hepatomas and hepatocellular carcinomas) was observed at the intermediate dose, but a similar increase at the highest dose was not significant (Tóth et al., 1979).

In a study of the carcinogenicity of TCDD in two rodent species, groups of 50 Osborne-Mendel rats and 50 B6C3F₁ mice of each sex were given TCDD by gavage twice a week for 104 weeks at a dose of 0, 0.01, 0.05 or 0.5 µg/kg bw per week for rats and male mice (equal to 0, 0.0014, 0.0071 and 0.071µg/kg bw per day) and 0, 0.04, 0.2 or 2 µg/kg bw per week for female mice (equal to 0, 0.006, 0.03 and 0.3 µg/kg bw per day). Decreased body-weight gain was seen in male and female rats at the highest dose, and an increased incidence of hepatic lesions described as ‘toxic hepatitis’ was seen in both species at the highest dose. Increased incidences of follicular-cell adenomas or carcinomas of the thyroid were found in male rats at the two higher doses (16% and 22%, respectively), with a non-significant increase (13%) in female rats. Furthermore, 24% of female rats at the highest dose had neoplastic nodules in the liver and 6% had hepatocellular carcinoma. The incidence of hepatocellular carcinoma was also increased in male (34%) and female (13%) mice at the highest dose. Females at this dose had an increased incidence of follicular-cell adenomas of the thyroid (11%) and an increased incidence (with a dose-related trend) of histiocytic lymphomas in the haematopoietic system (National Toxicology Program, 1982).

Groups of 10 male Sprague-Dawley rats were maintained on diets containing TCDD at a concentration of 0, 0.001, 0.005, 0.05, 0.5, 1, 5, 50, 500 or 1000 ng/kg for up to 78 weeks, equal to weekly doses of 0, 0.0003, 0.001, 0.01, 0.1, 0.4, 2, 24, 240 and 500 µg/kg bw or daily doses of 0.00004, 0.00014, 0.0014, 0.014, 0.057, 0.29, 3.4, 34 and 71 µg/ kg bw. All animals at the five higher doses died, the time to 100% mortality ranging from week 3 for animals at 24 µg/kg bw per week to week 31 for animals at 0.4 µg/kg bw per week. Further deaths (40–50% of animals) occurred at 0.001, 0.01 and 0.1 µg/kg bw per week before the end of the study. Neoplasms were found at multiple sites in 57% of animals at doses > 0.0001 µg/kg bw per week. Among those reported were ear-duct carcinoma, renal adenocarcinoma, skin angiosarcoma, Leydig-cell adenoma, fibrosarcoma, squamous-cell carcinoma of the skin and lung, glioblastoma, astrocytoma, cholangiocarcinoma and lymphocytic leukaemia. No tumours were found at the lowest dose (Van Miller et al., 1977). The Committee noted that the small number of animals used and the high mortality rates limit interpretation of this study.

Groups of 50 Sprague-Dawley rats of each sex (86 rats of each sex as vehicle controls) were fed diets formulated with TCDD to provide a dose of 0, 0.001, 0.01 or 0.1 µg/kg bw per day for 2 years. Body weight and food consumption were measured throughout the study; haematological examinations and urinary analyses were performed on eight rats of each sex per group after 3, 12 and 23 months. Serum samples were collected twice during the study. The biochemical and histopathological examinations were extensive. Increased incidences of hepatocellular carcinoma, squamous-cell carcinomas of the lung, hard palate and tongue were observed at the highest dose. TCDD not only affected the incidence rates of cancer but had additional toxicological effects, particularly at the highest dose, which included increased mortality (females only), decreased body-weight gain, splenic and thymic atrophy, hepatic degeneration and necrosis. On the basis of increased urinary excretion of porphyrins and delta-aminolaevulinic acid and hyperplastic nodules in the liver in females at 0.01 µg/kg bw per day, the NOEL was 1 ng/kg bw per day, which, at termination, resulted in a concentration of TCDD of 540 ng/kg in fat and liver (Kociba et al., 1978b).

As part of a study of reproductive toxicity, groups of female rhesus monkeys were given diets containing TCDD at 5 or 25 pg/g diet for 3.5–4.0 years, providing doses of 0.15 and 0.67 ng/kg bw per day. Animals at the higher dose showed marginal signs of toxicity (Bowman et al., 1989; see section 2.2.5 for details).

2.2.4 Genotoxicity

Previous WHO expert groups have evaluated the genotoxicity of PCDDs, PCDFs and PCBs (IARC, 1987, 1997; van Leeuwen & Younes, 2000). Several short–term assays for genotoxicity with TCDD covering various end-points gave primarily negative results. Furthermore, TCDD did not bind covalently to mouse liver DNA.

TCDD did not induce mutations in Salmonella typhimurium strain TA98, TA100, TA1535, TA1537 or TA1538 with or without the addition of an exogenous metabolic activation system (Geiger & Neal, 1981; Mortelmans et al., 1984). Assays for Tk^+/– mutation in mouse lymphoma L5178Y cells gave variable results, the outcome depending on the mutant selection protocol used, methotrexate or thymidine selection leading to a positive response and ouabain or arabinose C selection leading to a negative response. Thioguanine selection resulted in a weakly positive response (Rogers et al., 1982; McGregor et al., 1991).

Sister chromatid exchange and micronuclei were found in human lymphocytes treated with TCDD, in the absence or presence of alpha-naphthoflavone (Nagayama et al., 1993, 1994).

In assays for cell transformation in C3H10T1/2 mouse and rat tracheal epithelial cells, TCDD increased the formation of foci in cells initiated with N-methyl-N-nitro-N-nitrosoguanidine (Abernethy et al., 1985; Tanaka et al., 1989). TCDD also transformed Ad12-SV40-immortalized cells but not primary human epidermal keratinocytes, as revealed by growth in soft agar, and increased foci formation, cell density and the carcinogenic response in nude mice. The transformed cells caused a 100% incidence of squamous-cell carcinomas when injected into nude mice and 0% in control mice (Yang et al., 1992).

TCDD did not induce unscheduled DNA synthesis in normal mammary epithelial cells (Eldridge et al., 1992).

OCDD did not induce mutations in S. typhimurium (Zeiger et al., 1988), and 1,2,3,6,7,8-HeCDD and 1,2,3,7,8,9-HeCDD did not transform C3H10T1/2 mouse cells (Abernethy & Boreiko, 1987).

TCDD did not bind to mouse liver DNA (Turteltaub et al., 1990), but it induced DNA single-strand breaks in the liver and in peritoneal lavage cells in rats (Wahba et al., 1988, 1989; Alsharif et al., 1994).

When administered concomitantly with 12-O-tetradecanoylphorbol 13-acetate, TCDD increased the cell transforming capacity of peritoneal macrophages in mice in a dose-dependent manner (Massa et al., 1992).

TCDD enhanced alpha-naphthoflavone-induced sister chromatid exchange frequency in cultured rat lymphocytes (Lundgren et al., 1986). It did not induce sister chromatid exchange, micronuclei or chromosomal aberrations in mouse bone marrow (Meyne et al., 1985) or in lymphocytes of persons who had been exposed to high concentrations of TCDD (Reggiani, 1980; Tenchini et al., 1983; Zober et al., 1993).

TCDD increased the mutagenic and recombinogenic activity of N-ethyl-N-nitrosourea in the mouse spot test by twofold (Fahrig, 1993).

In lacI transgenic rats, TCDD increased neither the mutation frequency nor the mutation spectrum in the liver (Thornton et al., 2001). Similarly, TCDD did change the spontaneous spectrum of H-ras codon 61 point mutations in mouse liver, nor did it affect the mutation spectrum of H-ras mutations in hepatocellular adenomas and carcinomas after treatment of mice with vinyl carbamate (Watson et al., 1995).

2.2.5 Reproductive toxicity

In a three-generation study of reproductive toxicity, male and female Sprague-Dawley rats (16 males and 32 females in the control and high-dose groups; 10 males and 20 females at the low and intermediate doses) were maintained on diets containing TCDD designed to deliver a dose of 0, 0.001, 0.01 or 0.1 µg/kg bw per day. After 90 days on diet, F₀ rats were bred to produce the F_1a generation and then again, 33 days after weaning, to produce the F_1b generation. The F_1b and F₂ litters were mated when the animals were about 130 days of age to produce the F₂ and F₃ generations, respectively. Fertility, litter sizes and neonatal survival were severely decreased for animals at the highest dose at the F₀ matings and in ensuing generations at the intermediate dose. Although slight effects were seen on pup survival and renal morphology at the low dose, they did not occur consistently across all generations. The NOEL was 0.001 µg/kg bw per day (Murray et al., 1979). The steady-state body burdens of TCDD of the rats at the two lower doses were estimated to have been 0.029 µg/kg bw and 0.29 µg/kg bw, respectively (Peterson et al., 1993).

Groups of 30 pr