T-2 and HT-2 toxins are type-A trichothecene mycotoxins, which are closely-related epoxy sesquiterpenoids. Surveys have revealed the presence of T-2 and HT-2 toxins in grains such as wheat, maize, oats, barley, rice, beans, and soya beans as well as in some cereal-based products. T-2 and HT-2 toxins have been reported to be produced by Fusarium sporotrichioides, F. poae, F. equiseti, and F. acuminatum. The most important producer is F. sporotrichioides, a saprophyte (i.e. not pathogenic to plants) which grows at –2 to 35 °C and only at high water activities (above 0.88). In consequence, T-2 and HT-2 toxins are not normally found in grain at harvest but result from water damage to the grain such as may occur when it remains for extended periods in the field at or after harvest, especially in cold weather, or in grain that becomes wet during storage.

T-2 toxin is the trivial name for 4beta,15-diacetoxy-3alpha,dihydroxy-8alpha-[3-methylbutyryl-oxy]-12,13-epoxytrichothec-9-ene, which is shown in Figure 1. Corresponding to the molecular formula C₂₄H₃₄O₉, its relative molecular mass is 466.5 g/mol. 15-Acetoxy-3alpha,4beta-dihydroxy-8alpha-[3-methylbutyryloxy]-12,13-epoxytrichothec-9-ene is the systematic name of HT-2 toxin. The molecular formula is C₂₂H₃₂O₈, and the relative molecular mass is 424.5 g/mol. The structures of T-2 and HT-2 toxins differ only in the functional group at the C-4 position. As T-2 toxin is readily metabolized to HT-2 toxin, these two mycotoxins were evaluated together.

Neither T-2 nor HT-2 toxin has been evaluated previously by the Committee.

2.1.1 Absorption, distribution, and excretion

T-2 toxin is metabolized extensively in the small intestine. [³H]T-2 toxin at a dose of 5 nmol/L (2.3 µg) or 500 nmol/L (230 µg T-2 toxin plus 2.3 µg [³H]T-2 toxin), both in 0.5 ml of an ethanol:saline solution, was injected into semi-intact isolated jejunal loops from female Sprague-Dawley rats. Blood draining from the loops was collected and analysed by gas–liquid chromatography–mass spectroscopy at various times. Both T-2 and HT-2 toxin were found in the blood within 1 min after injection of T-2 toxin into the lumen of the intestinal loop. Only 2% of the dose of 5 nmol/L was recovered in plasma as unchanged T-2 toxin after 50 min, while 25% was recovered as HT-2 toxin, 4–7% as 3’-OH-HT-2 toxin, and smaller amounts as 3’-OH-T-2 toxin, T-2 tetraol, and 4-deacetylneosolaniol. No glucuronide or sulfate conjugates were detected. Similarly, less than 1% of the dose was recovered as T-2 toxin in the intestinal lumen, while 15% was recovered in the intestinal lumen as HT-2 toxin and 5% as 3’-OH-HT-2 toxin. The dose of 500 nmol/L caused extensive tissue damage, but the ability to metabolize T-2 toxin was retained. Intestinal fluid extracted from the jejunal loops was incubated with T-2 toxin at either 5 or 500 nmol/L for 50 min. Less metabolism was seen than in intact preparations: after 50 min of incubation with intestinal fluid alone, 14% of the administered dose was recovered as HT-2 toxin at the 5 nmol/L concentration and 12% at 500 nmol/L. The rest of the dose was recovered as T-2 toxin, and no other metabolites were observed. Filtration (0.45-µm pore) to remove bacteria from the intestinal fluid did not appreciably reduce the amount of T-2 toxin that was deacetylated to HT-2 toxin in 50 min (Conrady-Lorck et al., 1988).

T-2 toxin was also deacetylated to HT-2 toxin in sheep rumen. When T-2 toxin at a dose of 0.02 mg/mL was incubated with sheep rumenal fluid for 0.5, 1, 2, or 3 h, it was deacetylated at a rate of 1.7 mg/L-h. The proportion of a 100-µg dose of T-2 toxin recovered as HT-2 toxin increased over 0.5, 1, 2, and 3 h of incubation with sheep rumenal fluid, to approximately 50% HT-2 toxin by 3 h. Gas chromatography (with a flame ionization detector) was used to detect T-2 toxin and HT-2 toxin. HT-2 toxin was the only T-2 toxin metabolite analysed in the culture medium (Kiessling et al., 1984).

Intestinal de-epoxidation of T-2 toxin also occurs, with deacetylation to HT-2 toxin in rats. De-epoxy HT-2 toxin was the predominant metabolite observed when T-2 toxin (at 0.1 mg/mL) was incubated with rat caecal microorganisms for 4 days. T-2 tetraol, 4-deacetylneosolaniol, T-2 toxin, HT-2 toxin, and T-2 triol were not found in the culture medium after 4 days (Swanson et al., 1987).

No studies of the systemic bioavailability of T-2 or HT-2 toxin were available. As T-2 toxin is extensively metabolized in the small intestine, however, the bioavailability of unchanged T-2 toxin may be quite low and the predominant absorbed chemical species may be HT-2 toxin (Conrady-Lorck et al., 1988; Kiessling et al., 1984; Swanson et al., 1987).

Placental transfer of T-2 toxin to fetal tissues was observed after intravenous injection of [¹⁴C]T-2 toxin to dams. Preferential distribution of T-2 toxin to thymus and spleen rather than liver was observed in fetuses after intraperitoneal administration of T-2 toxin to dams (Lafarge-Frayssinet et al., 1990).

In 6-week-old broiler chicks fed a diet containing T-2 toxin at 2 mg/kg for 5 weeks and then intubated with a single dose of [³H]T-2 toxin at 0.5 mg/kg bw, the radiolabel reached a maximum concentration in most tissues 4 h after dosing; the exceptions were muscle, skin, and bile, in which the maximum level was reached after 12 h. After 48 h, the chicks contained the equivalent of 39 µgkg of T-2 toxin and/or its metabolites in the muscle and 40 µg/kg in liver, as calculated on the basis of the specific activity of the radiolabelled T-2 toxin administered (Chi et al., 1978a).

In a weanling cross-bred pig (weighing 7.5 kg bw) intubated with [³H]T-2 toxin at a dose of 0.1 mg/kg bw, the percentage of administered radiolabel 18 h after dosing was 0.7% in muscle, 0.43% in liver, 0.08% in kidney, 0.06% in bile, 22% in urine, and 25% in faeces. The percentage of administered radiolabel 18 h after dosing in another pig intubated with T-2 toxin at 0.4 mg/kg bw was 0.7% in muscle, 0.29% in liver, 0.08% in kidney, 0.14% in bile, 18% in urine, and 0.86% in faeces (Robison et al., 1979a).

Four hours after intravenous administration of [³H]T-2 toxin to pigs, the largest amount of radiolabel was located in the gastrointestinal tract (15–24% of the dose), and 4.7–5.2% of the dose was found in the remaining tissues; muscle accounted for 2.9–3.2% of the dose and liver for 0.7–1.7% (Corley et al., 1986).

The fate and distribution of an intramuscular dose of 1.04 mg/kg bw of [³H]T-2 toxin was studied in guinea-pigs. Except in the large intestine and bile, the amount of radiolabel had peaked by 30 min and rapidly declined, with no measurable long-term accumulation. The tissue distribution was expressed in intervals over a 30-min to 672-h period, with six animals per time, as picomoles per milligram of tissue. Three hours after dosing, the distribution of T-2 toxin-derived radiolabel corresponded to 850 pmol/mg of tissue (wet weight) in kidney, 970 pmol/mg in liver, 490 pmol/mg in lung, 570 pmol/mg in spleen, 280 pmol/mg in adrenals, 630 pmol/mg in fat, 230 pmol/mg in heart, 430 pmol/mg in muscle, 440 pmol/mg in testis, and 160 pmol/mg in brain (equal to 400, 450, 230, 260, 130, 290, 110, 200, 200, and 77 µg/g of tissue). At 3 h, the distribution of T-2 toxin-derived radiolabel corresponded to 290 pmol/ml of plasma and 2700 pmol/ml of bile. At 72 h after dosing, the distribution of T-2 toxin-derived radiolabel corresponded to 90 pmol/mg in kidney, 100 pmol/mg in liver, 61 pmol/mg in lung, 59 pmol/mg in spleen, 49 pmol/mg in adrenals, 39 pmol/mg in fat, 36 pmol/mg in heart, 51 pmol/mg in muscle, 41 pmol/mg in testis, and 24 pmol/mg in brain (equal to 43, 47, 28, 28, 23, 18, 17, 24, 19, and 11 µg/g of tissue). At this time, the distribution of radiolabel corresponded to 88 pmol/ml of plasma and 39 000 pmol/ml of bile (equal to 41 and 18 000 µg/L) (Pace et al., 1985).

In pigs given T-2 toxin at 1.2 mg/kg bw by intra-aortal injection, the plasma and tissue concentrations of T-2 toxin decreased rapidly. By 4 h after dosing, no T-2 toxin was detected in any of the tissues examined (analysis by gas–liquid chromatography; limit of quantification, 40 ng/g). However, at 1, 2, and 3 h after injection, larger amounts of T-2 toxin were seen in spleen than in either kidney or muscle. T-2 toxin was not detected in liver at any time (Beasley et al., 1986).

A lactating Jersey cow weighing 375 kg was given 180 mg of T-2 toxin orally, equal to 0.48 mg/kg bw per day, by capsule for 3 days and was then given 160 mg of [³H]T-2 toxin, equal to 0.42 mg/kg bw. Although almost all the administered dose was eliminated within 72 h, measurable tritium remained in the bile, liver, and kidney (equivalent, respectively, to 27, 18, and 14 µg/kg of tissue) 3 days after dosing. These concentrations are higher than those in whole blood (13 µg/kg), plasma (10 µg/kg), and other tissues, including the spleen (9.4 µg/kg), heart (10 µg/kg), mammary gland (11 µg/kg), ovaries (11 µg/kg), muscle (8.8 µg/kg), and fat (4.7 µg/kg) (Yoshizawa et al., 1981).

Excretion was measured in faeces and urine collected for 6 days after oral, intravenous, and dermal administration of [³H]T-2 toxin to rats at a dose of 0.15 or 0.6 mg/kg bw. After oral administration, > 95% of the administered radiolabel was excreted within 72 h. After intravenous administration, similarly rapid elimination of the dose of 0.15 mg/kg bw was seen, but < 80% of the dose of 0.6 mg/kg bw was eliminated within 72 h. Extensive vascular damage was seen at the higher dose in these animals. After dermal application, < 60% of either dose was excreted within 72 h. More radiolabel was excreted in faeces than in urine with all routes and both doses. After oral administration, faecal excretion accounted for approximately 80% of the administered dose. A similar ratio (80:20, faeces:urine) was seen with the dose of 0.15 mg/kg given intravenously (Pfeiffer et al., 1988).

In 6-week-old broiler chicks given [³H]T-2 toxin by crop intubation at a dose of 0.5 mg/kg bw, the radiolabel peaked at 4 h in most tissues but at 12 h in muscle, skin, and bile. Bile contained the highest concentration of radiolabel of the tissues examined over the 48-h observation period. The pattern of distribution and excretion indicated that biliary excretion dominated (Chi et al., 1978a).

Biliary excretion was also found after intramuscular injection of [³H]T-2 toxin at a dose of 1 mg/kg bw to guinea-pigs. The concentration in bile at 12 h was 540 000 pmol/ml (equal to 250 000 µg/L), whereas that in plasma was 290 pmol/ml (equal to 140 µg/L). Over the first 5 days 75% of the administered radiolabel was excreted in urine and faeces at a ratio of 4:1. The amount peaked in urine at 24 h; 99% of the administered dose was eliminated by 28 days (Pace et al., 1985).

Radiolabel was transmitted into the eggs of laying hens that had been intubated gastrically with a single or several doses of [³H]T-2 toxin. In birds given a single dose of 0.25 mg/kg bw, the maximum residues were found in eggs 24 h later; the yolk contained 0.04% of the total dose and the white contained 0.13%. In birds given 0.1 mg/kg bw per day for 8 days, radiolabel accumulated in eggs until the fifth day of dosing, remained unchanged until the last day of dosing, and rapidly decreased thereafter. The maximum percentage of administered radiolabel recovered in egg white and yolk after the repeated doses was 0.41% and 0.28%, respectively. Assuming that birds weighing 1.6 kg consumed 100 g of the diet containing T-2 toxin at 1.6 mg/kg each day, the concentration of residues (T-2 toxin and/or its metabolites) in the contaminated eggs would be about 0.9 µg/egg (Chi et al., 1978b).

A pregnant Holstein cow was given 182 mg of T-2 toxin, equal to 0.5 mg/kg bw per day, for 15 days, and milk was sampled on days 2, 4, 5, 8, 10, and 12 after initiation of treatment. T-2 toxin was detected at concentrations of 10–160 ng/kg of milk. A sow was given T-2 toxin in feed at a concentration of 12 mg/kg, equivalent to 0.48 mg/kg bw per day, for 220 days, and its milk was analysed on day 190 of treatment. T-2 toxin was found at a concentration of 76 ng/g. No analysis for metabolites was reported (Robison et al., 1979b). The Committee noted that the purity and source of the T-2 toxin were not reported.

T-2 toxin was found at a concentration of 2 ng/ml in cows’ milk after administration of 160–180 mg/day for 4 days (equivalent to 0.42–0.48 mg/kg bw per day) (Yoshizawa et al., 1981).

2.1.2 Biotransformation

Metabolic transformations of T-2 toxin and HT-2 toxin that have been demonstrated in animals are shown in Table 1.

Male Sprague-Dawley rats received [³H]T-2 toxin at a dose of 0.15 or 0.6 mg/kg bw by oral, intravenous, or dermal administration, and 16 peaks in urine and faecal extracts were compared with known metabolite standards by high-performance liquid chromatography (HPLC) to identify radiolabel associated with T-2 toxin, HT-2 toxin, 3’-OH-HT-2, 3’-OH-T-2, neosolaniol, T-2 tetraol, and 4-deacetylneosolaniol. Averaged over all doses and routes, 68% of the recovered radiolabel in urine was associated with T-2 toxin (5.6%), HT-2 toxin (8.9%), 3’-OH-HT-2 (29%), 3’-OH-T-2 (3.2%), and T-2 tetraol (21%). On the basis of retention times, de-epoxy 3’-OH-T-2 triol, de-epoxy 3’-OH-HT-2, and 3’-OH-T-2 triol were also tentatively identified in excreta. The relative conversion rates to the identified metabolites were not affected by dose, but higher relative rates of conversion to T-2 tetraol, HT-2 toxin, and de-epoxy tetraol and lower rates of conversion to 3’-OH-HT-2 were seen after intravenous than after oral administration. More de-epoxy 3’-OH-HT-2 was found in orally dosed animals than those treated intravenously. In general, de-epoxidation appeared to be an important route of detoxication. T-2 toxin represented a greater percentage of the recovered radiolabel in urine over time, suggesting slow release from cutaneous fat stores. An increase over time in the percentage of recovered radioactivity as the de-epoxy metabolites was also observed (Pfeiffer et al., 1988).

C-4 deacetylation of T-2 toxin to HT-2 toxin was demonstrated in microsomal preparations of liver, kidney, and spleen from various species. The reaction rates of hepatic microsomes from various species, expressed in nmol/mg protein per 10 min, were: rabbit, 3000; human, 330; mouse, 75; chicken, 55; rat, 36; and guinea-pig, 14 (equal to 1400, 150, 35, 26, 17, and 6 µg/mg of protein) (Ohta et al., 1977; Johnsen et al., 1986).

Metabolism of T-2 toxin to hydrolysates at the C-4, C-8, and C-15 positions and hydroxylation at the C-3´ position by liver homogenates from rats, mice, and monkeys were demonstrated in vitro. Hydrolytic transformation was also observed in hepatic homogenates from rabbits, pigs, and cows (Yoshizawa et al., 1984, 1985b).

Enzymic conversion of T-2 toxin to HT-2 toxin was examined in liver homogenates from male Wistar rats, in studies in which 0.5 µmol (230 µg) of T-2 toxin were added to 2 ml of a cytosol/microsomal fraction. Complete conversion to HT-2 toxin was demonstrated within 60 min, and no other metabolites were reported (analysis by gas chromatography (GC)–mass spectrometry (MS)). There was much less conversion to HT-2 toxin in liver cytosol, and no metabolism was reported in plasma. Co-incubation of T-2 toxin in a liver homogenate with paraoxon showed that inhibition of serine esterases completely blocked conversion of T-2 toxin to HT-2 toxin. Further analysis with serine esterases showed that carboxylesterase but not cholinesterase was effective in converting T-2 toxin to HT-2 toxin (Johnsen et al., 1986)

Inhibition of carboxylesterase with tri-ortho-cresyl phosphate (TOCP) in groups of 10 mice (strain and sex unspecificed) given T-2 toxin at 0.5, 1, 2, 3, or 4 mg/kg bw intravenously led to the death of all animals at 2, 3, and 4 mg/kg bw, but not at 0.5 or 1 mg/kg bw (Johnsen et al., 1986).

Glucuronide-conjugated HT-2 toxin was the main metabolite recovered from bile collected from isolated, perfused male Wistar rat livers. Only trace amounts of T-2 toxin were recovered as the glucuronide conjugate. Analysis was performed with gas–liquid chromatography–mass spectroscopy. beta-Glucuronidase was used to determine glucuronide conjugates (Gareis et al., 1986).

Glucuronide conjugates of T-2 toxin and metabolites were identified in the urine and bile of two sows after intravenous administration of [³H]T-2 toxin at 0.15 mg/kg bw. Urine was collected hourly for 4 h through a catheter, and bile was collected after the animals were killed at 4 h. High-performance thin-layer chromatography with comparison to standards was used to identify and quantify the metabolites. Glucuronide conjugates were determined with beta-glucuronidase for T-2 toxin, 3’-OH-T-2, neosolaniol HT-2, 3’-OH-HT-2, T-2 triol, 4-deacetylneosolaniol, and T-2 tetraol. De-epoxy derivatives were not determined. Less than 1% of the recovered T-2 toxin and < 10% of the recovered HT-2 toxin was unconjugated in either bile or urine. The main metabolite recovered in bile was conjugated T-2 toxin, followed by conjugated HT-2 toxin. Conjugated metabolites represented an average of 77% of the recovered radiolabel in bile, and all metabolites accounted for an average of 92% of the radiolabel. In urine, conjugated metabolites represented an average of 63% of the recovered radiolabel, with an average of 90% of the recovered radiolabel accounted for by metabolites (Corley et al., 1985).

2.1.3 Effects on enzymes and other biochemical parameters

(a) Effect on nutrients

The effect of T-2 toxin on intestinal absorption of monosaccharides was studied in rats. The absorption of 3-O-methylglucose was reduced one- to threefold after injection of T-2 toxin into the jejunal lumen or after intravenous injection. Jejunal function was impaired by specific damage to the active transport and diffusional movement of monosaccharides (Kumagai & Shimizu, 1988).

Three groups of 1-day-old chicks (10 chicks per group) were given diets containing T-2 toxin at a concentration of 0.5–15 mg/kg, equivalent to 0.06–1.9 mg/kg bw, for 3 weeks. Plasma vitamin E activity and hepatic vitamin A content were measured. A dose-dependent decrease in plasma vitamin E activity was observed, with a 65% decrease from that of controls in chicks fed the diet containing 15 mg/kg. This decrease was considered to be due to a reduction in the plasma concentration of lipoproteins, which are required for the transport of vitamin E (Coffin & Combs, 1981).

Several authors reported changes in the serum concentrations of glucose and essential elements in animals given T-2 toxin in the feed (for example, Rafai et al., 1995a; Weaver et al., 1978). The Committee noted that, as feed refusal was observed at all doses tested and decreased feed intake would result in changes in metabolism and energy use, observations such as these are difficult to interpret. Experimental designs that control for feed intake are needed before changes in metabolism and energy use can be assessed.

(b) Inhibition of protein synthesis

The effects of T-2 toxin on protein synthesis were studied in Swiss mice and hepatoma cell cultures. T-2 toxin was given as an intraperitoneal dose of 0.75 mg/kg bw per day for 3 or 7 days. Protein synthesis was inhibited in the animals and in cells obtained from bone marrow, spleen, and thymus. Protein synthesis was also inhibited in vitro in hepatoma cell cultures and phytohaemagglutinin-stimulated lymphocytes (Rosenstein & Lafarge-Frayssinet, 1983).

Chinese hamster ovary and African green monkey kidney (Vero) cell cultures were exposed to T-2 toxin at 0.01 or 1 ng/ml for 1 or 12 h. The cells showed morphological changes indicative of inhibition of protein synthesis, including disassociation of polysomes and matrix density, ballooning of the intracristal space, and malalignment of cristae in mitochondria. The Chinese hamster ovary cells had bleb formations of the plasma membrane, which indicates inhibition of protein synthesis (Trusal, 1985).

Protein inhibition was also observed in muscle, heart, liver, and spleen of rats that received intraperitoneal injections of T-2 toxin at 0.3, 0.75, or 2 mg/kg bw (Thompson & Wannemacher, 1990).

The effects of T-2 toxin on rat hepatocytes in culture were studied by adding it at 0.01 or 1 ng/ml for 1 or 12 h. The lower concentration caused 75% inhibition of protein synthesis within 1 h. At the higher concentration, the hepatocytes recovered from a 1-h but not a 12-h exposure. Cell damage (release of lactate dehydrogenase) lagged behind inhibition of protein synthesis, which was 90% at 1 ng/ml. Ultra-structural alterations were present in the endoplasmic reticulum and mitochondria. The rough endoplasmic reticulum showed degranulation, and mitochondria had translucent foci and electron-dense cores (Trusal & O’Brien, 1986).

T-2 toxin induced disaggregation of polysomes in HeLa cells (Liao et al., 1976). It interacted with the peptidyl transferase centre on the 60S ribosomal subunit and inhibited transpeptidation of peptide-bond formation. These results were consistent with the effects of an inhibitor of prolongation or termination of protein synthesis (Stafford & McLaughlin, 1973).

The ribosomal subunits of Myrothecium verrucaria, a producer of macrocyclic trichothecenes, were resistant to T-2 toxin. The authors concluded that the 60S subunits of eukaryotes are responsible for the sensitivity to T-2 toxin (Hobden & Cundliffe, 1980).

(c) Inhibition of nucleic acid synthesis

The incorporation of [³H]thymidine into DNA in cell lines from thymus was strongly inhibited by T-2 toxin at 10 ng/ml and slightly inhibited at 0.1–10 ng/ml. Concentrations of 0.1–1 ng/ml caused a transient increase in DNA polymerase and alpha- and beta-terminal deoxynucleotidyl transferase activity, whereas concentrations > 1 ng/ml caused strong inhibition (Munsch & Mueller, 1980)

A single dose, three daily doses, or seven daily doses of T-2 toxin at 0.75 mg/kg inhibited DNA synthesis ex vivo in cell cultures from the spleen, thymus, and bone marrow of treated mice. T-2 toxin also inhibited DNA synthesis in vitro in cultures of hepatoma cells and in phytohaemagglutinin-stimulated lymphocytes (Rosenstein & Lafarge-Frayssinet, 1983).

The effects of T-2 toxin on DNA synthesis in phytohemagglutinin-stimulated human peripheral blood lymphocytes was assayed by incorporation of [³H]thymidine. Total inhibition was obtained with 8 ng /ml and 80% inhibition with 1.5 ng/ml (Cooray, 1984).

In synchronously dividing Tetrahymena cells, incorporation of radiolabelled thymidine and uracil into DNA and RNA, respectively, was inhibited. Neither DNA nor RNA synthesis nor RNA hybridase activity was altered by T-2 toxin in vitro in isolated nuclei from normal cells or from cells pretreated with T-2 toxin (Iwahashi et al., 1982).

Thymidine uptake and incorporation into DNA was biphasic, with increased uptake at 0.4 pg/ml reduced uptake at 4 pg/ml, and reduced incorporation at 40 pg/ml (Bunner & Morris, 1988).

(d) Alterations of cellular membranes

At a concentration of T-2 toxin of 20 µg/ml, no entry of [¹⁴C]sucrose or [³H]inulin was observed in bovine erythrocytes in vitro. Very little radiolabel was bound to bovine erythrocytes, and the binding was independent of the T-2 toxin concentration. The toxin had no effect on the entrapment of sucrose or inulin. Carrier erythrocytes retained 85% of [¹⁴C]sucrose and only 18% of [³H]T-2 toxin. Thus, T-2 toxin diffused from carrier cells more rapidly than sucrose. The authors concluded that the interaction of T-2 toxin with bovine erythrocytes was minimal and intercalation with the inner bilayer was unlikely, because the increase in cell volume that would have resulted did not occur (DeLoach et al., 1987).

The effects of T-2 toxin on membrane function were studied in L-6 myoblasts. The minimal effective concentration of T-2 toxin for reducing uptake of calcium and glucose and for reducing uptake of leucine and tyrosine and their incorporation into protein was 4 pg/ml. The uptake of rubidium was increased at 0.4 pg/ml and reduced at 4 pg/ml or more. Calcium efflux was reduced after 1, 5, and 15 min of exposure to T-2 toxin at a concentration of 40 pg/ml. The authors concluded that that T-2 toxin has multiple effects on membrane function at low concentrations and that these effects are independent of inhibition of protein synthesis (Bunner & Morris, 1988).

The effect of T-2 toxin on lipid peroxidation and the antioxidant status of liver and blood was examined in three species of fowl. As measured by malondialdehyde concentrations in liver homogenate and blood plasma, lipid peroxidation was increased in ducks, chickens, and geese given feed containing T-2 toxin at concentrations of 0.2–0.6 mg/kg (Mezes et al., 1999).

2.2.1 Acute toxicity

The LD₅₀ values for T-2 toxin and HT-2 toxin in several animal species are summarized in Tables 2 and 3. Strain and sex differences in death rates were seen.

Table 3. Results of studies of the acute toxicity of HT-2 toxin

Groups of five to seven male and female mice of strains ICR:CD1, BALB/c, C57Bl/6, and DBA/2 were given single doses of T-2 toxin at 2.5, 5, or 10 mg/kg bw by oral gavage. T-2 toxin was more lethal in C57Bl/6 and BALB/c strains and more lethal in the females of those strains. Five of six C57Bl/6 females and three of seven BALB/c females at 10 mg/kg bw but none of the males at this dose had died by 24 h, whereas one of six male and female DBA/2 mice at 10 mg/kg bw, one of six female BALB/c mice at 5 mg/kg bw, and none of the ICR:CD1 mice had died by 24 h. At 48 h, none of the male DBA/2 mice given the same doses of T-2 toxin had died. The mitotic index of intestinal crypt epithelial cells was depressed at all doses in all strains, with no difference by sex, strain, or dose. The apoptotic index in these cells varied by dose, was higher in C57Bl/6 mice than in the other three strains, and was reportedly generally greater in females (Li et al., 1997; Shinozuka et al., 1997a,b).

Sex differences were also observed after exposure by inhalation. When 9-week-old Swiss ICR mice weighing 15–20 g were exposed to T-2 toxin at a concentration of 120 µg/L of air for 10 min (dose per kg bw not estimated), adrenal lesions in the zona fasciculata were observed in 11 of 11 females and in none of 10 males. Extensive necrosis of cortical lymphocytes and necrosis of lymphoid cells in the follicles of the spleen were observed in both males and females (Thurman et al., 1986).

Adaptation to the local and systemic acute effects of T-2 toxin was observed in groups of eight CD-1 mice after dermal application to shaved areas on the back of 10 or 25 µg per animal weekly for 22 weeks. Initially, T-2 toxin caused necrosis, scarring, and sloughing of skin. Application of 10 µg caused reactions that healed within 2–3 weeks, and application 4 or 7 days later and weekly thereafter caused minor or no irritation. Furthermore, a lower acute mortality rate than expected was observed in the group given the higher dermal dose: at 25 µg, only 2/20 mice died after an intraperitoneal LD₅₀ dose (0.4 mg/kg bw), while 10/16 mice given the same intraperitoneal LD₅₀ dose died after having received 10 µg of T-2 toxin (Lindenfelser et al., 1974).

Newborn mice appeared to be more sensitive than adults to a single subcuta-neous dose of T-2 toxin (Ueno et al., 1973a); however, the LD₅₀ after oral adminis-tration did not differ with age in poultry. The birds died within 48 h of receiving T-2 toxin. Within 4 h, they showed asthenia, inappetence, diarrhoea, and panting. The abdominal cavities of birds given lethal doses contained a white chalk-like material that covered much of the viscera (Chi et al., 1977b).

Inhaled T-2 toxin was at least 10 times more toxic than orally administered material (Creasia et al., 1987). Surprisingly, no epithelial necrosis was reported after exposure to T-2 toxin by inhalation, although inflammation and necrosis were seen after dermal application or ingestion of high doses. The LC₅₀ of an aerosolized saline solution of T-2 toxin was 0.035 mg/L of air in mice (Creasia & Thurman, 1993).

Intraperitoneal injection of T-2 toxin caused myocardial damage in rats. A single dose of 2 mg/kg bw or four doses of 0.3 mg/kg bw per day caused focal necrosis of endothelial cells and other histological signs of cell damage (Yarom et al., 1987) Two months after the last of 10 daily intraperitoneal injections of T-2 toxin at 0.3 mg/kg bw, hypertrophy, focal fibrosis, and abundant cellularity were seen (Yarom et al., 1983).

The LD₅₀ of T-2 toxin dissolved in ethanol and administered intravenously was 1.2 mg/kg bw in normal, healthy, cross-bred pigs weighing 3–50 kg. Soon after administration, emesis was followed by eager consumption of feed, moderate posterior paresis, staggering gait, extreme listlessness, and frequent defaecation of normal stools. Between 1 and 6 h, severe posterior paresis, knuckling-over of the rear feet, and extreme lethargy were observed. These signs were followed by severe posterior paresis, frequent falling because of hind-quarter weakness, and dragging of both rear legs. Twenty-four hours after administration, the surviving pigs appeared normal (Weaver et al., 1978; Coppock et al., 1985).

Similar clinical signs were observed in pigs exposed to T-2 toxin by inhalation. Necrosis was present in the epithelial cells of the mucosa and in the crypt cells of the jejunum and ileum, the Peyer’s patches of the ileum, the lymphoid elements of the caecum, the lymphoid follicles in the spleen, and the germinal centre of the mesenteric lymph node (Weaver et al., 1978; Pang et al., 1988).

Eighteen white, cross-bred female pigs weighing 40–60 kg, immunized against Erysipelothrix rhusiopathiae, were given purified T-2 toxin dissolved in 70% ethanol intravenously at a dose of 0 (five pigs), 0.6 (five pigs), 1.2 (one pig), 4.8 (five pigs), or 5.4 (two pigs) mg/kg bw. The animals at 4.8 or 5.4 mg/kg bw died 5–10.5 h later, and the other animals were killed 12–24 h after treatment. Gross lesions were seen in pigs given doses > 1.2 mg/kg bw, consisting of oedema, congestion, and haemorrhage of the lymph nodes and pancreas and congestion and haemorrhage of the gastrointestinal mucosa, subendocardium, adrenal glands, and meninges. The histological alterations parallelled the gross lesions. Other lesions were widespread degeneration and necrosis of lymphoid tissue and the surface and crypt epithelium of the intestines. Scattered foci of necrosis were present in the pancreas, myocardium, bone marrow, adrenal cortex, and the tubular epithelium of the renal medulla. Most of the lesions were dose-dependent. The T-2 toxin-induced lesions in the lymphoid and gastrointestinal tract of pigs were similar to those described in other species. The heart and pancreas were additional target organs in pigs (Pang et al., 1987a).

Groups of 17 castrated male, cross-bred, specific pathogen-free pigs, 9–11 weeks of age, were used to characterize pulmonary and systemic responses to nebulized T-2 toxin (mixed with 100–200 µCi of technetium) at a dose of 9 mg/kg bw given by endotracheal intubation. The animals were exposed to aerosols in pairs, one animal receiving T-2 toxin and the other acting as a control. The pigs retained 20–30% of the T-2 toxin. Five pairs of animals were killed 1, 3, and 7 days after dosing; two pairs in which one treated pig died and the other was killed in a moribund state 0–10 h after dosing were designated 0.33-day groups. The treated pigs vomited after exposure and showed cyanosis, anorexia, and lethargy; they then became laterally recumbent. Alveolar macrophages showed reduced phagocytosis, and the blastogenic responses of pulmonary lymphocytes, but not peripheral blood lymphocytes, to mitogen were reduced. The lesions in the pigs that died included multifocal interstitial pneumonia, necrosis of lymphoid tissue, necrohaemorrhagic gastroenteritis, oedema of gall-bladder mucosa, and multifocal areas of necrosis in the heart and pancreas. Inhalation of T-2 toxin produced a clinical and morphological syndrome resembling that caused by intravenously administered T-2 toxin at doses > 1.2 mg/kg bw (approximate LD₅₀) or death. The lesions produced after inhalation were more severe than those seen after intravenous administration (Pang et al., 1987a).

2.2.2 Short-term studies of toxicity

Wistar rats fed T-2 toxin (purity not specified) at a concentration of 5, 10, or 15 mg/kg of feed, equivalent to 0.25, 0.5, and 0.75 mg/kg bw per day, for 4 weeks showed gastric lesions, which were diffuse and severe in the rats at the highest dose, focal but definite in those at 0.5 mg/kg bw per day, and negligible in rats at the lowest dose (Ohtsubo & Saito, 1977). The Committee noted that few experimental details were provided, e.g. on the purity of the T-2 toxin used, the way in which it was incorporated into the diet, and the number of rats per dose.

Twenty-four male Wistar rats received subcutaneous injections of T-2 toxin at 0.05 mg/kg bw per day for 28 days, and groups of six were examined at 7, 14, 21, and 28 days. Changes ranging from dystrophia or necrosis to hyperplasia were observed in the liver, kidney, and heart, with progression of severity with duration of exposure. Necrotic changes were observed from the third week. The changes in the kidney appeared to be more severe and occurred earlier (Sinovec & Jovanovic, 1993).

Groups of five to six female Holtzman albino rats were given diets containing T-2 toxin (purity not specified) for 3 weeks to 8 months at a concentration of 5 or 15 mg/kg (equivalent to 0.25 and 0.75 mg/kg bw per day) for up to 3 weeks and 10 mg/kg (equivalent to 0.5 mg/kg bw per day) during alternate 4-week periods for 8 months (4 weeks on the T-2 toxin-containing diet alternating with 4 weeks on control diet). The body weight of rats fed a diet containing 15 mg/kg feed for 19 days was markedly reduced. Slight growth depression was reported in rats fed a diet containing T-2 toxin at 5 mg/kg for 3 weeks. No gastric lesions were observed in any of the treated animals. Focal changes and cytoplasmic degradation (but no macroscopic abnormalities) were seen in the livers of four rats fed diets containing 5 mg/kg feed for 3 weeks followed by 15 mg/kg feed for 3 weeks. Severe inflammation around the nose and mouth were also seen at this time. After 8 months of alternating 4-week exposures to 10 mg/kg of diet, histopathological examination of the livers showed no evidence of toxic hepatitis, cirrhosis, neoplasia, or hyperplasia of either hepatocytes or cholangioles (Marasas et al., 1969).

Chickens were fed a diet containing T-2 toxin at a concentration of 1–16 mg/kg for 3 weeks. The T-2 toxin was extracted from a F. tricinctum culture and purified by the method of Burmeister (1971) to yield a crystalline product melting at 148–150 °C. Birds at 4, 8, and 16 mg/kg of diet showed reduced growth and developed yellow–white lesions in the mouth consisting of a fibrinous surface layer and a heavy infiltration of the underlying tissues by granular leukocytes. Escherichia coli and Staphylococcus epidermis were isolated from the lesions (Wyatt et al., 1972).

Groups of 36 broiler chicks aged 1 day to 9 weeks received a diet containing T-2 toxin (purity not specified) at a concentration of 0.2, 0.4, 2, or 4 mg/kg, equivalent to 0.025, 0.05, 0.25, and 0.5 mg/kg bw per day. Those at the highest concentration had reduced body-weight gain and feed consumption and developed oral lesions characterized by circumscribed, proliferating, yellow caseous plaques at the margin of the beak, the mucosa of the hard palate, and the tongue and the angle of the mouth. No lesions were observed in the bone marrow or in peripheral blood (Chi et al., 1977a).

In 1-day-old broiler chicks fed a diet containing T-2 toxin at 1, 2, 4, 8, or 16 mg/kg (equivalent to 0.125, 0.25, 0.5, 1, and 2 mg/kg bw per day) for 3 weeks, the growth rate, the weight of the pancreas, and the weight of the spleen were decreased at concentrations > 4 mg/kg. The T-2 toxin was extracted from F. tricinctum culture and purified by the method of Burmeister (1971) to yield a crystalline product melting at 148–150 °C. Oral lesions were seen at concentrations > 1 mg/kg (Wyatt et al., 1973).

T-2 toxin was administered to 10 laying hens at a concentration of 20 mg/kg of feed for 3 weeks, equivalent to 0.86–0.91 mg/kg bw on the basis of the reported weekly feed consumption during the study. The T-2 toxin was extracted from F. tricinctum culture and purified by the method of Burmeister (1971) to yield a crystalline product melting at 148–150 °C. In comparison with untreated controls, the T-2 toxin-treated hens had reduced feed consumption, body weight, and egg production and thinner egg shells (Wyatt et al., 1975).

Single-comb white Leghorn hens were fed a diet containing T-2 toxin (purity not specified) at 0.5, 1, 2, 4, or 8 mg/kg for 8 weeks. Feed consumption, egg production, and shell thickness were significantly decreased in hens fed the highest concentration. Furthermore, the hatchability of fertile eggs of hens fed 2 or 8 mg/kg of diet was lower than that of hens fed the control diet (Chi et al., 1977b).

Feed consumption and egg production were also reduced in groups of 10 laying hens, 33 weeks old, fed a diet containing T-2 toxin (purity not specified) at 2 mg/kg for 24 days. The total amount of T-2 toxin consumed per chicken per day was 0.2 mg on the basis of feed intake rates and the concentration of T-2 toxin in the feed. Body weights were not reported; however, the authors stated that there was no reduction in body weight (Diaz et al., 1994).

T-2 toxin was administered to four groups of four to six cats orally in gelatin capsules on alternate days at a dose of 0.06, 0.08, or 0.1 mg/kg bw per day, until death. The animals survived for 6–40 days. Emesis, anorexia, bloody diarrhoea, and ataxia were observed. The cats lost weight and became emaciated. The gross lesions observed included multiple petechiae to ecchymotic haemorrhages of the intestinal tract, lymph nodes, and heart. The lumen of the gut contained copious amounts of dark-red material. The microscopic lesions included haemorrhages in the gut, lymph nodes, heart, and meninges, necrosis of the gastrointestinal epithelium, and decreased cellularity of the bone marrow, lymph nodes, and spleen. The mean survival time was inversely related to the dose of T-2 toxin (Lutsky et al., 1978). The Committee noted that, given the substantial detoxication of T-2 toxin by glucuronide conjugation in rats, dogs, and pigs (Corley et al., 1985; Gareis et al., 1986; Sintov et al., 1986, 1987), the severe effects in cats at this low dose were likely to be a result of deficient glucuronide conjugation in this species. For example, as stated in Annex 1, reference 123, p. 40: "The urinary metabolites of benzoic acid were determined after oral administration of ¹⁴C-benzoic acid, mostly at a dose of 50 mg/kg bw, to a large variety of species, including primates, rodents, carnivores, reptiles, and birds.… Substantial amounts of the glucuronide of benzoic acid, in addition to hippuric acid, were detected in the urine of carnivores, except in cats (Bridges et al., 1970)." and "An outbreak of poisoning affected 28 cats that had eaten meat containing 2.39% benzoic acid. The effects were nervousness, excitability, and loss of balance and vision. Convulsions occurred, and 17 cats died or were killed. Autopsies showed damage to the intestinal mucosa and liver. The sensitivity of cats may be due to their failure to form benzoyl glucuronide, … (Bedford & Clarke, 1971)."

Groups of 10 pigs were fed a diet containing T-2 toxin at 0.0, 0.5, 1, 2, 3, 4, 5, 10, or 15 mg/kg for 3 weeks. On the basis of feed intake and weekly measurement of the T-2 toxin concentration in the feed, the 3-week average daily intakes were equal to 0.029, 0.062, 0.10, 0.13, 0.1, 0.08, 0.09, and 0.23 mg/kg bw per day, respectively. The T-2 toxin was extracted from a culture of F. tricinctum and determined to be > 90% pure by gas and liquid chromatography. Feed was refused at all concentrations tested. Decreased weight gain was observed at concentrations > 1.0 mg/kg of feed. Hyper- and parakeratosis, acanthosis, erosion, and inflammatory-cell infiltration were observed on histological examination of the skin near the mouth and snout of pigs at all doses. Clinical haematological parameters, such as serum enzyme activities and urea and glucose concentrations, were affected at 0.029 and 0.062 mg/kg bw per day (Rafai et al., 1995a). The Committee noted that the changes in clinical haematological parameters were not clearly dose-related and may have been due in part to reduced feed intake.

Nine young male pigs were fed a diet containing T-2 toxin (purity, 99%) at a concentration of 8 mg/kg for 30 days, equivalent to 0.64 mg/kg bw per day. Feed intake, weight gain, and haemoglobin concentrations were reduced in treated pigs. Serum alkaline phosphatase activity was also significantly decreased (Harvey et al., 1994).

Groups of five male Suffolk-Finn-Columbian lambs were fed diets containing T-2 toxin (purity, 99%) to give a dose of 0.0, 0.3, or 0.6 mg/kg bw per day for 21 days. All treated lambs developed focal hyperaemia and dermatitis at the mucocutaneous junction of the commissure of the lips, diarrhoea, leukopenia, lymphopenia, and lymphoid depletion of the mesenteric lymph nodes and spleen (Friend et al., 1983a).

Four calves were given T-2 toxin at a dose of 0.08, 0.16, 0.32, or 0.6 mg/kg bw per day orally in capsules for 30 days. The calf at the highest dose developed a hunched stance and died on day 20. Some evidence of mild enteritis was seen at all doses. Bloody faeces were observed at doses > 0.32 mg/kg bw per day. At necropsy, abomasal ulcers were present in the calf at 0.16 mg/kg bw per day and rumenal ulcers in the two calves given the two higher doses. Prothrombin times and the activity of serum aspartate aminotransferase were increased in calves at the two higher doses (Pier et al., 1976).

2.2.3 Long-term studies of toxicity and carcinogenicity

No standardized long-term studies of HT-2 toxin were available.

Groups of 50 male and 50 female weanling CD-1 mice were fed a semi-synthetic diet containing T-2 toxin (purity, 99%) at 1.5 or 3 mg/kg, equivalent to 0.22 and 0.45 mg/kg bw per day, for 71 weeks. The survival rate (reported in a graph) was consistently lower in the control groups of both males and females, beginning from about week 35; the survival rate of treated males at 71 weeks was 75% or more, whereas that of the controls was about 62%. No statistically significant differences among the groups were found in feed consumption or body-weight gain. A dose-related increase in heart weight was seen in males receiving T-2 toxin, being statistically different from controls at the high dose. No changes were seen in the heart weights of females, and no treatment-related weight changes were reported for other organs in either males or females. No treatment-related changes were reported in haematological parameters or in the response to sheep red blood cell challenge. A dose-related increase in the frequency of squamous mucosa hyperplasia was found in the forestomach of both male and female mice, with an increased frequency of hyperkeratosis. The incidence of pulmonary adenoma increased in a dose-related manner in males (10, 15, and 23% for controls and at the low and high dose, respectively) but not in females. The incidences of pulmonary adenomas and hepatic adenomas in males at the high dose were statistically significantly higher than in controls. The incidence of pulmonary adenocarcinoma was 5% in control males and 6% in males at the high dose (Schiefer et al., 1987). The Committee noted the lower survival rate of control rats.

Dermal application of 100 µg of 7,12-dimethylbenz[a]anthracene (DMBA) to 45 BALB/c mice, followed 1 week later by T-2 toxin at 0.5 µg, three times a week for 26 weeks, resulted in papillomas in eight mice and a carcinoma in one, with one papilloma in 15 mice that received DMBA alone. In the same study, 35 BALB/c mice were given dermal applications of 5 µg of T-2 toxin for 6 days, followed 1 week later by application of 12-O-tetradecanoylphorbol 13-acetate (TPA) or acetone three times a week for 26 weeks. No tumours were observed in either group. In 21 BALB/c mice, no papillomas were seen after DMBA or T-2 toxin alone [dosing regimen not translated from Chinese], but papillomas occurred in two of 22 mice treated with DMBA followed by T-2 toxin and four of 21 mice treated with T-2 toxin followed by TPA. One skin carcinoma was observed in mice treated with DMBA followed by T-2 toxin (Yang & Xia, 1988a,b).

Dermal application of 5, 10, or 20 µg of T-2 toxin to the backs of 20 mice (strain not specified) followed after 2 weeks by croton oil twice weekly for 10 weeks did not produce papillomas. Two of 20 mice developed papillomas after dermal treatment with DMBA followed 2 weeks later by 10 µg of T-2 toxin twice weekly for 10 weeks; however, the papilloma rate was not statistically different from that in DMBA-treated controls (Marasas et al., 1969). The Committee noted that the statistical test and the results for DMBA-treated mice were not reported.

In a promotion–initiation study in groups of eight CD-1 mice, an initial dose of DMBA, aflatoxin B₁, or T-2 toxin (25 µg) was applied to shaved areas on the back and was followed 4 days later by weekly applications of 10 or 25 µg of T-2 toxin to the same area for 22 weeks. One of eight mice treated with DMBA once and 25 µg of T-2 toxin for 22 weeks developed papillomas. No papillomas were observed in any other T-2 toxin-treated groups. Adaptation to the dermal effects of T-2 toxin was observed.: the initial T-2 toxin treatment caused severe skin reactions (necrosis and sloughing), while applications 4 or 7 days later and weekly thereafter caused only minor or no irritation. The authors concluded that T-2 toxin was not initiating but was a weak promoter (Lindenfelser et al., 1974)

Fifty male Kunming mice received T-2 toxin in ethanol:saline at a dose of 0.1 mg/kg bw per day three times per week by oral gavage for up to 25 weeks. Forestomach papillomas occurred in five of 35 treated animals, with one each at weeks 6 and 20 and three at week 25 of exposure. No papillomas of the forestomach were observed in 30 control mice (Yang & Xia, 1988a). The Committee noted that few experimental details were provided.

Groups of 16–22 female DDD mice were given feed containing T-2 toxin at a concentration of 0, 10, or 15 mg/kg, equivalent to 0, 1.5, and 2.2 mg/kg bw per day, for 12 months. Two mice from each group were killed at 3, 6, and 9 months. At 12 months, all controls were killed, and five mice in each treated group were fed a T-2 toxin-free diet for 3 months. Lesions were observed in the oesophageal region of the stomach of mice fed T-2 toxin, which included hyperkeratosis, acanthosis, and papillomatosis with inflammatory-cell infiltration of the squamous epithelium. These changes were found 13 weeks after the start of treatment and persisted throughout the 12-month feeding; however, most had subsided by 3 months after cessation of treatment. One adenocarcinoma of the glandular stomach and two hepatocellular carcinomas were observed in mice at the high dose. No papillomas of the forestomach were found (Ohtsubo & Saito, 1977). The Committee noted that few experimental details were provided, including the purity of the T-2 toxin used and the way in which it was incorporated into the diet.

A working group convened by IARC (1993) evaluated the experimental data on the carcinogenicity of T-2 toxin and concluded that it was not classifiable as to its carcinogenicity to humans (Group 3).

2.2.4 Genotoxicity

The results of studies on the genotoxicity of T-2 toxin are summarized in Table 4.

Hydroxyurea did not alter unscheduled DNA synthesis in cells treated with T-2 toxin or HT-2 toxin at 6 ng/ml; however, the combination of rat liver microsomes and hydroxyurea increased the rate of unscheduled DNA synthesis in cells exposed to HT-2 toxin at 100 µg/L. The authors concluded that the microsomal drug-metabolizing enzyme system participates in the induction of DNA damage by T-2 toxin (Agrelo & Schoental, 1980).

2.2.5 Reproductive toxicity

No data were available on the reproductive toxicity of HT-2 toxin.

(a) Multigeneration study

A two-generation study of reproductive and developmental toxicity was conducted in which 90 female CD-1 mice were fed a semi-synthetic diet containing T-2 toxin (purity, 99%) at a concentration of 1.5 or 3 mg/kg, equivalent to 0.22 and 0.45 mg/kg bw per day. The body-weight gain of dams was similar in all groups. T-2 toxin had minimal, if any, effects on female reproduction and fetal development and was not teratogenic or fetotoxic. Offspring of dams at the higher concentration (through milk) had an initial depression of weight gain, but the weights by 6 weeks of age were reported to be within the normal range for Swiss outbred mice. The weights of the spleen of male offspring of treated dams were greater than that of male offspring of control dams (Rousseaux et al., 1986).

(b) Developmental toxicity

Groups of five female B6C3F₁ mice received T-2 toxin by gavage at a dose of 1.2 or 1.5 mg/kg bw per day on days 14–17 of gestation. On day 18, fetal liver cells were collected and pooled per dam for staining and flow cytometry. Depletion of cells expressing CD44 and CD45 antigens was observed. Subsequent analysis of a prolymphocyte-enriched culture of fetal liver cells exposed to T-2 toxin also showed selective elimination of a subpopulation of lymphocytes suggested to be of the CD45⁺ B-lineage. Similar reductions were found in CD44^lo and CD45R⁺ bone-marrow cells of adult mice exposed to T-2 toxin by gavage at 1.8 mg/kg bw per day, suggesting that B-cell precursors are a sensitive target for T-2 toxin (Holladay et al., 1995).

T-2 toxin was injected intraperitoneally at a dose of 0.5, 1, or 1.5 mg/kg bw into pregnant mice on day 7, 8, 9, 10, or 11 of gestation (number of mice per dose and per gestation day not reported). The two higher doses caused significant maternal mortality, fetal deaths, and fetal body-weight loss. Gross malformations were seen in 37% of the fetuses of dams given 1 mg/kg bw (eight litters) or 1.5 mg/kg bw (four litters) on day 10. The most frequent anomalies were bent, shortened, or missing tails and limb malformations including oligodactyly and syndactyly. Exencephaly, open eyes, retarded jaw, and skeletal malformations of the rib or vertebrae were also found (Stanford et al., 1975). The Committee noted that intraperitoneal injection of T-2 toxin might have resulted in high concentrations in the conceptus and that the malformations occurred at doses that were maternally toxic.

Pregnant CD-1 mice (18 litters per treatment group) were given T-2 toxin dissolved in propylene glycol intraperitoneally at a dose of 0.5 mg/kg bw on day 8 or 10 of gestation. Treatment induced grossly malformed fetuses, principally with tail and limb anomalies. A higher incidence of malformations was observed when T-2 toxin was combined with ochratoxin A at 4 mg/kg bw (Hood et al., 1978). The Committee noted that intraperitoneal injection of T-2 toxin might have resulted in high concentrations in the conceptus.

T-2 toxin dissolved in a 1:1 mixture of propylene glycol and 0.1 N sodium bicarbonate was administered intraperitoneally at a dose of 0.5 mg/kg bw alone or in combination with rubratoxin B at 0.4 mg/kg bw to pregnant CD-1 mice on day 1 of gestation. Only T-2 toxin caused gross malformations. The combination of toxins increased the adverse effects on fetal body weight and mortality rate but not the incidence or severity of the gross malformations (Hood, 1986). The Committee noted that intraperitoneal injection of T-2 toxin might have resulted in high concentrations to the conceptus.

Groups of 10–29 CD-1 mice received crystalline T-2 toxin dissolved in propylene glycol orally by gavage at a dose of 0, 0.5, 1, 2, 3, 3.5, or 4 mg/kg bw on day 9 of gestation. One control group was fed normally and another was starved for 36 h after injection of the vehicle. Fetal morphology and implantation loss were examined on day 18 of gestation. Three of 20 starved controls and 0/19, 1/10, 0/20, 3/20, 9/25, 10/16, and 21/29 of mice given T-2 toxin at 0.0, 0.5, 1, 2, 3, 3.5, and 4 mg/kg bw, respectively, died. T-2 toxin at doses of 3.5 and 4 mg/kg caused more maternal deaths than in the starved controls. Over the 9-day period, reduced feed consumption and weight gain were seen at 4 mg/kg bw, and reduced weight gain alone was seen at 3.5 mg/kg bw. The rate of fetal resorption was 6% in the starved control group and 100%, 73%, and 4% at 4, 3.5, and 0 mg/kg bw, respectively. More skeletal abnormalities were seen in the starved controls and T-2 toxin-treated mice at 3 mg/kg bw.

In a second experiment, a single dose of T-2 toxin at 3 mg/kg bw was given by oral gavage on day 6, 7, 8, 9, 10, 11, or 12 of gestation to CD-1 mice. Fetal morphology and implantation loss were examined on day 18 of gestation. Control groups for each treated group were starved for 36 h after administration of the vehicle. The fetuses of 279 dams were examined, but the number of dams per treated and control group and the number of animals that died before examination were not reported. The treated females lost more fetuses than controls, and there were more dead fetuses among litters treated on day 9 of gestation than on other days. Major skeletal defects (not defined) were reported to be more numerous in mice treated on day 7 of gestation than in those treated on other days or in starved controls. The authors concluded that a single oral dose of T-2 toxin in propylene glycol was primarily maternally toxic and embryolethal; the defective development was possibly secondary to the maternal toxicity (Rousseaux & Schiefer, 1987).

Wag rat dams were treated on days 14–20 of gestation with T-2 toxin at 0.1, 0.2, or 0.4 mg/kg bw per day by intraperitoneal injection or at 0.1 or 0.4 mg/kg bw per day in the diet. Feed intake, weight gain, and the numbers of treated and control dams were not reported. Four to 29 rat pups per dose and time of sacrifice were examined; no mention was made of efforts to control for litter effects. Differences in thymus weight were seen in all treated groups. In rat pups examined 1 day after birth, the thymus weights, expressed as a percentage of total body weight, were 30–39% lower in treated rats, with the exception of those treated at 0.1 mg/kg bw per day intraperitoneally, which had thymus weights 11% lower than those of controls. Liver weight as a percentage bw was reported to be higher in 1-day-old pups of dams dosed with 0.4 mg/kg bw per day intraperitoneally. No differences were seen in liver or spleen weights at other doses and times. One week after parturition, thymic atrophy was less marked, suggesting that the effect was transient. The lymphoblastic response of spleen T and B cells (but not thymus T cells) to phytohaemagglutinin, concanavalin A, and lipopolysaccharide was reduced in 4- and 6-day-old offspring of dams that received T-2 toxin at 0.2 mg/kg bw per day intraperitoneally on days 18–20 of gestation (Lafarge-Frayssinet et al., 1990). The Committee noted the lack of detail in reporting and the lack of statistical analysis of the any of the reported differences. In general, the differences were difficult to interpret.

(c) Reproductive endocrine effects

Groups of 10 New Zealand white rabbits were fed a diet consisting of ‘naturally infected’ wheat containing T-2 toxin at a concentration of 0.19 mg/kg, equivalent to 0.008 mg/kg bw per day, for 32 days. A control group was fed uncontaminated wheat. The treated animals were then given gonadotropin-releasing hormone to induce false gestation plus T-2 toxin for a total duration of T-2 toxin treatment of 50 days; progesterone levels were monitored during this time. The concentrations of T-2 toxin and zearalenone were determined by HPLC; no other toxins were assayed. Two animals died during the 32-day treatment period, and one animal died during the subsequent treatment, each of Staphylococcus aureus infection. No control animals died. No gross morphological differences were found in three treated and three control animals killed after 32 days. Three of the five animals given T-2 toxin and gonadotropin-releasing hormone showed abnormal progression of progesterone concentrations, and one of these animals died about 1 week after initiation of hormone treatment. Serum creatinine and alanine aminotransferase activity were higher in treated than in control animals, and the serum cholinesterase concentration was decreased. No differences in feed intake were seen. Body weights were not reported (Fekete & Huszenicza, 1993). The Committee noted the poor description of the experimental design, that naturally infected feed was used, and that the process of infection and the organism used were not described.

Perturbation of progesterone progression after stimulation of ovarian activity was reported in groups of four to five ewes given T-2 toxin (purity, 95%) at 0.005 or 0.015 mg/kg bw per day for 21 days orally by intubation. The authors concluded that progesterone cycling was affected at the higher dose (Huszenicza et al., 2000). The Committee noted that the report did not permit evaluation of the results. Examples of progesterone profiles were given, but no statistical analysis was reported.

Four heifers fed a diet that stimulated ruminal acidosis were given T-2 toxin (purity, 95%) at a dose of 0.025 mg/kg bw per day by oral intubation for 20 days after initiation of ovulation. Rumenal acidosis was induced in order to investigate an increased rate of abortions that had been observed in a dairy herd on a similar diet contaminated with T-2 toxin. Three heifers were fed the acidosis-stimulating diet without T-2 toxin; no heifers were fed diets that did not stimulate rumenal acidosis. The ovarian activity of the heifers was synchronized by administering 25 mg of prostaglandin F_2 intramuscularly 23 and 12 days before T-2 toxin and on the day of initiation of T-2 toxin treatment. Plasma progesterone was sampled on alternate days starting 12 days before T-2 toxin treatment and continuing 10 days afterwards. The authors reported that ultrasonography showed no difference in the number of antral follicles or the size of the dominant follicle. All the treated heifers ovulated, but the mean time to ovulation was significantly greater in T-2 toxin-treated animals when compared with their pre-treatment ovulation time and with the ovulation time of controls that did not receive T-2 toxin. The progesterone concentrations remained low for about 2 days longer after the last prostaglandin F_2 injection in the T-2 toxin-treated heifers, suggesting that, under acidotic conditions, a low dose of T-2 toxin can retard follicle maturation and ovulation (Huszenicza et al., 2000).

T-2 toxin inhibited testosterone secretion in gerbil testicular interstitial cells in vitro. The median inhibitory dose was 0.042 nmol/L, equivalent to 0.02 ng/mL (Fenske & Fink-Gremmels, 1990).

2.2.6 Special studies

(a) Cell proliferation and apoptosis in dermal tissues

Hairless WBN/ILA-Ht rats were given single applications of 10 µL of a 0.5 mg/mL solution of T-2 toxin on each of four dorsal skin areas. Control animals were treated with the vehicle, a 20% ethanol solution. Biopsies of treated skin were taken 3, 6, 12, and 24 h after application, and the number of cells staining for proliferating cell nuclear antigen (PCNA) was used as a measure of proliferation. Less epidermal basal-cell proliferation was seen in treated than control animals 3 h after treatment, and the number continued to decrease during the observation period. No degranulation of mast cells was seen, but the number of mast cells increased during the observation period, especially around the small vessels of the dermis. Basal cells showing acidophilic degeneration were noted 12 h after treatment. DNA fragmentation was analysed by tritiated thymidine-mediated dUTP-biotin nick end-labelling (TUNEL). Basal cells showing acidophilic degeneration were generally positive, indicating apoptosis. The number of these cells increased at the 12- and 24-h observation times (Albarenque et al., 1999).

(b) Haematological effects

Groups of five male CD-1 mice were given T-2 toxin orally by gavage at a dose of 0.1, 0.5, or 2.5 mg/kg bw per day for 2 weeks. Increased formation of macrophage colonies from cultured bone marrow was observed, but no change was seen in the number of granulocyte or granulocyte–macrophage (GM) colonies. At 0.5 mg/kg bw per day, no change in macrophage colonies was observed in bone-marrow culture, but an increased number of granulocyte colonies and a decreased number of GM colonies were observed. At 2.5 mg/kg bw per day, there was no change in the number of granulocyte colonies, but an increased number of macrophages and a decreased number of GM colonies were seen. Increased spleen weight and decreased thymus weight (both relative to body weight) and a decreased red blood cell count were observed at the highest dose. No change was seen in the leukocyte count at any dose. Exposure of gGM progenitor cells from unexposed mice to T-2 toxin inhibited their proliferation into granulocyte or GM colonies at concentrations > 1 nmol/L (0.47 ng/ml) and inhibited their proliferation to macrophage colonies at concentrations > 3 nmol/L (1.4 ng/ml) (Dugyala et al., 1994).

Groups of 10 male BALB/c mice received T-2 toxin by subcutaneous injection at a dose of 0.17, 0.3, 0.44, 0.66, 1, or 1.5 mg/kg bw 1 h before an intraperitoneal injection of ⁵⁹Fe. Blood was drawn 24 and 72 h after ⁵⁹Fe injection. Dose-dependent inhibition of ⁵⁹Fe uptake into erythrocytes was seen at doses of T-2 toxin > 0.3 mg/kg bw at both 24 and 72 h. The mean response to the lowest dose was also lower than that of controls but was not statistically significant. The leukocyte count was affected in a dose- and time-dependent manner. In a separate experiment, groups of four to six male BALB/c mice received T-2 toxin at the same doses, followed 3, 24, or 72 h later by withdrawal of blood for leukocytre counting. Dose-dependent leukocytosis was observed 3 h after injection of T-2 toxin, with increases of several hundred per cent in the count at doses > 0.66 mg/kg bw. Conversely, 24 h after injection, the leukocyte count was less than 50% that of controls at the same doses. By 72 h after T-2 toxin injection at doses > 0.3 mg/kg bw, the leukocyte count was numerically lower but was not significantly lower than that of controls. The erythrocyte count was not affected at the doses tested, nor was the serum Fe concentration changed by treatment, suggesting that the effect on Fe incorporation occurred at the level of erythrocyte formation. The lowest statistically significant level of effect on ⁵⁹Fe uptake in circulating erythrocytes was 0.3 mg/kg bw (Faifer & Godoy, 1991).

The proliferation of colony-forming units (CFU) GM colonies was assessed in the bone marrow of groups of eight to ten male BALB/c mice that received a single subcutaneous injection of T-2 toxin at 0.17, 0.3, 0.6, or 1 mg/kg bw. All doses caused a decrease in the number of colonies 1 h after dosing. The effect was transient, with a slight increase 72 h after exposure. Significant inhibition of ⁵⁹Fe uptake in circulating erythrocytes was seen at doses > 0.3 mg/kg bw 1 h after dosing, with non-significant inhibition at 0.17 mg/kg bw. A significant rebound to levels of ⁵⁹Fe uptake above that of controls was seen even at the lowest dose 72 h after treatment (Faifer et al., 1992).

Recovery after T-2 toxin treatment occurred more readily in spleen than in bone marrow in male BALB/c mice. A single subcutaneous dose of 2 mg/kg bw reduced ⁵⁹Fe uptake into erythrocyte precursors in bone marrow and spleen. The uptake into spleen returned to control levels by 3 days, but that into bone marrow was depressed for 15–21 days. The cellularity of spleen and femur marrow had decreased 1 day after T-2 toxin injection, but that of the femur returned to normal and that of the spleen was increased to 200% of the control value by day 6. By day 35, the cellularity of the spleen had returned to normal (Velazco et al., 1996).

Repeated doses of T-2 toxin at 2 mg/kg bw per day for 3 days to groups of five or more male BALB/c mice did not cause additional damage to their blood-forming capacity over that seen with single doses. The repeated doses caused similar cellular depletion in the spleen and femur marrow, with a similar recovery period (Velazco et al., 1996). Furthermore, repeated doses did not inhibit the haematopoietic response to bleeding, consisting in removal of approximately one-third of the total blood volume on each of two successive days, the volume being replaced by saline. Rather, there was an increased response, measured as total nucleated cellularity and total erythroid cellularity in spleen up to 35 days after T-2 toxin treatment (Godoy et al., 1997).

Twenty male weanling outbred Swiss mice were fed a dry diet containing purified crystalline T-2 toxin at a concentration of 20 mg/kg for 41 days, and another group of four animals received the same diet for 21 days, followed by control diet for 7 days. One control group of 20 animals was maintained on restricted diet; a second control group consisting of eight animals was given the diet ad libitum; and 12 animals were killed at day 0. Haematological examinations were made weekly of treated and control animals. During the first 3 weeks of treatment, lymphoid tissues, bone marrow, and splenic red pulp became hypoplastic, resulting in anaemia, lymphopenia, and eosinopenia. Subsequently, during continued treatment, regeneration occurred, leading to hyperplasia of the haematopoietic cells by 6 weeks. All treated animals also developed perioral dermatitis and ulceration of the gastric mucosa. The authors concluded that T-2 toxin is irritating and suppresses haematopoiesis; however, the haematopoietic effects were transient at the dose tested and did not lead to haematopoietic failure (Hayes et al., 1980).

Incubation of CFU-GM from the bone marrow of rats with T-2 toxin and HT-2 toxin at 1 nmol/L (equivalent to 0.47 ng/ml) for 7, 10, or 14 days inhibited the growth of the cells (Parent-Massin & Thouvenot, 1995).

Hartley guinea-pigs (number not stated) given T-2 toxin dissolved in ethanol by intramuscular injection at the 24-h LD₅₀ dose of 1 mg/kg bw showed decreased activities of all coagulation factors except fibrinogen. Platelet aggregation in the whole blood response to ADP and collagen was depressed. The animals also showed an initial rise, followed by a fall in erythrocyte volume fraction, leukocytosis, and a fall in the platelet count. These changes, which were found within a few hours of administration, reached a maximum at 24 h and returned to normal over the next 2 days. Pretreatment with vitamin K₁ did not prevent the effects of T-2 toxin on coagulation. Addition of the toxin to plasma and blood of untreated guinea-pigs at a concentration of 1 mg/mL did not affect clotting times or platelet aggregation, indicating that T-2 toxin itself did not have a direct effect on the activity of coagulation factors (Cosgriff et al., 1984).

Eight New Zealand white rabbits were given T-2 toxin dissolved in dimethyl sulfoxide by intravenous injection at 0.5 mg/kg bw; and five rabbits were given a single oral dose of 2 mg/kg bw by gavage. The rabbits treated intravenously showed reductions in both packed cell volume and total leukocyte count, but no significant alterations in haematological parameters were seen in rabbits given T-2 toxin orally. In another study, nine New Zealand white rabbits were given a single intravenous injection of T-2 toxin dissolved in dimethyl sulfoxide at a dose of 0.5 mg/kg bw; five animals received daily subcutaneous injections of vitamin K at a dose of 0.5 mg/kg bw per day for 5 days before administration of the same dose of T-2 toxin for the subsequent 4 days. Two groups of eight rabbits served as controls. Blood samples were taken from each animal before treatment with dimethyl sulfoxide and 6–96 h later. The concentrations of coagulation factors VII, VIII, IX, X, and XI were decreased by about 40% within 6 h of administration of T-2 toxin, and the fibrinogen content was elevated at 24 h. The reduction in coagulation factors did not induce clinical haemorrhage, however, and administration of vitamin K did not alter the effects of T-2 toxin, indicating that the effect of the toxin on coagulation was not due to antagonism of vitamin K (Gentry & Cooper, 1981).

Cats given T-2 toxin subcutaneously or in the feed showed leukocytosis followed by leukopenia. The frequency of dosing and the dose were different for individual cats, and there were few cats per dose, so that the dose–response relationship could not be analysed. When three cats were given T-2 toxin subcutaneously at 0.05 mg/kg bw per day for 12 days, a transient increase in leukocyte count was seen in two cats within the first few days. This transient increase was followed by a decrease in all cats, to < 20% of the initial values by the last day of dosing. Of the three cats, two died within 35 days after the last injection. Deaths occurred at all doses tested (Sato et al., 1975).

Extracted, purified T-2 toxin was administered to cats in gelatin capsules every 2 days at a dose of 0.08 mg/kg bw per day (six cats) or 0.1 mg/kg bw per day (four cats). Pancytopenia and death occurred within 6–24 days for all cats (Lutsky et al., 1978).

Ten male cats were anaesthetized with ketamine and given T-2 toxin orally by capsule at a dose of 0.08 mg/kg bw every 48 h until death; two cats received the vehicle only. Blood was drawn at each administration. All 10 treated cats but neither of the controls died within 32 days of initiation of treatment, with an average survival time of 21 days. The clinical signs included vomiting, bloody faeces, weakness, lassitude, ataxia, dyspnoea, dehydration, loss of weight, and pre-terminal anorexia. Progressive decreases were observed in total leukocyte count, erythrocte volume fraction, haemoglobin, and thrombocyte count during treatment. Initial leukocytosis was observed in most cats. On gross examination, subcutaneous petaechiae, haemorrhagic lymph nodes, and multiple haemorrhagic erosions were observed in the gastric and intestinal mucosa. Petaechial haemorrhages and ecchymoses were observed on the myocardium (Lutsky & Mor, 1981).

The effects of T-2 toxin on blood coagulation were studied in groups of 40-day-old chickens fed diets containing T-2 toxin at a concentration of 1, 2, 4, 8, or 16 mg/kg, equivalent to 0.12, 0.25, 0.5, 1, and 2 mg/kg bw. Forty birds served as controls. The activities of factor X, prothrombin, and fibrinogen were reduced only at the highest concentration. The activity of factor VII was reduced at the three higher concentrations (Doerr et al., 1981).

In a study of the effects of ochratoxin and T-2 toxin, nine young pigs were given feed containing T-2 toxin (purity, 99%) at 8 mg/kg, equivalent to 0.64 mg/kg bw per day, for 30 days. Treatment reduced the haemoglobin concentration and serum alkaline phosphatase activity. No effect was reported on the erythrocte volume fraction or mean cell volume (Harvey et al., 1994).

The erythrocyte volume fraction was reduced in young pigs fed a diet containing T-2 toxin extracted from a culture of F. tricinctum (> 90% pure by gas and liquid chromatography) at a concentration of 3 mg/kg of diet, equivalent to 0.13 mg/kg bw per day. Groups of two 7-week-old pigs (sex unspecified) received diets containing T-2 toxin at concentrations equivalent to doses of 0, 0.029, 0.062, 0.10, or 0.13 mg/kg bw per day for 21 days. A dose-related reduction in leukocyte count was observed at all doses. The haemoglobin concentration was also decreased in a dose-related manner, at doses ž 0.062 mg/kg bw per day. A reduction in the erythrocyte count was observed at 0.10 and 0.13 mg/kg bw per day (Rafai et al., 1995b). The Committee noted that the changes observed might have been due to reduced feed intake.

Two calves were given T-2 toxin by stomach tube at a dose of 0.2 mg/kg bw per day for 11 days. There were no controls. The treated animals developed clinical signs of weakness and inappetence, and one died. Prothrombin time was prolonged in both animals, and one had marked neutrophilia. No haemorrhagic syndrome was found (Patterson et al., 1979).

Nine male cynomolgus monkeys received a single intramuscular injection of T-2 toxin at a dose of 0.65 mg/kg bw (LD₂₀ dose). Three monkeys served as controls. Haematological parameters were measured before injection and 0, 6, 12, and 24 h and 2, 3, and 7 days after treatment. Samples of 8 ml of blood were drawn through a heparinized catheter implanted surgically 7 days before treatment. The monkeys wore leather restraints for the duration of surgery, recovery, and the observation period; they were observed for signs of toxicity and particularly for evidence of haemorrhage. Three of the treated monkeys died < 24 h after treatment, and two more died during the observation period; none of the controls died. The animals that died were necropsied, and necrosis of lymphoid tissues and petaechial haemorrhage of the colon and heart were observed. A parallel decline in erythrocyte volume fraction (10–25% over 3 days) was observed in both the surviving treated and control monkeys. Transient leukocytosis was seen, the neutrophil and lymphocyte counts in treated animals being four to five times those before treatment and in controls. The lymphocyte counts returned to normal within 1 day and those of neutrophils within 3 days. Slight lymphopenia was seen at days 2 and 3. Prolongation of prothrombin and activated thromboplastin times and decreased activities of multiple coagulation factors were also observed within hours of administration, which reached a maximum at 24 h and returned to normal over the next 3 days. Fibrin–fibrinogen degradation products were not detected at any time. The platelet counts of treated animals varied nonsignificantly over the observation period but were significantly increased in controls. Despite the changes in haematological parameters, none of the surviving animals had clinical signs of haemorrhage (Cosgriff et al., 1986).

Three male and two female adult rhesus monkeys were given T-2 toxin in 20 ml of milk, initially at 1 mg/kg bw per day for 4 days and at 0.5 mg/kg bw per day on days 5–15. Three males and three females served as controls. All three treated males died of respiratory failure between days 0 and 15. After 30 days’ recovery, the two treated females and two additional males received T-2 toxin at 0.1 mg/kg bw per day for 15 days. All monkeys given 1 mg/kg per day showed signs of toxicity similar to those of alimentary toxic aleukia in humans, i.e. vomiting, apathy, and weakness of the lower limbs. The signs were more severe in males, which also developed petaecheal haemorrhages on the face. All males developed severe leukocytopenia, follicular atrophy of the spleen and lymph nodes, and pneumonia, suggesting involvement of the immune system. Bone-marrow changes were not found at necropsy. All animals at 0.1 mg/kg per day developed leukocytopenia and mild anaemia after 15 days of treatment (Rukmini et al., 1980).

Development of burst-forming unit–erythroid (BFU-E) colonies was scored in cells harvested from human umbilical cord after addition of T-2 toxin or HT-2 toxin to the culture medium for 14 days. The size of the colonies was measured microscopically, and the degree of cell differentiation was measured from the total porphyrin and haemoglobin content. The concentrations tested were 0.1, 0.5, 2.2, and 10 nmol/L (equivalent to 0.047, 0.23, 1.1, and 4.7 ng/ml) for T-2 toxin and 0.1, 0.5, 2.5, and 100 nmol/L (equivalent to 0.042, 0.21, 1.1, and 42 ng/ml) for HT-2 toxin. Significant differences in colony growth were observed at 0.1–2.5 nmol/L of HT-2 toxin, with no difference in porphyrin or haemoglobin concentrations, even though growth was inhibited. No colonies survived at the highest dose of HT-2 toxin (100 nmol/L). No differences in growth were observed with T-2 toxin up to a concentration of 2.2 nmol/L, but the highest concentration caused complete loss of BFU-E colonies. Intermediate doses did change the porphyrin or haemoglobin concentrations, although no dose–response relationship was evident. At the lowest dose of T-2 toxin (0.1 nmol/L), the haemoglobin concentration was greater than that of controls, and at 0.5 nmol/L a decrease in porphyrin concentration was observed (Rio et al., 1997). The Committee noted that the number of individuals on which the mean values were based was not reported.

In cells harvested from human umbilical cord, inhibition of CFU-GM colony growth was observed after 7 days of culture with T-2 toxin at a concentration of 0.1 nmol/L (equivalent to 0.047 ng/ml); however, no inhibition was observed after 10 and 14 days. At 2 nmol/L, T-2 toxin inhibited colony growth at 7, 10, and 14 days. With HT-2 toxin, a concentration of 1 nmol/L (equivalent to 0.42 ng/ml) inhibited growth at 7 days of culture, with no inhibition at day 10 or 14. At 10 nmol/L (equivalent to 4.2 ng/ml), HT-2 toxin inhibited growth at 7, 10, and 14 days (Parent-Massin et al., 1994).

Similar results were obtained for growth of rat and human CFU-GM colonies. GM progenitors were collected from the marrow of rats and from the umbilical cord of humans and were cultured and incubated with T-2 toxin and HT-2 toxin for 14 days. The median inhibitory concentrations (IC₅₀) for rat CFU-GM at 14 days were 2.6 nmol/L for T-2 toxin (equivalent to 1.2 ng/ml) and 2.2 nmol/L for HT-2 toxin (equivalent to 0.93 ng/ml), and the values for human CFU-GM were 1.4 nmol/L (equivalent to 0.65 ng/ml) for T-2 toxin and 1.8 nmol/L (equivalent to 0.76 ng/ml) for HT-2 toxin (Lautraite et al., 1995, 1996).

When platelets isolated from 12 healthy volunteers were incubated with T-2 toxin at doses of 5–500 µg/10⁹ platelets for 20 min, a concentration-related inhibition of platelet aggregation was seen with various activators, including adrenaline, arachidonic acid, and collagen, with release of dense bodies consisting mainly of serotonin-containing granules. There was also a change in membrane permeability but no change in shape. No parallel inhibition of thromboxane synthesis or significant alterations in platelet calcium content were observed. The microtubular system was unaffected (Yarom et al., 1984a).

(c) Effect on vascular parameters

Intravenous administration of T-2 toxin to pigs at a dose of 4 or 8 mg/kg bw resulted in a shock syndrome characterized by reductions in cardiac output and blood pressure and increased plasma concentrations of adrenaline, noradrenaline, thromboxane B2, 6-keto-prostaglandin F_1a, and lactate. The pigs given the higher dose showed signs including persistent vomiting, watery diarrhoea, abdominal straining, cold extremities, coma, and death (Lorenzana et al., 1985).

In the bovine ear perfusion system in vitro, T-2 toxin caused dose-dependent vasoconstriction of the peripheral vasculature but was a less potent vasoconstrictor than histamine or noradrenaline. The presence of known histamine or noradrenergic antagonists did not affect the response to T-2 toxin (Wilson & Gentry, 1985). T-2 toxin administered systemically markedly increased peripheral vascular resistance in conscious rats. The cardiac output gradually decreased, eventually resulting in cardiovascular collapse and death (Feuerstein et al., 1985).

Lethal intravenous doses of T-2 toxin to male Sprague-Dawley rats reduced blood flow and increased vascular resistance in hindquarter, mesenteric, and renal vascular beds. The mean arterial pressure and heart rate were not significantly altered after an intravenous dose of 1 mg/kg bw. Two of five rats died within 5 h, and only one animal survived to 24 h after treatment. The maximum fall in blood flow in mesenteric and renal vascular beds occurred 4 h after injection of T-2 toxin (Siren & Feuerstein, 1986).

(d) Immunotoxicity

A single dose of T-2 toxin at 10 mg/kg bw given to female ICR:CD1 mice by oral gavage induced apoptosis in thymus and spleen, as measured by TUNEL. The spleen and thymus weights were decreased, as were the lymphocyte and platelet counts in blood. Hypocellularity was observed in bone marrow and spleen, with specific depletion of myelocytes in bone marrow due to loss of immature granulocytes, erythroblasts, and lymphocytes (Shinozuka et al., 1997a,b, 1998, 1999). TUNEL, indicating apoptosis, was also seen after treatment of female ICR:CD1mice with T-2 toxin at 2.5 mg/kg bw by oral gavage (Shinozuka et al., 1997a,b) or intraperitoneal injection of female BALB/c mice at 2.5 or 5 mg/kg bw (Ihara et al., 1997; Sugamata et al., 1998).

Thymic atrophy and apoptosis were observed in groups of female BALB/c mice after a single intraperitoneal dose of 1.8 or 3.5 mg/kg bw. A dose of 0.35 mg/kg bw per day did not affect thymus weight or cellularity. The percentage of DNA fragmentation, as a measure of apoptosis, was increased with T-2 toxin treatment at 1.8 mg/kg bw per day but was reduced in animals that received a protein synthesis inhibitor (cycloheximide) 5 min after T-2 toxin, suggesting that protein synthesis may be necessary for the toxicity of T-2 toxin to thymus cells (Islam et al., 1998a).

Depletion of fetal liver B lymphocytes was observed in the offspring of female B6C3F₁ mice exposed on days 14–17 of gestation to T-2 toxin by oral gavage at a dose of 1.2 or 1.5 mg/kg bw per day for 4 days. Fetal livers were collected on day 18 of gestation for cell flow cytometric analysis. A specific subpopulation of CD44⁺ lymphocytes (CD44^lo) was reduced in fetal livers of dams at 1.5 mg/kg bw per day. The number of CD45R⁺ lymphocytes was decreased at both 1.2 and 1.5 mg/kg bw per day. Subsequent analysis of a prolymphocyte-enriched culture of fetal liver cells exposed to T-2 toxin in vitro suggested selective elimination of a subpopulation of B-lineage lymphocytes, CD45R⁺. Similar reductions were seen in CD44^lo and CD45R⁺ bone-marrow cells in adult mice exposed to T-2 toxin by gavage at 1.8 mg/kg bw per day, suggesting that B-cell precursors represent a sensitive target for T-2 toxin (Holladay et al., 1993).

The proliferative response of lymphocytes to phytohaemagglutinin and lipopoly-saccharide was examined in male French IC mice treated with T-2 toxin (purity unspecified) at doses representing one-half or one-quarter of the LD₅₀ for 2 days or one-twelfth of the LD₅₀ for 15 days. No other information on doses was reported. Stimulation of both T and B cells was inhibited reversibly, and the ability to synthesize antibodies to sheep red blood cells was suppressed. The effects on lymphocytes and fibrosarcoma cells in culture included a direct cytostatic action at high concentrations and stimulation at low concentrations. The histological observations included severe lymphoid damage in the thymus and spleen. The immune system therefore appears to be sensitive to the trichothecenes and is impaired at doses that do not inhibit other organs (Lafarge-Frayssinet et al., 1979).

The proliferative response of spleen lymphocytes of female BALB/c mice to concanavalin A, phytohaemagglutinin, pokeweed mitogen, and lipopolysaccharide was examined in vitro and in vivo. Spleen lymphocytes from five mice were incubated with T-2 toxin at a concentration of 0, 0.25, 0.5, 0.75, 1, or 2.5 nmol/L (equivalent to 0.12, 0.23, 0.35, 0.47, and 1.2 ng/ml) and the mitogens. The lymphoproliferative response in vitro depended on the concentration of T-2 toxin and the mitogen; the response was increased with T-2 toxin at 0.5 nmol/L and concanavalin A and decreased with T-2 toxin at 2.5 nmol/L alone or with concanavalin A; decreased with T-2 toxin at concentrations > 0.75 nmol/L T-2 toxin and phytohaemagglutinin, pokeweed mitogen, or lipopolysaccharide. For exposure in vivo, groups of three mice were given intraperitoneal injections of T-2 toxin at 0, 0.8, or 1.6 mg/kg bw per day on days 0, 7, and 14; the spleens were harvested on day 15, and lymphocytes cultured with mitogen. In T-2 toxin treated groups, more lymphoproliferation was seen at 0.08 mg/kg bw per day (a 1000% increase) and less at 1.6 mg/kg bw per day, when compared with controls. When lymphocytes were cultured with mitogen, the lymphoproliferative response was decreased with T-2 toxin at 1.6 mg/kg bw per day plus phytohaemagglutinin, pokeweed mitogen, or lipopolysaccharide. In a separate experiment in which groups of three mice were immunized with keyhole limpet haemocyanin and Bordetella pertusis antigen by subcutaneous injection on days 1 and 8 and treated as in the initial experiment, the lymphoproliferative response was increased with T-2 toxin at 1.6 mg/kg bw per day and concanavalin A or phytohaemagglutinin (Paucod et al., 1990).

Male Swiss mice were fed a diet containing T-2 toxin (purity, 99%) at 5, 10, or 20 mg/kg, equivalent to 0.75, 1.5, and 3.0 mg/kg bw per day, for up to 48 days. A control group fed the same amount of food as that consumed by the mice at 20 mg/kg of diet was used to determine the effects of dietary restriction caused by feed refusal in the treated groups; an additional control group was fed ad libitum. Eight mice from each control and treated group were killed and examined after 7, 14, 21, 28, and 48 days of treatment. After 7 days, the spleen cell count was significantly lower in the pair-fed control group than in the group fed T-2 toxin at 20 mg/kg of diet, and was lower in all T-2 toxin-treated groups than in controls fed ad libitum. At subsequent times, the mean count in pair-fed controls was no different from that of the group given T-2 toxin at the highest concentration, and both were lower than in controls fed ad libitum. The lymphoproliferative response of the spleen to concanavalin A and lipopolysaccharide at the same times was significantly lower at 20 mg/kg of diet and in pair-fed controls than in the group fed ad libitum. The mean proliferative response to both mitogens was lower in the group given T-2 toxin at 20 mg/kg of diet than in the pair-fed control group, but the difference was not significant (Friend et al., 1983b).

The T-2 toxin metabolites 3’-OH-T-2 , HT-2 toxin, 3’-OH-HT-2, neosolaniol, and T-2 tetraol were tested for their ability to induce apoptosis in the thymus, assessed by DNA fragmentation analysis after an intravenous dose of 1.6 mg/kg bw to female BALB/c mice. 3’-OH-T-2, T-2 toxin, HT-2 toxin, and 3’-OH-HT-2 caused a significant increase in DNA fragmentation. On this basis, 3’-OH-T-2 was as effective as T-2 toxin in causing thymic apoptosis, while HT-2 toxin and 3’-OH-HT-2 were less effective. Neosolaniol and T-2 tetraol did not cause thymic cell apoptosis (Islam et al., 1998b).

Four male Swiss IC mice received T-2 toxin by intraperitoneal injection at a dose of 0.75 mg/kg bw per day for 7 days. On day 3 of treatment, the mice were immunized with sheep erythrocytes, and all animals were killed 5 days later. T-2 toxin decreased the antibody titres, as measured by haemagglutination stimulation, and reduced the thymic weight. In a second study, seven groups of five mice received T-2 toxin intraperitoneally at a dose of 0.5, 0.6, 0.75, 1, or 2 mg/kg bw per day for 7 days. The mice were immunized on day 3 and killed 5 days later. Antibody-producing cells from the spleen were counted as the number of plaque-forming cells on sheep erythrocytes. A dose-dependent inhibition of plaque-forming cells was observed in T-2 toxin-treated mice, with total suppression of the immune response at 2 mg/kg bw per day. A dose-dependent reduction in thymus weight and antibody titre to sheep red blood cells was seen at 0.5–1 mg/kg bw per day. The immune suppressive effect disappeared within 6 days after cessation of treatment.

In another experiment, 36 Swiss IC mice were treated intraperitoneally with T-2 toxin at 0.75 mg/kg bw per day for 7 days, and groups of four mice were killed at nine times over the subsequent 83 days. The mice were immunized with sheep red blood cells 5 days before being killed. The thymus weight and antibody titre to sheep red blood cells titre returned to the range seen in controls as early as 6 days after cessation of T-2 toxin treatment (Rosenstein et al., 1979).

Inhibition of cellular immunity by trichothecenes included effects on grafting. The mean length of survival of skin grafted from C57Bl/6 mice onto Swiss mice was 8.7 days in control recipients and 12 days in recipients treated with T-2 toxin at 0.75 mg/kg bw per day for 7 days before skin grafting and then three times a week for 20 days, indicating that T-2 toxin suppressed immunity, resulting in allograft rejection. The areas of the graft in T-2 toxin-treated mice lacked the typical cellular infiltrates of a cell-mediated immune response of macrophages and lymphocytes (Rosenstein et al., 1979).

Delayed hypersensitivity is an immune response mediated by sensitized T lymphocytes. The effect of T-2 toxin on T lymphocyte responses was studied in female BDF₁ mice sensitized by subcutaneous injection of sheep red blood cells followed by measurement of foot-pad swelling. No appreciable effect on delayed hypersensitivity was observed when mice received T-2 toxin at 3 mg/kg bw before or on the day of sensitization. However, wh