U. S. Food and Drug Administration
Center for Veterinary Medicine
November 9, 2001

The purpose of this document is to provide the scientific support behind our (CVM's) recommended maximum levels for fumonisins in animal feed (Table I). Fumonisins are environmental toxins produced by molds and found primarily in corn. The major types of fumonisins are B₁ (FB₁), B₂ (FB₂) and B₃ (FB₃).

Our goal was to identify fumonisin levels in feed that are adequate to protect animal and human health and that are achievable with the use of good agricultural and good manufacturing practices. We wish to emphasize that the recommended levels are intended to provide guidance that may change following public input and are not to be considered tolerances. Future research and/or different interpretations of existing research could change the recommended values.

These recommendations are the result of reviewing the published literature to determine the effects of fumonisins when fed to various animals, including horses, rabbits, catfish, ruminants, poultry and mink. There were many gaps in the literature regarding the feeding of low levels of fumonisins to animals. Although this compelled some extrapolation of the data to establish draft guidance levels for fumonisins in the diets of various species, all calculations are derived from factors found in the literature.

In six instances, we grouped species together because the animals seemed to have a similar sensitivity to fumonisins. This is an attempt to avoid a multitude of guidance levels and does not necessarily imply that the species are biologically similar.

Horses and rabbits were grouped together as the most sensitive species. Corn and corn by-products used in rations of horses and rabbits should contain less than 5 ppm of FB₁ + FB₂ + FB₃ and comprise no more than 20% of the dry weight of the total ration (Table I). The total ration should contain less than 1 ppm of FB₁ + FB₂ + FB₃ (0.2 x 5 ppm FB₁ + FB₂ + FB₃ = 1 ppm of FB₁ + FB₂ + FB₃).

Catfish and swine were grouped together as intermediate in sensitivity to fumonisins. Corn and corn by-products used in rations of catfish and swine should contain less than 20 ppm of FB₁ + FB₂ + FB₃ and comprise no more than 50% of the dry weight of the total ration (Table I). The total ration should contain less than 10 ppm of FB₁ + FB₂ + FB₃ (0.5 x 20 ppm of FB₁ + FB₂ + FB₃ = 10 ppm of FB₁ + FB₂ + FB₃).

Ruminants, mink and poultry were considered more resistant than horses, rabbits, catfish and swine to fumonisin; however, there was no data found in ruminants and mink at total dietary levels between 25 and 100 ppm of total fumonisins, while the data in poultry at these levels was more robust. Due to this data gap, we were more conservative in our recommendations for ruminants and mink than in poultry.

Corn and corn by-products used in rations of ruminants that are at least 3 months old and fed for slaughter and in rations of mink fed for pelt production should contain less than 60 ppm of FB₁ + FB₂ + FB₃ and comprise no more than 50% of the dry weight of the total ration (Table I). The total ration should contain less than 30 ppm of FB₁ + FB₂ + FB₃ (0.5 x 60 ppm of FB₁ + FB₂ + FB₃ = 30 ppm of FB₁ + FB₂ + FB₃).

Corn and corn by-products used in rations of poultry fed for slaughter should contain less than 100 ppm of FB₁ + FB₂ + FB₃ and comprise no more than 50% of the dry weight of the total ration (Table I). The total ration should contain less than 50 ppm of FB₁ + FB₂ + FB₃ (0.5 x 100 ppm of FB₁ + FB₂ + FB₃ = 50 ppm of FB₁ + FB₂ + FB₃).

The National Center for Toxicological Research (NCTR in Jefferson, AR) recently completed a chronic dietary bioassay with purified FB₁. This study showed clear evidence of kidney tumors in male rats and of liver tumors in female mice at dietary levels of 50 ppm and above.

We believe 15 ppm of FB₁ + FB₂ + FB₃ in the total ration of mink, ruminant and poultry breeding stock should provide adequate protection against any potential carcinogenic effects in these animals. This recommendation is based upon the NCTR chronic study where 15 ppm FB₁ produced the same or fewer kidney and liver tumors compared to the controls. Corn and corn by-products used in the rations of mink, ruminant and poultry breeding stock should contain less than 30 ppm of FB₁ + FB₂ + FB₃ and comprise no more than 50% of the dry weight of the total ration (Table I). If the recommended total fumonisin level in the total ration for a species was less than 15 ppm, we did not believe that the breeding stock of the species needed additional protection from possible carcinogenic effects.

The last grouping was of animal species/classes not mentioned above (e.g. dogs, cats). Often there was no published dietary study with fumonisins in these animals and no historical indication/association of problems from feeding corn. We believe 5 ppm of FB₁ + FB₂ + FB₃ in the total ration should provide adequate protection against any potential acute and/or carcinogenic effects in these animals. This recommendation is based largely upon the NCTR chronic study where 5 ppm FB₁ appeared to be the no-observed-adverse-effect level. Corn and corn by-products used in the rations of these animals should contain less than 10 ppm of FB₁ + FB₂ + FB₃ and comprise no more than 50% of the dry weight of the total ration (Table I).

We acknowledge that extensively validated "quick" or confirmation tests are not commercially available for total rations. However, the Association of Official Analytical Chemists International has established an official method (995.15) for determining fumonisins B₁, B₂ and B₃ in corn. In addition, the United States Department of Agriculture's Grain Inspection, Packers and Stockyards Administration (GIPSA) announced on June 5, 2001, that two test kits have been approved for official testing of fumonisins in the national grain inspection system. GIPSA authorized the use of the Veratox Quantitative Fumonisin Test kit, manufactured by Neogen Corporation, to determine fumonisins in corn, corn meal, popcorn, rough rice, corn/soy blend, and wheat; and RIDASCREEN^® FAST Fumonisin test kit, manufactured by r-Biopharm Inc., for fumonisins in corn, corn meal, sorghum, corn gluten meal, corn germ meal, and corn/soy blend. We believe that the recommended fumonisin levels will stimulate additional interest in developing and certifying/validating confirmatory tests and "quick tests" for determining fumonisins in corn, corn by-products, and complete animal feed rations.

The following is a summary of pivotal studies that were crucial in our decision making. While we reviewed each publication listed in the reference section, no attempt was made to summarize each article.

Experimental Reproduction of ELEM: A Study to Determine the Minimum Toxic Dose in Ponies

Wilson et al, Mycopathologia, 1992; 117: 115-120.

8 ppm FB₁ + 2.56 ppm FB₂ = 10.56 ppm FB₁ + FB₂ (fum) in concentrate (conc)
Phase 1 Dose: 10.56 mg fum/kg conc X .008 kg conc/kg bw = .085 mg fum/kg bw
Phase 2 Dose: 10.56 mg fum/kg conc X .016 kg conc/kg bw = .169 mg fum/kg bw

All 5 ponies consumed all their rations throughout the entire trial with few exceptions. Clinical chemistry parameters for the entire group were within normal ranges for the entire period. All 5 gained weight during Phase 1 (8 ppm FB₁ at 0.8% bw), but did not gain weight during Phase 2 (8 ppm FB₁ at 1.6% of bw).

One pony was euthanized on day 92 of Phase 1. It had exhibited mild symptoms of confusion, circling, incoordination, agitation in the stall, and facial twitching periodically during Phase 1.

The 4 remaining ponies were euthanized over a period of 2 weeks after Phase 2 was complete. During Phases 1 and 2, these ponies also exhibited periodic episodes of confusion, apathy, dullness, abnormal reactions to minor changes in handling patterns, hyperexcitability, incoordination, stupidity, head shyness, and depression. On necropsy, no significant gross lesions were noted in any ponies.

All 5 ponies had similar histopathological lesions including (1) mild, focal to diffuse, random, hepatocyte vacuolation, (2) mild, focal, interstitial infiltrates of mononuclear cells in the renal cortex and medulla, (3) focal, hemorrhagic foci (often perivascular) in transverse sections of the brain stem at the level of the vestibular, olivary, and hypoglossal nuclei (these foci were deep in the neuropile as well as adjacent to the floor of the fourth ventricle), and (4) reactive astrocytes, glial cells, and edema.

Clinical and Diagnostic Veterinary Toxicology (Buck et al, 1976, p 13) indicates growing colts weighing 90 kg can consume from 3.1-3.8% of their body weight (bw) in feed/day and mature horses can consume 1.5-2.0% of their bw in feed/day.

.085 mg fum/kg bw ÷ .038 kg of feed/kg bw = 2.24 mg fum/kg of feed = 2.24 ppm of fum in ration for growing colts.

.085 mg fum/kg bw ÷ .020 kg of feed/kg bw = 4.25 mg fum/kg of feed = 4.25 ppm of fum in ration for mature horses.

The above calculations suggest that growing colts could be poisoned with 2.24 ppm of fumonisins in a complete feed, while mature horses could be adversely effected by about 4.25 ppm of fumonisins in a complete feed.

The other pivotal study we used was by Ross et al in 1991 (J Vet Diagn Invest; 3: 238-241). This study analyzed feeds associated with 45 cases of equine leukoencephalomalacia (ELEM) from 1984-5 and 1989-90 for FB₁. Seven of 9 ELEM cases from 1984-5 and 32 of 36 cases from 1989-90 had at least one feed sample containing FB₁ at levels of > 10 ppm. The study also analyzed 19 horse feed samples representing 10 cases that were not associated with any disease problems and found FB₁ at levels ranging from <1 to 9 ppm, with only 2 samples containing >5 ppm FB₁.

Conclusions for Horses

Lack of Embryotoxicity of Fumonisin B₁ in New Zealand White Rabbits

The lowest observed adverse effect level for rabbits, based on the decrease in male kidney weights, was 0.1 mg of FB₁/kg bw/day (Table II). This gavage dose is approximately equal to 2.29 ppm of FB₁ in the diet as these 5-6 month old New Zealand White does averaged 3.9 kg and consumed 170 grams of chow per day (0.1 mg of FB₁/kg bw/day x 3.9 kg bw ÷ 0.17 kg of feed = 2.29 ppm FB₁ in the diet).

There also appear to be individual differences in maternal susceptibility to the effects of purified FB₁. The one rabbit (out of five in the dose-range finding study) treated with 1.75 mg/kg bw/day that survived to gestation day (GD) 29 showed no pathological lesions in the liver and only mild proliferative glomerulopathy in the kidney; serum ALT and AST were well within normal limits. Of the four other rabbits given this dose, two died, one aborted her entire litter, and the fourth was euthanized on GD 20 for Sa/So analysis. The rabbit that aborted was not examined further, but the two that died had pathological lesions in the liver, kidney and brain. The brain lesions consisted of leukoencephalomalacia and hemorrhage (Bucci et al, Natural Toxins, 1996: 4: 51-52). The animal that was sacrificed on GD 20 had elevated Sa/So ratios in serum and kidney while serum ALT and AST were over 10-fold higher than the upper limit of normal range. This animal also demonstrated pathological changes in the liver and kidney, but there were no changes in the brain.

Conclusions for Rabbits

The above calculation results in a concentration of 2.29 ppm fumonisin in the diet that could be harmful to rabbits. This level is very close to the 2.24 ppm of fumonisins in the diet of young colts that could theoretically be hazardous. Consequently, rabbits were grouped with horses.

Thus, as with horses, we recommend that if corn and corn by-products are used in rabbit rations, then the corn and corn by-products should contain less than 5 ppm of FB₁ + FB₂ + FB₃ and comprise no more than 20% of the dry weight of the total ration. Thus, the total ration should contain less than 1 ppm of FB₁ + FB₂ + FB₃ (0.2 x 5 ppm of FB₁ + FB₂ + FB₃= 1 ppm of FB₁ + FB₂ + FB₃). We believe that rabbits, like horses, should be considered sensitive to fumonisins and do not recommend that corn screenings be used in their rations.

Growth, Hematology, and Histopathology of Channel Catfish, Ictalurus punctatus, Fed Toxins from Fusarium moniliforme.

Lumlertdacha et al, Aquaculture, 1995; 130: 201-218.

Experimental diets were formulated to contain 0.3, 20, 80, 320, and 720 mg of FB₁/kg of diet by substituting ground corn cultured with F. moniliforme strain MRC 826 for ground, autoclaved noncultured corn in various proportions. The noncultured corn contained 0.7 mg of FB₁/kg while the cultured corn contained FB₁ and FB₂ at concentrations of 1,600 and 365 mg/kg, respectively. Diets contained 32% crude protein, 30% moisture and 3.1 kcal DE/g.

The 1-year fish (fry) were hatched from a single egg mass and reared in the hatchery on a starter diet for about 1 month. The fry, averaging 1.2 g, were fed the control diet for 4 days and then 50 fish were randomly assigned to each of four replicate aquaria for each of the experimental diets. The fry were fed to satiation four times daily for 10 weeks.

Survival was 100% for controls and fish fed 20 ppm FB₁, 99% for fish fed 80 ppm FB₁ and <30% in fish fed >320 ppm FB₁. At 20 ppm FB₁ for 10 wk, weight gain was significantly (P<.05) decreased (15% less than controls) and liver lesions were noted histologically. These lesions included foci of swollen hepatocytes with centrally located nuclei and foci of hepatocytes containing clear spherical vacuoles near the hepatic veins. When the livers were post fixed in osmium tetroxide, the vacuoles were osmiophilic, consistent with lipid. Foci of hepatocellular necrosis and shrunken hepatocytes were noted at >80 ppm FB₁.

Catfish Growth Not Affected by Low Levels of Mycotoxins Produced by the Mold Fusarium moniliforme, New Research Indicates.

Li and Robinson, Catfish J, 1995; June: 16.

Eight practical-type diets (32% protein; 2.8 kcal DE/g diet) were prepared to contain 0.7 (basal), 2.5, 5.0, 10.0, 20.0, 40.0, 80.0 and 240.0 mg FB1/kg. The basal diet contained 31% cooked corn that was substituted with F. moniliforme strain 826 cultured corn to give the desired FB₁ levels. The FB₁ level of corn was determined using HPLC, but there was no data provided on FB₂ or FB₃.

[Note: Assuming the FB₃ levels in the cultured corn from the Lumlertdacha et al (1995) study were 10% of the FB₁ levels, then one would estimate the 20 ppm FB₁ diet contained about 26.6 ppm of FB₁ + FB₂ + FB₃. Assuming the FB₂ and FB₃ levels from the Lumlertdacha et al (1995) study apply to the cultured corn in the Li and Robinson (1995) study (both studies cultured F. moniliforme strain MRC 826 on corn), then one would estimate the 10 ppm FB₁ diet in the Li and Robinson (1995) study contained about 13.3 ppm FB₁ + FB₂ + FB₃).]

Each of the 8 treatment groups was assigned 20 catfish each (average initial weight of 6.1 g/fish) in a completely randomized design. The fish were fed twice daily to approximate satiation for 12 weeks.

Significantly decreased (P<.05) weight gain, feed consumption and feed conversion ratio at >40 ppm FB₁.
Significantly decreased hematocrit at >80 ppm FB₁.
No apparent histologic changes in fish fed <20 ppm FB₁.
Significantly decreased survival rates at 20, 40 and 240 ppm FB₁. Fish fed 80 ppm FB₁ had survival rates that were lower than, but not statistically different than fish fed <10 ppm FB₁. The differences in survival rate may not have been related to dietary FB₁ levels.

Finally, Goel et al (Toxicology, 1994; 14: 207) measured sphingolipids in channel catfish dose with F. moniliforme culture material. The lowest dietary concentrations of fumonisin associated with significantly elevated ratios of sphinganine to sphingosine in kidney, serum, liver and muscle were 10, 20, 40 and 80 mg of FB₁/kg of diet, respectively.

Conclusions for Catfish

Due to these data gaps, we recommend that corn and corn by-products used in catfish rations contain less than 20 ppm of FB₁ + FB₂ + FB₃ and that the corn and corn by-products comprise no more than 50% of the dry weight of the total ration. Thus, the total ration should contain less than 10 ppm of FB₁ + FB₂ + FB₃ (0.5 x 20 ppm of FB₁ + FB₂ + FB₃= 10 ppm of FB₁ + FB₂ + FB₃).

Temporal and Dose-Response Features in Swine Fed Corn Screenings Contaminated with Fumonisin Mycotoxins

Thirty, 6-13 kg, weanling SPF male castrated crossbred pigs were used in this 14-day dietary study. Six nutritionally balanced test diets were prepared. Diets 1 to 5 contained 60, 30, 15, 7.5 and 0% of the 'contaminated' corn screenings. Each of diets 2-5 contained 'clean' corn screenings sufficient to bring the total corn screenings to 60% of the diet. Diet 6 contained ground corn. The concentrations of FB₁ + FB₂ in diets 1 through 6 were 175, 101, 39, 23, 5 and <1 ppm (below detection limit), respectively. This study had 5 replicates of 6 pigs each with 1 animal in each replicate randomly assigned to 1 of the 6 test diets.

The 'no-observed-adverse-effect level' (NOAEL) was determined by using linear regression to estimate the lowest fumonisin concentration which gave an estimated response just exceeding the upper 95% confidence limit on the fumonisin free control diet (diet 6, Table III).

Three of 5 pigs fed diet 1 developed clinical signs of respiratory distress on day 4 to 6 and were euthanized. The gross findings and histologic lesions were consistent with porcine pulmonary edema (PPE). The feed consumption (as percentage body weight ± SD) was decreased in pigs fed diet 1 at 3.6 ± 1% body weight compared to 5.6 ± 1.8% body weight in animals fed the control diet 6.

Liver injury was detected histologically in all pigs fed diets 1 and 2, in 3 of 5 pigs fed diet 3, and in 1 of 5 pigs fed diet 4 (23 ppm). Liver injury was characterized by moderate disorganization of hepatic cords and individual hepatocyte necrosis with nuclear pyknosis, karyorrhexis or karyolysis, and cytoplasmic hypereosinophilia. Hepatocytes often contained homogenous eosinophilic cytoplasmic inclusion bodies and occasionally were undergoing mitosis. There were scattered small accumulations of inflammatory cells, consisting primarily of macrophages.

The liver, lung, kidney and serum from pigs fed diets 1-4 had elevated sphinganine/sphingosine (Sa/So) ratios. The text indicates that 42% (8/19) of the tissue and serum samples from diet 5 had elevated Sa/So ratios, while the accompanying table only shows that 39% (7/18) were elevated. In the controls (diet 6), elevated Sa/So ratios were present in 17% (4/23) of the tissue and serum samples (Riley et al, Toxicol Appl Pharmacol, 1993; 118: 105-112).

We believe the NOAEL for the Motelin et al (1994) study could be increased by approximately 10% for the FB₃ that was likely present in the corn screenings but was apparently not measured. We believe the NOAEL for ALP of 12 ppm more likely represents statistical happenstance than biological significance. There were no statistically significant differences from control values for ALP throughout the study and ALP does not appear to us to be the most sensitive clinical pathology parameter.

We believe the Motelin et al (1994) study could be used to establish guidance at about 15 ppm FB₁ + FB₂ + FB₃ in the total diet of swine; however, we recognize that this was only a 14-day study. Also, Rotter et al (Natural Toxins, 1996; 4: 42-50 and Can J Anim Sci, 1997; 77: 465-470) have reported equivocal changes in cholesterol levels, carcass quality, and in overall weight gain in males in diets containing up to 10 ppm of purified fumonisin B₁ and fed for 8-11 weeks. Rotter et al (1996) reported significant increases (p<.000001) in the Sa:So ratios of the lung, liver and kidney in the 10 ppm FB₁ group. Lastly, the oral bioavailability of radiolabeled FB₁ in swine appears to vary widely as it ranged from zero (no radioactivity detected in serum) to almost 6% in the study by Prelusky et al (Nat Toxins, 1994; 2(2): 73-80).

Conclusions for Swine

Based on these studies, we recommend that corn and corn by-products used in swine rations contain less than 20 ppm of FB₁ + FB₂ + FB₃ and that the corn and corn by-products comprise no more than 50% of the dry weight of the total ration. Thus, the total ration should contain less than 10 ppm of FB₁ + FB₂ + FB₃ (0.5 x 20 ppm of FB₁ + FB₂ + FB₃ = 10 ppm of FB₁ + FB₂ + FB₃).

Effects of Fumonisin-Contaminated Corn Screenings on Growth and Health of Feeder Calves

Osweiler et al, J Anim Sci, 1993; 71: 459-466.

Eighteen crossbred Limousine X Angus-Hereford steers from one herd were assigned to diets containing either control (<5 ppm), low (33 ppm FB₁ + FB₂ + FB₃; assumes FB₃ = 2 ppm), or high (148 ppm FB₁ + FB₂ + FB₃) levels of total fumonisins. The concentrate consisted of approx. 87.5% corn (control group) or corn screenings (low and high fumonisin groups) and 12.5% of a pelleted supplement (Yoder Feeds) containing 38% crude protein in the control group.

Two supplies of corn screening (one containing 30-50 ppm and the other containing 150-200 ppm fumonisins), which were naturally contaminated with fumonisins, were available for the low and high fumonisin groups. These concentrates were fed at 2.0% of body weight (bw) for the 1^st week, then at the rate of 2.25% of bw for the remainder of the study. Each calf was individually penned and also offered alfalfa hay at about 0.75% of bw once daily.

The total diet of the control group contained <5 ppm total fumonisins. The total diet of the low fumonisin group contained about 24.6 ppm total fumonisins. The total diet of the high fumonisin group contained about 110.3 ppm total fumonisins (see Table IV below).

Gross Lesions/Histology on d 31: n=2 from control and high dose groups only.

Gross Lesion: Gastrohepatic lymph nodes in high dose group were mildly enlarged and edematous and contained petechial hemorrhages throughout. No significant lesions were found in control animals.

Histology: Microscopic lesions were not observed in controls. In the high dose group, the livers had undergone mild hydropic degeneration and cloudy swelling in a periacinar pattern throughout the liver. Red pulp in the spleen of both principals was depleted or red and white cells and contained sinusoidal-like spaces, in some cases filled with a protein-like material. These sinusoidal spaces were often surrounded by bundles of hypertrophied smooth muscle and elastic fibers. Changes observed were mild and considered reversible.

Baker and Rottinghaus (J Vet Diagn Invest, 1999; 11: 289-292) fed a corn-culture material mixture containing 328 ppm FB₁, 89 ppm FB₂ and 23 ppm FB₃ to three Holstein steers weighing 86-127 kg. Because only minimal effects were evident when this mixture was fed at 0.72% of body weight for 154 days, the amount of this mixture was slowly increased to 1.07% of body weight on day 161. The amount of FB1 consumed initially was 2.36 mg/kg bw and the dose was increased to 3.54 mg/kg bw after 23 weeks until necropsy on days 239-253. Assuming steers consume 3.0% of their BW per day, then the levels in this study equate to about 79 ppm FB₁ (106 ppm FB₁ + FB₂ + FB₃) in the diet during the first 154 days and 118 ppm FB₁ (158 ppm FB₁ + FB₂ + FB₃) in the diet for the last 78-92 days.

Gurung et al (J Anim Sci, 1998; 76: 2863-2870) added lyophilized cultures of Fusarium moniliforme M-1325 at approx. 2.5% to a basal diet so that the treated diet contained 95 ppm of FB₁. They fed this diet to 4 weanling (15 kg) Angora female goats for 112 days.

Edrington et al (J Anim Sci, 1995; 73: 508-515) added Fusarium moniliforme culture material to 1,000 mL of water and gavaged 4 Suffolk X Rambouillet wether lambs for 4 consecutive days at 11.1 mg of FB₁ + FB₂ + FB₃/kg body weight (BW). This daily gavage dose equals about 256 ppm in the diet for the 32 kg lambs.

Smith and Thakur (Adv Exp Med Biol, 1996; 392: 39-55) added Fusarium moniliforme M1293 culture material at about 7.4% so the treated diet contained approx. 194.2 ppm of FB₁ and 63.5 ppm of FB₂. The treated diet was fed at 2.0% of body weight to 3 Holstein steers (210-234 kg) for 30 days. Assuming steers consume 3.0% of their BW per day, then the levels in this study equate to 129.5 ppm FB₁ and 42.3 ppm FB₂ in the total diet.

Most of the major findings from the high dose group in the Osweiler et al study were also reported in the papers by Baker and Rottinghaus, Gurung et al, Edrington et al, and Smith and Thakur. Tubular nephrosis was reported in lambs, but not steers. No histology was reported by Gurung et al.

Conclusions for Ruminants

We recommend that corn and corn by-products used in rations of ruminants that are >3 months old and fed for slaughter contain less than 60 ppm of FB₁ + FB₂ + FB₃ and that they comprise no more than 50% of the dry weight of the total ration. Thus, the total ration should contain less than 30 ppm of FB₁ + FB₂ + FB₃ (0.5 x 60 ppm of FB₁ + FB₂ + FB₃ = 30 ppm of FB₁ + FB₂ + FB₃).

Dietary Fumonisins Disrupt Sphingolipid Metabolism in Mink and Increase the Free Sphinganine to Sphingosine Ratio in Urine but not in Hair.

Thirty-six adult, pastel female mink were randomly allocated into 3 groups and provided a basal mink diet supplemented with either 0% (control), 1.45% (low dose) or 2.90% (high dose) F. moniliforme M-1325 culture material containing 7300, 2200 and 697 mg/kg FB₁, FB₂ and FB₃, respectively. Samples of the culture material and diets were analyzed by HPLC/fluorescence for FB₁, FB₂ and FB₃ at the National Veterinary Service Laboratory (NVSL), Ames, IA. The mink were fed the control and fumonisin treated diets ad libitum for 100 days.

Analysis of the mink diets yielded FB₁ + FB₂ + FB₃ levels of <0.5 ppm (detection limit) for the control diet, 115 ppm for the low dose diet, and 254 ppm for the high dose diet. No clinical signs of toxicity were observed in the mink during the trial.

Urine sphinganine and sphingosine levels were increased markedly on Day 4 and 7 in the low dose group and on all 3 days of urine collection (Days 2, 4 and 7) in the high dose group. Urine sphinganine levels were 2.6-22.0 times higher than controls and urine sphingosine levels were 1.6-3.3 times higher than controls at these time points. Mean urine Sa/So ratios were 2.1-10.9 X greater than controls on Days 2, 4 and 7 in both treated groups.

Chronic Toxicity of Fumonisins from Fusarium moniliforme Culture Material (M-1325) to Mink

Sixteen non-sibling, 9-month-old, pastel, female mink (Mustela vison) were fed a basal diet that contained either no added culture material (controls) or a quantity of F. moniliforme (M-1325) culture material for 87 d. A sample of the control diet and of the F. moniliforme-containing diet (each consisting of 6 blended subsamples) were analyzed by HPLC/fluorescence for FB₁, FB₂ and FB₃ concentrations by the NVSL in Ames, IA. Levels of FB₁ + FB₂ + FB₃ in the control and fumonisin treated diets were <0.5 ppm and 119 ppm.

Feed consumption was not significantly different during the first 2 weeks and was not collected thereafter. Slight lethargy was noted in some mink in the fumonisin-treated group during the latter weeks of the trial. Although not statistically different, the mink fed the fumonisin-treated diet lost body weight over the 87-day trial while controls gained weight.

Several hematologic parameters (mean corpuscular hemoglobin concentration, plasma total solids, and lymphocyte concentration) and serum chemical concentrations (globulin, phosphorus, potassium, blood urea nitrogen, creatinine, bilirubin, and cholesterol) and activities (alkaline phosphatase, alanine aminotransferase, amylase, and aspartate aminotransferase) were significantly greater in the mink fed fumonisins than in the controls. Serum albumin/globulin and sodium/potassium ratios and chloride concentrations were significantly lower in the fumonisin-fed mink than in the controls.

The concentrations of free sphinganine and the ratio of free sphinganine to sphingosine in the liver and kidneys of the fumonisin-treated mink were greater than in the control mink. The mean sphinganine concentrations in the liver and kidneys of the fumonisin-treated mink were about 2.5 X and 50 X higher than in controls.

The brain, liver, spleen, heart, esophagus, lungs, kidneys, and adrenal glands were collected and weighed. Mean kidney weight as a % bw in treated mink was significantly less than controls (.521 vs. .572). Samples from the brain, liver, kidneys, spleen, lungs, heart, and esophagus were processed according to routine histologic procedures. No remarkable gross or histologic alterations were associated with fumonisin treatment.

Conclusions for Mink

We recommend that corn and corn by-products used in rations of mink fed for pelt production contain less than 60 ppm of FB₁ + FB₂ + FB₃ and that the corn and corn by-products comprise no more than 50% of the dry weight of the total ration. Thus, the total ration should contain less than 30 ppm of FB₁ + FB₂ + FB₃ (0.5 x 60 ppm of FB₁ + FB₂ + FB₃ = 30 ppm of FB₁ + FB₂ + FB₃).

Effects of Feeding Fusarium moniliforme Culture Material, Containing Known Levels of Fumonisin B₁, in the Young Turkey Poult.

Three hundred and sixty day-old female Nicholas Large White poults were allotted randomly to pens in a stainless steel chick battery and allowed to consume feed and water ad libitum. The experimental design consisted of 10 dietary treatments with six pen replicates of six birds allotted randomly to each dietary treatment. The day-old poults were fed experimental diets from hatching to 21 d of age.

Dietary treatments were prepared by substituting ground F. moniliforme M-1325 culture material (CM) for ground corn in a typical corn-soybean meal basal diet. Fumonisin culture material contained 6,125 mg FB₁/kg, 1,400 mg FB₂/kg, and 535 mg FB₃/kg by analysis and made up 0, 0.41, 0.82, 1.23, 1.64, 2.87, 4.10, 5.33, 6.56, and 7.79% of the respective diets. Thus, the diets were calculated to contain 0, 33, 66, 99, 132, 231, 330, 429, 528, and 627 mg/kg of FB₁ + FB₂ + FB₃, respectively.

Liver histopathology was graded in a blind fashion. Hepatocellular hyperplasia was mild at 99 and 132 ppm, moderate to severe at 330 ppm, and severe at 429 to 627 ppm. Mild biliary hyperplasia was also observed in poults fed 627 ppm. No liver lesions were mentioned in poults fed 33 and 66 ppm.

At 33 ppm of FB₁ + FB₂ + FB₃ in the diet, poults had statistically poorer feed conversion (1.57 vs. 1.48 g of feed per g of gain), increased liver weights (LW) (16.65 g vs. 14.50 g), and increased Sa/So liver ratios (approx. 1.0 vs. 0.4) compared to controls. At 66 ppm of FB₁ + FB₂ + FB₃ in the diet, poults had only statistically increased liver weights (16.68 g vs. 14.50 g) compared to controls; however, the Sa/So liver ratios were higher than controls (approx. 0.85 vs. 0.4) and appeared to approach statistical significance.

The statistically poorer feed conversion at 33 ppm may have been due to spillage as the feed intake for this group was statistically higher than controls and 6.6% greater than the next highest group. The only other group to have a poorer feed conversion than controls was the 627 ppm group and they consumed 33.5% less than controls.

While some scientists may interpret the increased liver weights and increased liver Sa/So ratios as injurious to health. It is our opinion that tissue and serum Sa/So ratios should be interpreted as biomarkers for fumonisin exposure and increased liver weights without any hematology, serum chemistry or histology changes may not be biologically significant. The increased liver weights may be culture material (but not fumonisin) related.

We believe the Ledoux et al (1996) study could be used to establish guidance at 66 ppm FB₁ + FB₂ + FB₃ in the total diet of turkey poults; however, we recognize that this was only a 21-day study.

Effects of Feeding Fusarium moniliforme Culture Material, Containing Known Levels of Fumonisin B₁, on the Young Broiler Chick

Weibking et al, Poult Sci, 1993; 72: 456-466.

One hundred ninety-two day-old female Arbor Acres X Peterson broiler chicks were used in this 21-day dietary study. The experimental design consisted of eight dietary treatments with four pen replicates of six birds allotted randomly to each dietary treatment. The day-old chicks were fed experimental diets from hatching to 21 days of age.

Dietary treatments were prepared by substituting ground F. moniliforme M-1325 culture material for ground corn in a typical corn-soybean meal basal diet. Fumonisin culture material contained 7,800 mg FB₁/kg by analysis (FB₂ and FB₃ levels not provided) and made up 0, 1.02, 2.04, 3.06, 4.08, 5.10, 6.12, and 7.14% of the respective diets. Thus, the diets were calculated to contain 0, 75, 150, 225, 300, 375, 450, and 525 mg of FB₁/kg. The total dietary fumonisin levels (FB₁ + FB₂ + FB₃) were reported as 0, 89, 190, 283, 389, 481, 592 and 681 ppm; however, the method used to analyze the diets was not provided.

Broilers fed diets containing 89 and 190 ppm of FB₁ + FB₂ + FB₃ showed no statistical differences from controls in feed intake, weight gain, feed conversion, liver weight (wt), kidney wt, heart wt, gizzard wt, proventriculus wt, bursa of Fabricius wt, hemoglobin, erythrocytes, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, glucose, total protein, albumin, AST, or GGT.

Broilers fed diets containing 89 ppm of FB₁ + FB₂ + FB₃ showed statistical increases in mean corpuscular hemoglobin concentration (MCHC) (26.3 g/dl vs. 24.1 g/dl in controls) and a statistically significant decrease in cholesterol (107 mg/dl vs. 134 mg/dl in controls). Both of these changes appeared to be statistical happenstance as the MCHC values in the 190, 283, 389, and 481 ppm groups and the cholesterol values in the 190, 283, 481, 592, and 681 ppm groups were not statistically different than controls.

Compared with controls, all chicks fed diets containing fumonisins had statistically increased (P<.05) serum sphinganine/sphingosine ratios [approx. values for the control, 89 and 190 ppm groups were 0.11, 0.27 and 0.31, respectively].

Histopathology of brain, kidney, proventriculus, duodenum, pancreas, jejunum, ileum, cecum, lung, bursa, thymus, spleen, proximal tibiotarsus, heart and skeletal muscle was unremarkable (no lesions or incidental findings) in all treatment groups. Isolated foci of hepatic necrosis with a mild heterophil and macrophage infiltration, moderate diffuse hepatocellular hyperplasia, mild biliary hyperplasia, and moderate to severe periportal granulocytic cell proliferation were noted only in broilers fed >283 ppm total fumonisins. No gross or histologic liver lesions were mentioned in the 89 and 190 ppm groups.

Effects of Fusarium moniliforme Culture Material Containing Known Levels of Fumonisin B₁ in Ducklings.

Bermudez et al, Avian Diseases, 1995; 39: 879-886.

Thirty 1-day-old male white Peking ducklings were used in this 21-day dietary study. A complete randomized design was used with two pen replicates of three or four ducklings assigned to each of four dietary treatments.

Dietary treatments were prepared by substituting ground F. moniliforme M-1325 culture material for ground corn in a typical corn-soybean meal type diet. Fumonisin culture material contained 6,500 mg FB₁/kg, 790 mg FB₂/kg, and 540 mg FB₃/kg by analysis and made up 0, 1.5, 3.1, and 6.2% of the respective diets. Thus, the diets were calculated to supply 0, 120.5, 240.9, and 481.8 mg/kg of FB₁ + FB₂ + FB₃, respectively.

Two of 8 ducklings fed 481.8 ppm of fumonisins died prior to termination of the study (one after 1 wk and one after 20 d). No mortality occurred in any other treatment groups.

Ducklings fed diets containing 120.5 ppm of FB₁ + FB₂ + FB₃ showed no statistical differences in feed intake (2031 g vs. 2131 g in controls), weight gain (1023 g vs. 1060 g in controls), feed conversion, liver weight (36.3 g vs. 35.4 g in controls), heart weight, pancreas weight, proventriculus weight, hemoglobin, erythrocytes, hematocrit, glucose, total protein, AST, or uric acid.

Ducklings fed diets containing 120.5 ppm of FB₁ + FB₂ + FB₃ showed statistical increases in mean kidney weights (11.8 g vs. 10.7 g in controls), liver sphinganine/sphingosine ratio (0.995 vs. 0.388 in controls), albumin (1.66 g/dL vs. 1.33 g/dL in controls), and GGT (11.0 U/L vs. 9.0 U/L in controls. Cholesterol values were markedly higher, but not statistically different than controls (283 mg/dL vs. 192 mg/dL).

No gross lesions were evident in the control ducklings and those fed a ration containing 120.5 ppm of FB₁ + FB₂ + FB₃. Histopathology of brain, proventriculus, duodenum, pancreas, jejunum, ileum, cecum, lung, bursa, thymus, spleen, proximal tibiotarsus, heart and skeletal muscle were unremarkable in all treatment groups. Mild to moderate hepatocellular hyperplasia was evident in ducklings fed 120.5, 240.9, and 481.8 ppm of FB₁ + FB₂ + FB₃ in the diet. Embryonic nephrons and foci of extramedullary hematopoiesis were rare in the kidneys from the controls, but prominent in the 481.8 ppm group.

Conclusions for Turkeys, Chickens and Ducks

Although chickens may be slightly more resistant to fumonisins than turkeys and ducklings, it appears that these three species should be considered fairly resistant to the toxic effects of fumonisins and should be grouped into one category (poultry fed for slaughter). In our overall assessment of poultry fed for slaughter, we took into consideration the fact that the critical studies were only 3 weeks in length.

We recommend that corn and corn by-products used in rations of poultry fed for slaughter contain less than 100 ppm of FB₁ + FB₂ + FB₃ and that they comprise no more than 50% of the dry weight of the total ration. Thus, the total ration should contain less than 50 ppm of FB₁ + FB₂ + FB₃ (0.5 x 100 ppm of FB₁ + FB₂ + FB₃ = 50 ppm of FB₁ + FB₂ + FB₃).

clear evidence of carcinogenic activity of FB₁ in male F344/N rats based on the increased incidence of renal tubule neoplasms at dietary levels of 50 ppm and above.

clear evidence of carcinogenic activity of FB₁ in female B6C3F₁ mice based on the increased incidence of hepatocellular neoplasms at dietary levels of 50 ppm and above.

Thus, we believe we must provide protection against the potential for liver and kidney cancers in mink breeding stock, ruminant breeding stock, and poultry breeding stock. Breeding stock means all males and females selected for reproductive purposes and includes laying hens and lactating dairy cows.

Conclusions for Ruminant, Poultry and Mink Breeding Stock

The guidance for the sensitive species (equids and rabbits) is likely too conservative, but the apparent no-observed-adverse-effect level (NOAEL) from a chronic bioassay in both sexes of mice and rats conducted at the NCTR (NTP TR 496; NIH Publication No. 99-3955) seems appropriate.

Some of the Non-Cancer Effects Noted at 15 ppm FB₁ in the Diet from the NCTR Chronic Bioassay

RATS--Incidences of apoptosis of the renal tubule epithelium were generally significantly increased in male rats exposed to FB₁ for up to 26 weeks. Kidney weights in females were less than the controls at 2 years.

MICE--Incidences of hepatocellular hypertrophy were significantly increased in males at 2 years.

Non-Cancer Effects at 5 ppm FB₁ in the Diet from the NCTR Chronic Bioassay

RATS--None.

MICE--Survival of females was significantly greater than controls.

Conclusions for All Other Animals

We believe that the recommended level of 10 ppm total fumonisins in corn and corn by-products intended for all other animal species or classes is a conservative measure intended to protect the health of these animals in the face of uncertainty. The recommended level represents a conservative extrapolation between animals that are quite tolerant of fumonisins (such as poultry for which our recommended level is 100 ppm total fumonisins in corn and corn by-products) and animals that are sensitive to fumonisins (such as horses and rabbits for which our recommended level is 5 ppm total fumonisins in corn and corn by-products).

The amount of corn used in dog and cat foods is largely limited by crude protein (CP). Corn typically contains about 9% CP. According to the Association of American Feed Control Officials Incorporated (Official Publication 2001, AAFCO Dog and Cat Food Nutrient Profiles, pages 124-140), dogs and cats require a minimum of 18-22% and 26-30% CP in their diet, respectively, depending on their life stage. If the diet of a growing kitten contained 50% corn, then the remainder of the diet would have to contain 51% CP to meet the kitten's minimum CP requirements ([50% of diet X 9% CP] + [50% of diet X 51% CP] = 30% CP total diet). For practical purposes, corn is limited to about 50% of the total diet in dogs and cats due to its low levels of CP.

rats

(Shephard, Thiel and Sydenham, Food Chem Toxicol, 1992, 30 (4): 277-9;

Shephard et al, Toxicon, 1992, 30 (7): 768-70;

Norred, Plattner and Chamberlain, Nat Toxins, 1993, 1 (6): 341-6; and

Shephard et al, Food Chem Toxicol, 1994, 32 (5): 489-91).

swine

(Prelusky, Trenholm, and Savard, Nat Toxins, 1994, 2 (2): 73-80; and

Prelusky, Miller and Trenholm, Food Addit Contam, 1996, 13 (2): 155-62).

vervet monkeys

(Shephard et al, Toxicon, 1994, 32 (6): 735-41;

Shephard et al, Food Chem Toxicol, 32 (1): 23-29; and

Shephard et al, Nat Toxins, 1995, 3 (3): 145-50).

Holstein cows

(Prelusky, Savard and Trenholm, Nat Toxins, 1995, 3 (5): 389-94).

Holstein steers

(Smith and Thakur, Adv Exp Med Biol, 1996, 392: 39-55).

Angora goats

(Gurung et al, J Anim Sci, 1998, 76 (11): 2863-70).