Impact of food processing on the safety assessment for proteins introduced into biotechnology-derived soybean and corn crops.

The food safety assessment of new agricultural crop varieties developed through biotechnology includes evaluation of the proteins introduced to impart desired traits. Safety assessments can include dietary risk assessments similar to those performed for chemicals intentionally, or inadvertently added to foods. For chemicals, it is assumed they are not degraded during processing of the crop into food fractions. For introduced proteins, the situation can be different. Proteins are highly dependent on physical forces in their environment to maintain appropriate three-dimensional structure that supports functional activity. Food crops such as corn and soy are not consumed raw but are extensively processed into various food fractions. During processing, proteins in corn and soy are subjected to harsh environmental conditions that drastically change the physical forces leading to denaturation and loss of protein function. These conditions include thermal processing, changes in pH, reducing agents, mechanical shearing etc. Studies have shown that processing of introduced proteins such as enzymes that impart herbicide tolerance or proteins that control insect pests leads to a complete loss of functional activity. Thus, dietary exposure to functionally active proteins in processed food products can be negligible and below levels of any safety concerns.

Results of a 90-day safety assurance study with rats fed grain from corn borer-protected corn.

The results of a 90-day rat feeding study with grain from MON 810 corn (YieldGard Cornborer -- YieldGard Cornborer is a registered trademark of Monsanto Technology, LLC) that is protected against feeding damage from corn and stalk boring lepidopteran insects are presented. Corn borer protection was accomplished through the introduction of cry1Ab coding sequences into the corn genome for in planta production of a bioactive form of Cry1Ab protein. Grain from MON 810 and its near-isogenic control was separately formulated into rodent diets at levels of 11% and 33% (w/w) by Purina Mills, Inc. (PMI). All diets were nutritionally balanced and conformed to PMI specifications for Certified LabDiet (PMI Certified LabDiet 5002 is a registered trademark of Purina Mills, Inc.) 5002. There were a total of 400 rats in the study divided into 10 groups of 20 rats/sex/group. The responses of rats fed diets containing MON 810 were compared to those of rats fed grain from conventional corn varieties. Overall health, body weight, food consumption, clinical pathology parameters (hematology, blood chemistry, urinalysis), organ weights, and gross and microscopic appearance of tissues were comparable between groups fed diets containing MON 810 and conventional corn varieties. This study complements extensive agronomic, compositional and farm animal feeding studies with MON 810 grain, confirming that it is as safe and nutritious as grain from existing commercial corn varieties.

Safety assessment of DHA-rich microalgae from Schizochytrium sp.

Schizochytrium sp. dried microalgae (DRM) contains oil rich in highly unsaturated fatty acids (PUFAs). Docosahexaenoic acid (DHA n-3) is the most abundant PUFA component of the oil. DHA-rich oil extracted from Schizochytrium sp. is intended for use as a nutritional ingredient in foods. As part of a comprehensive safety assessment program, the reproductive toxicity of DRM was examined in Sprague-Dawley-derived rats Crl:CD(SD)BR (30/sex/group) provided DRM in the diet at concentrations of 0, 0.6, 6.0, and 30%. These dietary levels corresponded to overall average dosages of approximately 400, 3900, and 17,800 mg/kg/day for F0 males (premating) and 480, 4600, and 20,700 mg/kg/day for F0 females, respectively. Prior to mating, males and females of the F0 generation were treated for 10 and 2 weeks, respectively. Treatment of males continued throughout mating and until termination (approximately 3 weeks after mating). Treatment of the females was continued throughout gestation and through lactation day 21. The females were killed after raising their young to weaning at 21 days of age. Food consumption was measured weekly throughout the study (except during mating) and body weights were recorded at least weekly during premating, gestation, and lactation. Reproductive parameters including estrus cycle duration, mating performance, fertility, gestation length, parturition, and gestation index were evaluated. Litter size and offspring body weights were recorded, offspring viability indices were calculated, and physical development (vaginal opening and preputial separation) was assessed for the F1 generation. All adult F0 and F1 animals were subjected to a detailed necropsy. DRM treatment had no effect on estrus cycles or reproductive performance including mating performance, fertility, gestation length, parturition, or gestation index. Litter size, sex ratio, and offspring viability indices were similarly unaffected and there were no effects of DRM treatment on the physical development of F1 animals.

Safety assessment of DHA-rich microalgae from Schizochytrium sp.

The purpose of this study was to determine the potential toxicity of docosahexaenoic acid-rich microalgae from Schizochytrium sp. (DRM), administered in the diet to rats for at least 13 weeks. DRM was administered in the diet to groups of 20 male and 20 female Sprague-Dawley derived rats (Crl:CD(SD)BR) to provide dosages of 0, 400, 1500, and 4000 mg/kg/day for at least 13 weeks. DRM contained high levels of fat (approximately 41% w/w) of which long-chain highly unsaturated fatty acids (PUFAs) were a major component. Vitamin E acetate was added to DRM at manufacture to provide supplementary dietary antioxidant given the highly unsaturated fat content of DRM. Untreated controls received the basal diet only. An additional group of 20 males and 20 females received basal diet mixed with fish oil (Arista) to provide a target dosage of 1628 mg/kg/day, an amount of fat comparable to that received by rats administered the highest dose of DRM. Vitamin E acetate was also added to the fish oil to provide a comparable level of dietary antioxidant provided to high-dose DRM rats. There were no treatment-related effects in clinical observations, body weights or weight gains, food consumption, hematologic or urinalysis values, gross necropsy findings, or organ weights and there were no deaths. The only treatment-related changes in clinical chemistry parameters were decreases in high-density lipoproteins and cholesterol in the DRM and fish oil groups when compared to the untreated controls. These changes were expected based on the high PUFA content of DRM and fish oil. There were no microscopic findings suggestive of toxicity. Periportal hepatocellular fat vacuolation (accumulation of fat) was observed only in the livers of female rats in both the DRM (all dosages) and fish oil groups. This finding was expected given the higher fat content of both the DRM and the fish oil diets compared to the basal diet fed to the untreated controls. A slight increase in the incidence, but not severity, of cardiomyopathy was observed only in the 4000 mg/kg/day DRM males. This finding was not considered adverse because cardiomyopathy occurs spontaneously in rats and especially male rats of the Sprague-Dawley strain when fed high levels of fat. Since cardiomyopathy does not develop in other species including primates fed high-fat diets, its occurrence in rats is considered to have little relevance to human health. This study demonstrates that administration of DRM did not produce any treatment-related adverse effects in Sprague-Dawley rats of relevance to humans at dosages up to 4000 mg/kg/day for 13 weeks.

Safety assessment of DHA-rich microalgae from Schizochytrium sp.

Schizochytrium sp. (DRM) contains oil rich in highly unsaturated fatty acids (PUFAs). Docosahexaenoic acid (DHA) is the most abundant PUFA component of the oil (approx. 35% w/w). DHA-rich extracted oil from Schizochytrium sp. is intended for use as a nutritional ingredient in foods. As part of a comprehensive safety assessment program, the developmental toxicity of DRM was assessed in Sprague-Dawley derived rats [25/group, provided DRM in the diet at 0.6, 6, and 30% on gestation days (GD) 6-15] and in New Zealand White (NZW) rabbits (22/group, dosed with DRM at levels of 180, 600, and 1800 mg/kg/day by oral gavage on GD 6-19). Fish oil was used as a negative control at dose levels to provide an equivalent amount of fat to that received by the high-dose DRM rabbits. Maternal food consumption, body weights, and clinical signs were recorded at regular intervals throughout these studies. Animals were sacrificed on GD 20 (rats) and GD 29 (rabbits) and examined for implant status, fetal weight, sex, and morphologic development. No clinical signs of toxicity were observed. Maternal exposure to DRM during organogenesis did not adversely affect the frequency of postimplantation loss, mean fetal body weight/litter, or external, visceral, or skeletal malformations in either the rat or the rabbit. In the rats, neither maternal nor developmental toxicity was observed at any dietary concentration of DRM. Thus, 22 g/ kg/day(1) of DRM administered in the feed to pregnant rats during organogenesis was the NOEL (no-observed-effect level) for both maternal and developmental toxicity. In rabbits, no maternal toxicity was expressed at DRM dose levels of 180 and 600 mg/kg/day. As a possible consequence of the high-fat content of the fish oil and DRM, reductions in food consumption and body weight gain and a slight increase in abortions occurred in the fish oil control and 1800 mg/kg/day DRM groups. Developmental toxicity was not observed at any DRM dose level. Based on the results of this study, the NOEL for maternal toxicity of DRM was 600 mg/kg/day, and the NOEL for developmental toxicity was 1800 mg/kg/day in NZW rabbits.

Safety and advantages of Bacillus thuringiensis-protected plants to control insect pests.

Plants modified to express insecticidal proteins from Bacillus thuringiensis (referred to as Bt-protected plants) provide a safe and highly effective method of insect control. Bt-protected corn, cotton, and potato were introduced into the United States in 1995/1996 and grown on a total of approximately 10 million acres in 1997, 20 million acres in 1998, and 29 million acres globally in 1999. The extremely rapid adoption of these Bt-protected crops demonstrates the outstanding grower satisfaction of the performance and value of these products. These crops provide highly effective control of major insect pests such as the European corn borer, southwestern corn borer, tobacco budworm, cotton bollworm, pink bollworm, and Colorado potato beetle and reduce reliance on conventional chemical pesticides. They have provided notably higher yields in cotton and corn. The estimated total net savings to the grower using Bt-protected cotton in the United States was approximately $92 million in 1998. Other benefits of these crops include reduced levels of the fungal toxin fumonisin in corn and the opportunity for supplemental pest control by beneficial insects due to the reduced use of broad-spectrum insecticides. Insect resistance management plans are being implemented to ensure the prolonged effectiveness of these products. Extensive testing of Bt-protected crops has been conducted which establishes the safety of these products to humans, animals, and the environment. Acute, subchronic, and chronic toxicology studies conducted over the past 40 years establish the safety of the microbial Bt products, including their expressed insecticidal (Cry) proteins, which are fully approved for marketing. Mammalian toxicology and digestive fate studies, which have been conducted with the proteins produced in the currently approved Bt-protected plant products, have confirmed that these Cry proteins are nontoxic to humans and pose no significant concern for allergenicity. Food and feed derived from Bt-protected crops which have been fully approved by regulatory agencies have been shown to be substantially equivalent to the food and feed derived from conventional crops. Nontarget organisms exposed to high levels of Cry protein are virtually unaffected, except for certain insects that are closely related to the target pests. Because the Cry protein is contained within the plant (in microgram quantities), the potential for exposure to farm workers and nontarget organisms is extremely low. The Cry proteins produced in Bt-protected crops have been shown to rapidly degrade when crop residue is incorporated into the soil. Thus the environmental impact of these crops is negligible. The human and environmental safety of Bt-protected crops is further supported by the long history of safe use for Bt microbial pesticides around the world.

Glyphosate-tolerant corn: the composition and feeding value of grain from glyphosate-tolerant corn is equivalent to that of conventional corn (Zea mays L.).

Glyphosate-tolerant (Roundup Ready) corn line GA21 has been developed by genetic modification to tolerate glyphosate, the active ingredient in Roundup herbicide. The purpose of this study was to evaluate the compositional and nutritional safety of corn line GA21 compared to that of conventional corn. Compositional analyses were conducted to measure proximate, fiber, amino acid, fatty acid, and mineral contents of grain and proximate, fiber, and mineral contents of forage collected from 16 field sites over two growing seasons. The nutritional safety of corn line GA21 was evaluated in a poultry feeding study conducted with 2-day old, rapidly growing broiler chickens, at a dietary concentration of 50-60% w/w. Compositional analysis results showed that, except for a few minor differences that are unlikely to be of biological significance, the grain and forage of GA21 corn were comparable in their composition to that of the control corn line and to conventional corn. Results from the poultry feeding study showed that there were no differences in growth, feed efficiency, adjusted feed efficiency, and fat pad weights between chickens fed with GA21 grain or with parental control grain. These data taken together demonstrate that Roundup Ready corn is as safe and nutritious as conventional corn for food and feed use.

The expressed protein in glyphosate-tolerant soybean, 5-enolpyruvylshikimate-3-phosphate synthase from Agrobacterium sp. strain CP4, is rapidly digested in vitro and is not toxic to acutely gavaged mice.

The safety of 5-enolpyruvylshikimate-3-phosphate synthase enzyme derived from Agrobacterium sp. strain CP4 (CP4 EPSPS) was assessed. CP4 EPSPS is the only protein introduced by genetic manipulation that is expressed in glyphosate-tolerant soybeans, which are being developed to provide new weed-control options for farmers. Expression of this protein in plants imparts high levels of glyphosate tolerance. The safety of CP4 EPSPS was ascertained by evaluating both physical and functional characteristics. CP4 EPSPS degrades readily in simulated gastric and intestinal fluids, suggesting that this protein will be degraded in the mammalian digestive tract upon ingestion as a component of food or feed, There were no deleterious effects due to the acute administration of CP4 EPSPS to mice by gavage at a high dosage of 572 mg/kg body wt, which exceeds 1000-fold tha anticipated consumption level of food products potentially containing CP4 EPSPS protein. CP4 EPSPS does not pose any important allergen concerns because this protein does not possess characteristics typical of allergenic proteins. These data, in combination with seed compositional analysis and animal feeding studies, support the conclusion that glyphosate-tolerant soybean are as safe and nutritious as traditional soybeans currently being marketed.

The feeding value of soybeans fed to rats, chickens, catfish and dairy cattle is not altered by genetic incorporation of glyphosate tolerance.

Animal feeding studies were conducted with rats, broiler chickens, catfish and dairy cows as part of a safety assessment program for a soybean variety genetically modified to tolerate in-season application of glyphosate. These studies were designed to compare the feeding value (wholesomeness) of two lines of glyphosate-tolerant soybeans (GTS) to the feeding value of the parental cultivar from which they were derived. Processed GTS meal was incorporated into the diets at the same concentrations as used commercially; diary cows were fed 10 g/100 g cracked soybeans in the diet, a level that is on the high end of what is normally fed commercially. In a separate study, laboratory rats were fed 5 and 10 g unprocessed soybean meal 100 g diet. The study durations were 4 wk (rats and dairy cows), 6 wk (broilers) and 10 wk (catfish). Growth, feed conversion (rats, catfish, broilers), fillet composition (catfish), and breast muscle and fat pad weights (broilers) were compared for animals fed the parental and GTS lines. Milk production, milk composition, rumen fermentation and nitrogen digestibility were also compared for dairy cows. In all studies, measured variables were similar for animals fed both GTS lines and the parental line, indicating that the feeding value of the two GTS lines is comparable to that of the parental line. These studies support detailed compositional analysis of the GTS seeds, which showed no meaningful differences between the parental and GTS lines in the concentrations of important nutrients and antinutrients. They also confirmed the results of other studies that demonstrated the safety of the introduced protein, a bacterial 5-enolpyruvyl-shikimate-3-phosphate synthase from Agrobacterium sp. strain CP4.

Safety assessment of the neomycin phosphotransferase II (NPTII) protein.

Two approaches were used to assess the safety of the NPTII protein for human consumption using purified E. coli produced NPTII protein that was shown to be chemically and functionally equivalent to the NPTII protein produced in genetically engineered cotton seed, potato tubers and tomato fruit. The NPTII protein was shown, as expected, to degrade rapidly under simulated mammalian digestive conditions. An acute mouse gavage study confirmed that the NPTII protein caused no deleterious effects when administered by gavage at a cumulative target dosage of up to 5000 mg/kg of body weight. This dosage correlates to at least a million fold safety factor relative to the average daily consumption of potato or tomato, assuming all the potatoes or tomatoes consumed contained the NPTII protein. These results, along with previously published information, confirm that ingestion of genetically engineered plants expressing the NPTII protein poses no safety concerns.

Effect of high doses of a sustained-release bovine somatotropin on antibody formation in dairy cows.

Eighty-two lactating Holstein cows received either one, three, or five concurrent, intramuscular injections of a unit dose (.6 g) of zinc methionyl bST (some-tribove) or five doses of the vehicle. Injections were administered at 14-d intervals from 60 d postpartum until the end of lactation or necropsy. Thirty-eight cows continued on the same treatment for a 2nd yr. Blood bST antibodies developed within the first 7 wk of treatment, and the number of cows with anti-bST binding generally declined with time. Thirteen out of 59 cows receiving bST developed binding activity > 25% (positives) during the 1st yr. At the .6-g dose level, no binding was detected after wk 15. Seven of the 13 positive cows were among the group randomly selected to continue on study during yr 2. In the 2nd yr, only 2 out of 24 bST-treated cows were positive. Binding activity was associated with the IgG fraction in serum. Binding capacities of antibodies ranged from .625 to 3.04 mg of bST/L, and affinities ranged from 1.14 x 10(8) to 3.14 x 10(8) L/mol. Cows considered to be clinically positive had performance similar to those of their herdmates having binding < 25%. No evidence of a pathologic effect of antibodies existed in treated cows, their calves, or fetuses. The presence of anti-bST antibodies did not affect milk production of the cow or growth of the calves conceived during bST treatment.

Response of dairy cows to high doses of a sustained-release bovine somatotropin administered during two lactations. 2. Health and reproduction.

Eighty-two lactating Holstein cows in their first, second, or third lactation received either one, three, or five concurrent i.m. injections of a unit dose (.6 g) of zinc methionyl bST (sometribove) or five doses of the vehicle. Injections were given at 14-d intervals from 60 +/- 3 d postpartum until the end of lactation or necropsy. Thirty-eight animals were continued on treatment for a 2nd yr. Sometribove did not affect the incidence of ketosis, milk fever, tetany, or pneumonia. Digestive disorders, primarily cows going off feed, were increased by bST during yr 1 only. The incidence of lameness was increased by bST in some time frames because of an increase in the 3.0-g bST group. Lameness was not associated with treatment-specific histopathologic changes or with abnormalities in cartilage or bone. Reproductive health generally was unaffected by treatment, but delayed conception and increased incidence of abortion were noted. Incidence of cystic ovaries was unaffected by bST. Pregnancy rates were decreased during the 100-d breeding interval of yr 1 but not during the 215-d interval of yr 2. The incidence of clinical mastitis was increased by bST, primarily at the 3.0-g dose. During the 2-yr study, 0, 3, 3, and 2 cows died or became moribund on 0, .6, 1.8, and 3.0 g of bST, respectively. Health issues identified for further evaluation included lameness and clinical mastitis for the 3.0-g group and early removal from the herd and decreased reproductive performance for all bST groups. Bovine somatotropin caused no treatment-specific toxic effects in dairy cows even at 3.0 g every 14 d.

Response of dairy cows to high doses of a sustained-release bovine somatotropin administered during two lactations. 1. Production response.

This study evaluated the effect of sometribove (zinc methionyl bST) in a sustained-release formulation administered to lactating cows at concentrations up to 3.0 g every 14 d over two lactations. Eighty-two lactating Holstein cows in their first, second, or third lactation were assigned to the study. Cows received .6, 1.8, or 3.0 g of bST in one, three, or five intramuscular injections of a unit dose (.6 g) every 2 wk. Controls received five injections of the vehicle (equivalent volume to the 3.0-g treatment) every 2 wk. Injections were administered from 60 +/- 3 d postpartum until dry-off or necropsy. Thirty-eight animals were continued on treatment for a second consecutive lactation. During the 1st yr of treatment, bST increased mean 3.5% FCM by 7.2, 9.4, and 8.4 kg/d over control production (21.1 kg/d). During the 2nd yr, milk response to .6, 1.8, and 3.0 g of bST averaged 10.6, 3.6, and 4.9 kg/d over controls (24.8 kg/d). The incidence of clinical mastitis increased in the 3.0-g group relative to controls during the 2nd yr. Thus, salable FCM averaged 8.1, 9.1, and 6.2 kg/d above controls (yr 1) and 12.1, 4.7, and -2.8 kg/d (yr 2) for the .6-, 1.8-, and 3.0-g groups. Salable FCM was unaffected by mastitis at a proposed commercial dose (.6 g). Milk fat, protein, lactose, calcium, phosphorus, zinc, magnesium, and ash concentrations were unaffected by bST treatment. Calculated energy, calcium, phosphorus, and protein balances also were unaffected except for early decreases of up to 5 Mcal/d, and 40, 20, and 600 g/d, respectively, until feed intake increased. Milk serum bST concentrations greater than the assay limit of sensitivity (1 ng/ml) were routinely measurable only at doses of 1.8 and 3.0 g. Results confirmed that bST concentrations in milk serum are exceedingly small. Overall, supraphysiological doses of sometribove increased milk production with little effect on composition. No toxic effects of bST were observed.

Food safety and pharmacokinetic studies which support a zero (0) meat and milk withdrawal time for use of sometribove in dairy cows.

Sometribove (SB) is a synthetic form of bovine somatotropin (BST) whose amino acid sequence is the same for 190 of the 191 amino acids in BST. Administration of 500 mg of SB to dairy cows every 14 d increases the efficiency of milk production. Regulatory agencies have authorized a zero (0) milk and meat withdrawal time for investigational use of SB. The scientific basis for this authorization is as follows: 1) BST and other non-primate somatotropins are not active in humans, due to differences in the amino acid sequence from human somatotropin, which limits the ability of BST to bind to receptors on human tissues. 2) SB is not orally active, as it is degraded like other proteins when eaten. Administration of 50,000 microgram/kg/d SB to rats for 90 d produced no growth response. 3) Residual levels of SB in meat/milk are very low (ppb) and comparable to endogenous BST levels. 4) Residual levels (ppb) of insulin-like growth factor I (IGF-I) in meat and milk are only marginally increased by SB treatment (somatotropin stimulates local production of IGF-I in tissues to mediate some of its biological effects. 5) IGF-I was not orally active when fed to rats at doses ranging from 200 to 2,000 microgram/kg for 14 d.

A review of the subchronic toxicity of butyl benzyl phthalate.

This review compares the subchronic toxicity of butyl benzyl phthalate (BBP) across several species. Data from the published literature as well as previously unpublished studies sponsored by Monsanto are presented. BBP-induced toxicity occurs only at relatively high levels of exposure and is dependent on the species, age and strain of test animals used. These factors should be considered in extrapolating findings from animal toxicology studies to humans when assessing the safety of BBP.

A review of toxicology studies on cyanurate and its chlorinated derivatives.

Chlorinated cyanurates are added to swimming pools as disinfectants. In the presence of water, these materials hydrolyze to yield cyanurate and hypochlorous acid. To evaluate the safety of exposure to these materials, a comprehensive testing program was undertaken. This review summarizes the results of acute and subchronic tests on chlorinated isocyanurates. Findings from acute, subchronic, reproduction, metabolism, mutagenicity, and chronic/carcinogenicity tests on cyanurate are also summarized. Results from these tests indicate that chlorinated isocyanurates are safe for use in swimming pools.

Teratogenicity studies of alkylaryl phosphate ester plasticizers in rats.

Santicizer 141 plasticizer (2-ethylhexyldiphenyl phosphate) and Santicizer 148 plasticizer (isodecyldiphenyl phosphate) were tested for teratogenic activity in Charles River COBS CD rats. Groups of 25 mated females were given 0, 300, 1000, or 3000 mg/kg/day by gavage on Days 6 through 15 (Santicizer 141) or 6 through 19 (Santicizer 148) of gestation. Mean maternal body weight gains were slightly and severely reduced at the mid- and high-dose levels of Santicizer 141, respectively. Body weights were not affected by treatment with Santicizer 148. Most malformations found in groups treated with either plasticizer occurred as single incidences and have been observed in historical controls. Thus, no teratogenic response was observed in rats after treatment with either of these two alkylaryl phosphates during the period of organogenesis.

Absence of mutagenic activity for monosodium cyanurate.

The mutagenic potential of monosodium cyanurate was evaluated using in vitro and in vivo tests. All in vitro tests were carried out in the presence and absence of metabolic activation. In each assay, the highest concentration tested generally exceeded the solubility of monosodium cyanurate in the incubation medium. In the Salmonella microbial assay, monosodium cyanurate was not mutagenic towards test strains TA 98, 100, 1535, and 1537 up to a concentration of 10,000 micrograms/plate. Monosodium cyanurate did not induce forward mutations at the TK locus of L5178Y mouse lymphoma cells up to a concentration of 2000 micrograms/ml. No significant increases in sister chromatid exchanges were observed when monosodium cyanurate was incubated with Chinese hamster ovary cells at concentrations up to 1500 micrograms/ml. In an in vivo test, rats were administered monosodium cyanurate by gavage at single dosages up to 5000 mg/kg and killed 24 and 48 hr after dosing. Bone marrow cells were collected and examined for chromosomal aberrations. At the time points examined, there was no evidence of monosodium cyanurate-induced chromosomal aberrations in rat bone marrow cells.

Modes of action of toxic agents.

The principal modes of action of toxic agents are discussed in relation to the type of chemical bond formed between the poison and the target constituent of tissues. Alteration of enzyme activity, interference with the binding of poisonous chemicals to proteins, intercalation with nucleic acids, disturbances in electrolyte balance and the disorganization of cellular water and membrane lipids are illustrated as toxic processes involving ionic or van der Waals forces. The reactions of heavy metals with tissue nucleophiles and of exogenous nucleophiles with tissue metals are given brief attention in connection with coordinate-covalent binding. Covalent binding of poisons can arise from the incorporation of an antimetabolite into a larger molecule or reactions of electrophiles or free radicals with tissue constituents. These modes of action are illustrated by chemicals that produce necrosis, allergy or cancer.