Lifetime Feeding Studies Research Articles

Un‐United Medial Epicondyle of the Humerus: Radiographic Prevalence and Association with Elbow Osteoarthritis in a Cohort of Labrador Retrievers

To longitudinally characterize the radiographic appearance of un-united medial epicondyle (UME) of the humerus, evaluate UME association with osteoarthritis (OA) and consider its relevance to the elbow dysplasia complex. Longitudinal cohort study. Labrador retrievers (n=48) from 7 litters. Forty-eight same-sex littermates were paired for this lifetime feeding study. One of each pair was control-fed; the pair mate was fed 25% less than the control each day. Elbows of 46 surviving dogs were radiographed at ages 6 and 8 years, and/or at end-of-life (EOL). Elbow histopathology was done EOL, although UME lesions were not evaluated histologically. Seven dogs (15%) had UME, representing 5 litters; 4 were control-fed, 3 diet-restricted. Six (86%) dogs had unilateral lesions; 1 was bilateral. UME was evident on craniocaudal (CrCd) radiographic projections by 8 years in all dogs. UME was detected in only 1 elbow by mediolateral radiographic projection. Elbow OA frequency in UME affected dogs was not significantly different from the remaining study population. Histopathologic lesions were bilateral in dogs with unilateral UME. UME may be more common than previously thought. Most cases were unilateral and diet restriction had no effect on frequency. The CrCd view was critical for diagnosis. Elbow OA was not directly associated with UME. Infrequent diagnosis of UME could result from infrequent radiography and use of only the flexed lateral radiographic projection required by the Orthopedic Foundation for Animals for elbow screening. Like hip evaluations, screening for UME should be continued for life, until genetics are better understood. Lack of association between UME and elbow OA suggests that UME is not likely a component of elbow dysplasia.

Glyphosate Results Revisited

With respect to the recent article by De Roos et al. (2005), we would like to a) comment on the authors’ incomplete genotoxicity review, which is inconsistent with conclusions reached by regulatory agencies; b) estimate the likely range of systemic doses and margins of exposure for farmers based on comprehensive glyphosate biomonitoring data published in 2004; and c) request further evaluation of confounding and selection bias in their analyses for multiple myeloma. In their discussion of genotoxicity, De Roos et al. focused on selected studies that conflict with the weight of evidence for glyphosate and Roundup brand (Monsanto Company, St. Louis, MO) agricultural herbicides containing glyphosate. They cited Williams et al. (2000) regarding the lack of a carcinogenic effect in rodent feeding studies with glyphosate but neglected to cite the extensive genotoxicity review in the same article in which Williams et al. concluded that Roundup and its components do not pose a risk for heritable or somatic mutations. This conclusion is in agreement with findings by the U.S. Environmental Protection Agency (U.S. EPA 1993), the World Health Organization (WHO 1994), the European Commission (2002), and regulatory agencies worldwide. None of the studies cited by De Roos et al. (2005) as presumptive evidence of genotoxicity were conducted under Good Laboratory Practices or according to international guidelines. Additionally, many of these studies used toxic dose levels and/or irrelevant routes of exposure. When evaluating epidemiologic findings, it can be helpful to compare the range of likely exposure levels to the exposure levels of toxicologic significance (Acquavella et al. 2003). The cancer no-effect levels for glyphosate, based on rat and mouse lifetime feeding studies, are 1,000 and 1,500 mg/kg/day, respectively (Williams et al. 2000). Acquavella et al. (2004) reported results of a biomonitoring study in which 48 farmers collected all of their urine over 5 consecutive days (before, during, and for 3 days after a glyphosate application). In this study, the maximum systemic dose resulting from application of glyphosate to areas as large as 400 acres was 0.004 mg/kg. The geometric mean systemic dose was 0.0001 mg/kg. Accordingly, in the worst-case situation, if a farmer made a similar application every day for a lifetime, the systemic dose would be at least 250,000-fold lower than the cancer no-effect level in rodents. Indeed, this very large margin of exposure combined with the lack of evidence for genotoxicity must be factored into an assessment of biologic plausibility. Finally, De Roos et al.’s Table 2 (De Roos et al. 2005) shows an age-adjusted relative risk (RR) of 1.1 [95% confidence interval (CI), 0.5–2.4] associating multiple myeloma and ever-use of glyphosate. The RR adjusted for selected demographic and lifestyle variables was 2.6 (95% CI, 0.7–9.4). The factors that account for the difference in these RRs are not well explained. Given the weak associations between the covariates and ever-use of glyphosate and the weak or nonexistent relation between these variables and risk of multiple myeloma, it is unlikely that the change in RR from 1.1 to 2.6 is attributable to confounding. The authors mention that only 75% of eligible subjects were included in the fully adjusted analysis and that this reduction in analytic sample size was due to the exclusion of subjects that were missing covariate data. Further, De Roos et al. (2005) did not find an association in the complete data set without adjustment for covariates (RR = 1.1), but they did find a positive association in the restricted data set without adjustment for covariates. The difference in association due simply to restricting the data set to those with covariate information was not quantified, although such quantification would help the reader understand what proportion of the change from 1.1 to 2.6 was attributable to adjustment for candidate confounders and what proportion was due to selection of subjects with more complete data. An analysis stratified by each covariate individually should have allowed the investigators to identify covariates for which missing data and/or adjustment made the biggest impact on the estimated RR. The identity of these covariates would help the reader weigh the potential for confounding versus selection bias to explain the change in RR from 1.1 to 2.6. Given that only 32 cases of multiple myeloma were observed and as few as 19 cases were included in some of the analyses, the authors should have explored the potential for the analysis of sparse data to result in estimates biased away from the null (e.g., see Greenland et al. 2000 for an example involving conditional logistic regression).

Safety evaluation and risk assessment of the herbicide Roundup and its active ingredient, glyphosate, for humans.

Reviews on the safety of glyphosate and Roundup herbicide that have been conducted by several regulatory agencies and scientific institutions worldwide have concluded that there is no indication of any human health concern. Nevertheless, questions regarding their safety are periodically raised. This review was undertaken to produce a current and comprehensive safety evaluation and risk assessment for humans. It includes assessments of glyphosate, its major breakdown product [aminomethylphosphonic acid (AMPA)], its Roundup formulations, and the predominant surfactant [polyethoxylated tallow amine (POEA)] used in Roundup formulations worldwide. The studies evaluated in this review included those performed for regulatory purposes as well as published research reports. The oral absorption of glyphosate and AMPA is low, and both materials are eliminated essentially unmetabolized. Dermal penetration studies with Roundup showed very low absorption. Experimental evidence has shown that neither glyphosate nor AMPA bioaccumulates in any animal tissue. No significant toxicity occurred in acute, subchronic, and chronic studies. Direct ocular exposure to the concentrated Roundup formulation can result in transient irritation, while normal spray dilutions cause, at most, only minimal effects. The genotoxicity data for glyphosate and Roundup were assessed using a weight-of-evidence approach and standard evaluation criteria. There was no convincing evidence for direct DNA damage in vitro or in vivo, and it was concluded that Roundup and its components do not pose a risk for the production of heritable/somatic mutations in humans. Multiple lifetime feeding studies have failed to demonstrate any tumorigenic potential for glyphosate. Accordingly, it was concluded that glyphosate is noncarcinogenic. Glyphosate, AMPA, and POEA were not teratogenic or developmentally toxic. There were no effects on fertility or reproductive parameters in two multigeneration reproduction studies with glyphosate. Likewise there were no adverse effects in reproductive tissues from animals treated with glyphosate, AMPA, or POEA in chronic and/or subchronic studies. Results from standard studies with these materials also failed to show any effects indicative of endocrine modulation. Therefore, it is concluded that the use of Roundup herbicide does not result in adverse effects on development, reproduction, or endocrine systems in humans and other mammals. For purposes of risk assessment, no-observed-adverse-effect levels (NOAELs) were identified for all subchronic, chronic, developmental, and reproduction studies with glyphosate, AMPA, and POEA. Margins-of-exposure for chronic risk were calculated for each compound by dividing the lowest applicable NOAEL by worst-case estimates of chronic exposure. Acute risks were assessed by comparison of oral LD50 values to estimated maximum acute human exposure. It was concluded that, under present and expected conditions of use, Roundup herbicide does not pose a health risk to humans.

Genotoxicity of garlic constituents

Reviews on the safety of glyphosate and Roundup herbicide that have been conducted by several regulatory agencies and scientific institutions worldwide have concluded that there is no indication of any human health concern. Nevertheless, questions regarding their safety are periodically raised. This review was undertaken to produce a current and comprehensive safety evaluation and risk assessment for humans. It includes assessments of glyphosate, its major breakdown product [aminomethylphosphonic acid (AMPA)], its Roundup formulations, and the predominant surfactant [polyethoxylated tallow amine (POEA)] used in Roundup formulations worldwide. The studies evaluated in this review included those performed for regulatory purposes as well as published research reports. The oral absorption of glyphosate and AMPA is low, and both materials are eliminated essentially unmetabolized. Dermal penetration studies with Roundup showed very low absorption. Experimental evidence has shown that neither glyphosate nor AMPA bioaccumulates in any animal tissue. No significant toxicity occurred in acute, subchronic, and chronic studies. Direct ocular exposure to the concentrated Roundup formulation can result in transient irritation, while normal spray dilutions cause, at most, only minimal effects. The genotoxicity data for glyphosate and Roundup were assessed using a weight-of-evidence approach and standard evaluation criteria. There was no convincing evidence for direct DNA damage in vitro or in vivo, and it was concluded that Roundup and its components do not pose a risk for the production of heritable/somatic mutations in humans. Multiple lifetime feeding studies have failed to demonstrate any tumorigenic potential for glyphosate. Accordingly, it was concluded that glyphosate is noncarcinogenic. Glyphosate, AMPA, and POEA were not teratogenic or developmentally toxic. There were no effects on fertility or reproductive parameters in two multigeneration reproduction studies with glyphosate. Likewise there were no adverse effects in reproductive tissues from animals treated with glyphosate, AMPA, or POEA in chronic and/or subchronic studies. Results from standard studies with these materials also failed to show any effects indicative of endocrine modulation. Therefore, it is concluded that the use of Roundup herbicide does not result in adverse effects on development, reproduction, or endocrine systems in humans and other mammals. For purposes of risk assessment, no-observed-adverse-effect levels (NOAELs) were identified for all subchronic, chronic, developmental, and reproduction studies with glyphosate, AMPA, and POEA. Margins-of-exposure for chronic risk were calculated for each compound by dividing the lowest applicable NOAEL by worst-case estimates of chronic exposure. Acute risks were assessed by comparison of oral LD50 values to estimated maximum acute human exposure. It was concluded that, under present and expected conditions of use, Roundup herbicide does not pose a health risk to humans.

Methodologies for interpretation of short-term test results which may allow reduction in the use of animals in carcinogenicity testing.

Assessment of the risk to humans posed by chemical substances currently relies primarily on experimental exposure of animals in lifetime feeding studies. Short-term tests for genotoxicity are much less costly and use fewer or no animals, but have not replaced the long-term animal bioassay because their results do not coincide completely. We have developed methodologies for interpretation of short-term tests which improve the usefulness of their results, and may allow them to replace the long-term animal bioassay in some circumstances.

Qualitative and quantitative cancer risk assessment of 2, 3, 7, 8-tetrachloro-dibenzo-p-dioxin (2, 3, 7, 8-TCDD)

The carcinogenicity of 2,3,7,8-TCDD at multiple organ sites in animals has been well established by several cancer bioassays. Results of two of the most notable of these, the Kociba et al. (1978) rat feeding study and the National Toxicology Program (1980) gavage study in rats and mice showed hepatocellular carcinomas in two strains of female rats and male and female mice. Other tumor sites included carcinomas of the lung, tongue, hard palate and nasal turbinates, thyroid, and subcutaneous tissue. The evidence for carcinogenicity of 2,3,7,8-TCDD in animals is regarded as “sufficient” using the classification system of the International Agency for Research on Cancer (IARC). Two Swedish epidemiologic case-control studies (Hardell and Sandstrom, 1979; Eriksson et al. 1979, 1981) reported a significant five- to sevenfold excess risk of soft-tissue sarcomas (STS) from occupational exposure to chlorinated phenoxyacetic acid herbicides and/or chlorophenols. Additionally, several small cohort studies collectively exhibited an unusual cluster of STS, significantly increased over combined expected incidence. Problems with these studies do not appear to be sufficient to discount this excess risk. The human evidence alone for the carcinogenicity of 2,3,7,8-TCDD is “inadequate” using the IARC classification. However, for 2,3,7,8-TCDD in combination with chlorinated phenoxyacetic acid herbicides and/or chlorophenols, the human evidence is considered to be “limited.” The overall evidence for carcinogenicity considering both animal and human studies would place 2,3,7,8-TCDD alone in the IARC category 2B, meaning that the substance is probably carcinogenic in humans. The overall weight of evidence for 2,3,7,8-TCDD in combination with chlorinated phenoxyacetic acid herbicides and/or chlorophenols is regarded as IARC category 2A, also meaning that they are probably carcinogenic for humans. Using current EPA methodology for quantitatively estimating cancer risks, several animal data sets have been analyzed. Comparing the results, the upper-limit incremental unit risk estimate is 1.6 × 10 −2 for a lifetime exposure of 1 ng/kg/day. This estimate is derived from a lifetime feeding study (Kociba et al., 1978) in which 2,3,7,8-TCDD induced tumors of the liver, lungs, hard palate, and nasal turbinates in female rats. Incremental unit cancer risks are also extrapolated for lifetime 2,3,7,8-TCDD exposures in water and air. Based on continuous lifetime exposure to 1 ng/L 2,3,7,8-TCDD in drinking water, the upper-limit estimate of extra cancer risk per individual is 4.5 × 10 −3. For lifetime exposure to 1 pg 2,3,7,8-TCDD/m 3 in the ambient air, the upper-limit individual risk is 3.3 × 10 −5.

Implications for the Future: Regulation of Chemicals and Prevention of Cancer

Lifetime feeding studies, when positive for cancer induction, do not provide information to differentiate between primary and secondary mechanisms of cancer induction. So many factors can influence the ultimate risk of incurring cancer in animals or humans that the observation of an increased incidence of cancer in association with exposure to any particular test substance should not lead automatically to the inference that the substance is a carcinogen per se. An increased incidence of cancer can result not only from exposure to “complete” carcinogens, but also from critical exposures to the following factors: those that decrease biological resistance to other independently occurring cancer risk factors, those that cause toxic hyperplasia, or those that act as classical promoters. Both initiation and promotion are prerequisites to cancer induction. Initiation can result independently from the effects of viruses, radiation, chemical initiators, and aging. For example: endogenously-formed nitrosamines are capable of effecting initiation in hepatic cells. Many non-genotoxic hepatic so-called “carcinogens” appear to be “promoters” of cancer induction rather than complete carcinogens. Hepatic cancer induction can be enhanced by classical promotion phenomena, by toxic hyperplasia, or by selective pressures on cell replication created by hormones. Substantial and consistent exposure doses are required to invoke secondary mechanisms of enhancing cancer induction; these secondary events occur in dosage ranges associated with observable histopathologic effects and thus can be detected and measured. By using combinations of in vitro and in vivo techniques, no-adverse-effect dosage levels can be determined whereby the risks for enhancing cancer induction can be avoided.

The lifetime feeding study in mice and rats — An examination of its validity as a bioassay for human carcinogens

Currently used designs for carcinogenic bioassay (lifetime feeding studies in mice or rats using maximum tolerated doses of the test compound) are examined to see if they meet the requirements of a bioassay, using the results of 170 compounds reported on as of June, 1980, by the National Cancer Institute. It is concluded that the lifetime feeding study has never been subjected to proper validation as an assay for human carcinogens. When an attempt is made to validate it on the basis of these reported studies and those in the literature, it appears to lack acceptable specificity and sensitivity. It is suggested that a drastically different design is needed and that such redesigning of the assay will require proper validation.

The Lifetime Feeding Study in Mice and Rats - An Examination of Its Validity as a Bioassay for Human Carcinogens

The Lifetime Feeding Study in Mice and Rats - An Examination of Its Validity as a Bioassay for Human Carcinogens

Long-term toxicity of ortho-toluenesulfonamide and sodium saccharin in the rat

In a two-generation lifetime feeding study, 50 male and 50 female weanling Sprague-Dawley rats were included in each of the following dietary treatment groups: control; 2.5 mg ortho-toluenesulfonamide ( o-TS, more than 99.9% pure)/kg/day; 25 mg o-TS/kg/day; 250 mg o-TS/kg/day; 250 mg o-TS/kg/day with 1% NH 4Cl in the drinking water: or 5% sodium saccharin, which contained no detectable amount of o-TS. After 3 months on test, the F 0 rats were bred and 50 pups of each sex, from every group, were weaned onto the parental diets which both generations received for their lifetime. Rats from both generations fed diets providing 250 mg o-TS/kg with 1.0% NH 4Cl in the drinking water or containing 5% sodium saccharin had decreased growth rates, but only the former two had lowered feed consumption. There were no treatment-related effects upon reproduction, longevity, or hematological parameters. The animals were free of the bladder parasite Trichosomoides crassicauda. A few animals developed grossly visible bladder and kidney stones but the incidence of these was not treatment related. The incidence of bladder tumours in male rats fed the 5% saccharin diet was significantly increased in both generations compared to their respective control groups. An evaluation of individual feed consumption data for animals fed saccharin-containing diets did not reveal any statistical difference between the amount of feed consumed by animals which had bladder tumors and those that did not. The incidence of bladder tumors in the o-TS-treated groups and in the female rats fed the 5% saccharin diet was not significantly different from that in control animals.

Lifespan carcinogenicity studies with hexachlorophene in mice and rats

Hexachlorophene (HCP) was tested for carcinogenicity in lifetime feeding studies in male Sprague--Dawley rats that were fed on a protein- and vitamin-deficient diet and in C57B1 and XVII/G mice that received the chemical incorporated into a complete diet. After 2 years, no significant carcinogenic effects were observed in these animals. In XVII/G mice injected subcutaneously at birth or receiving HCP via the mother's milk, the incidence of tumors was not statistically increased. No carcinogenic effect was observed in (C57B1 X C3H) F1 hybrid mice that received HCP transplacentally.