The Contribution of In Vivo Mammalian Studies to the Knowledge of Adverse Effects of Radiofrequency Radiation on Human Health.

The proliferation of cellular antennas and other radiofrequency radiation (RFR) generating devices of the last decades has led to more and more concerns about the potential health effects from RFR exposure. Since the 2011 classification as a possible carcinogen by the International Agency for Research on Cancer (IARC), more experimental studies have been published that support a causal association between RFR exposure and health hazards. As regard cancer risk, two long-term experimental studies have been recently published by the US National Toxicology Program (NTP) and the Italian Ramazzini Institute (RI). Despite important experimental differences, both studies found statistically significant increases in the development of the same type of very rare glial malignant tumors. In addition to carcinogenicity, reproductive organs might be particularly exposed, as well as sensitive to RFR. In this work, we reviewed the currently available evidence from in vivo studies on carcinogenicity and reproductive toxicity studies in order to summarize the contribution of experimental research to the prevention of the adverse effects of RFR on human health.

Differences in Types and Incidence of Neoplasms in Wistar Han and Sprague-Dawley Rats.

A substantial quantity of data on Sprague-Dawley (SD) and Hannover Wistar rats strains have been published concerning their source, diet, and housing conditions, as well as the incidences of nonneoplastic lesions and neoplasms observed in different laboratories. Differences between the commonly used rat strains provided by different breeders (i.e., CD (SD) vs. Harlan Sprague-Dawley strain or Crl: WI(Han) vs. Wistar Hannover (Han)-derived strain, continued breeding by RCC Ltd., Switzerland, thereafter continued breeding by Harlan) may include, but are not limited to, body weight, incidence, and onset of major nonneoplastic lesions and neoplasms, and these can impact the development of a nonclinical safety program. Fisher 344 (F344) and SD rat strains generally have the highest tumor incidences, exceeding that in Wistar rats. Certain tumors are more commonly observed in one strain, and for some, the difference in incidence may be so significant that the tumor may even be considered characteristic for a specific strain (e.g., thymoma in Wistar and amphophilic renal adenoma in SD).

Historical control data of neoplastic lesions in the Wistar Hannover Rat among eight 2-year carcinogenicity studies.

Incidences of neoplastic lesions were evaluated in untreated Hannover Wistar Rats RjHan: WI (470 males and 470 females) used as control animals in eight carcinogenicity studies. All these studies were performed in a similar environment either for the in vivo and the postmortem evaluation. The major neoplastic lesions were found in the endocrine, integumentary and reproductive systems. Pituitary adenoma was the most frequent neoplasm and occurred in 33.9% of the males and 54.6% of the female rats. The other most frequent tumors in males were thyroid C-cell adenoma (8.6%), pancreatic islet cell adenoma (8.1%), subcutaneous fibrosarcoma (6.6%), subcutaneous fibroma (4.7%), benign pheochromocytoma (3.4%), and cutaneous keratoacanthoma (3.4%). In females, the other highest incidences were mammary fibroadenoma (29%), uterine endometrial stromal polyp (18.1%), mammary adenocarcinoma (14.2%), mammary fibroadenoma with atypia (13.7%), thyroid C-cell adenoma (7.5%), benign thymoma (3.7%), and subcutaneous fibrosarcoma (3.6%). All these data were compared to previously published historical control data. This retrospective analysis was undergone in order to illustrate the result of a stable organization which guarantees a robust historical data base for neoplastic and non neoplastic findings.

Early occurrence of spontaneous tumors in CD-1 mice and Sprague-Dawley rats.

It is sometimes difficult to assess the relevance of tumors that occur in treated animals in short-term studies. This report is intended to establish a general profile of tumor occurrence in young control CD-1 mice and Sprague-Dawley rats. Data from 20 rat and 20 mouse carcinogenicity studies conducted between 1990 and 2002 at Huntingdon Life Sciences, UK. were collected and evaluated. The route of administration was either dietary oral gavage, and the analysis was confined to sporadic deaths (decedents) in control groups occurring during the first 50 weeks of study. In addition, tumor occurrence between 50-80 weeks were compared. In mice, the most common tumor was lymphoma, followed by bronchiolo-alveolar adenoma. In rats, the most common tumor was adenoma of the pituitary gland, followed by mammary fibroadenoma, and adenocarcinoma. When studies of up to 50 weeks, between 50 and 80 weeks, and at 2-year termination were compared, there was no great difference in tumor occurrence except in male rats, in which the most common tumor up to 50 weeks on study was lymphoma, whereas the most common tumor between 50-80 weeks and at 2 years was pituitary adenoma.

Prediction of rodent carcinogenesis: an evaluation of prechronic liver lesions as forecasters of liver tumors in NTP carcinogenicity studies.

The National Toxicology Program (NTP) developed the chronic 2-year bioassay as a mechanism for predicting the carcinogenic potential of chemicals in humans. The cost and duration of these studies has limited their use to small numbers of selected chemicals. Many different short-term methods aimed at increasing predictive accuracy and the number of chemicals evaluated have been developed in attempts to successfully correlate their results with evidence of carcinogenicity (or lack of carcinogenicity) are assessed. Using NTP studies, the effectiveness of correlating prechronic liver lesions with liver cancer encompassing multiple studies using mice (83 compounds) and rats (87 compounds). These lesions include hepatocellular necrosis, hepatocellular hypertrophy, hepatocellular cytomegaly, bile duct hyperplasia, and hepatocellular degeneration, along with increased liver weight. Our results indicate that pooling 3 of these prechronic data points (hepatocellular necrosis, hepatocellular hypertrophy, and hepatocellular cytomegaly) can be very predictive of carcinogenicity in the 2-year study (p < 0.05). The inclusion of increased liver weight as an endpoint in the pool of data points increases the number of rodent liver carcinogens that are successfully predicted (p < 0.05), but also results in the prediction of increased numbers of noncarcinogenic chemicals as carcinogens. The use of multiple prechronic study endpoints provides supplementary information that enhances the predictivity of identifying chemicals with carcinogenic potential.

Carcinogenic susceptibility to N-bis(2-hydroxypropyl)nitrosamine (DHPN) in rasH2 mice.

To evaluate the susceptibility of rasH2 mice to N-bis(2-hydroxypropyl)nitrosamine (DHPN), a potent carcinogen targeting the lung, liver, thyroid, and kidney, male, 6-week old, rasH2 mice and wild-type littermates (non-Tg mice) were given DHPN in drinking water at 0, 20 or 200 ppm, and 0 or 200 ppm, respectively, for 26 weeks. The experiment using rasH2 mice given 200 ppm DHPN and non-Tg mice given 200 and 0 ppm DHPN was completed at 20 weeks, since mortality in these groups was remarkably increased due to hemangiosarcomas of the liver. Histologically, tumors developed in the lung and liver in both rasH2 and non-Tg mice treated with DHPN. In addition, proliferative lesions were observed in the forestomach, urethra, and excretory duct of salivary glands in rasH2 mice given 200 ppm DHPN. RT-PCR analysis showed no marked difference in expression of mRNAs for the transgene and the endogenous mouse ras gene between the whole lung tissue containing a neoplasm and normal lung tissue. Our results suggest that rasH2 mice are highly susceptible to DHPN, the target organs including the forestomach, salivary gland and urethra, which have not been found to develop tumors in previous long-term carcinogenicity studies of DHPN in rats and mice.

Results of chronic dietary toxicity studies of high viscosity (P70H and P100H) white mineral oils in Fischer 344 rats.

Two-year dietary studies were conducted to determine the chronic toxicity and its reversibility, and the carcinogenicity of P70(H) and P100(H) white mineral oils in Fischer-344 rats (F-344). The studies were identical in design and followed the Organization for Economic Cooperation and Development, Guidelines for Testing Chemicals, Guideline 453, 1981. Additional endpoints evaluated were: (1) extent of mineral hydrocarbon deposition in liver, kidneys, mesenteric lymph nodes, and spleen of female rats at 3, 6, 12, 18 and 24 months, and (2) reversibility of effects following cessation of exposure. Dietary concentration were 60, 120, 240, and 1,200 mg/kg/day, adjusted periodically to account for bodyweight changes. Study results were consistent with preceding subchronic studies. No treatment-related mortality, neoplastic lesions, or changes in clinical health, hematology, serum chemistry, or urine chemistry were evident in any group administered either white oil. Statistically significant higher food consumption was noted in the 1,200 mg/kg group males and females exposed to either white oil and statistically significant higher body weights were noted in the 1,200-mg/kg males during the latter portion of the P100(H) study. Higher mesenteric lymph node weights were accompanied by increased severity of infiltrating histiocytes. This occurred to a greater extent with the P70(H) than the P100(H) oil. No other histopathology of significance was observed. Mineral hydrocarbons were detected in the liver following exposure to either oil. Maximal concentrations of mineral hydrocarbons in the liver were similar with both oils but occurred more rapidly with the P70(H) oil. Liver mineral hydrocarbon content returned to near-background levels during the reversibility phase. In conclusion, lifetime exposer of F344 rats to P70(H) and P100(H) white oils resulted in only minimal findings and with no consequence to clinical health. Thus, under the conditions of these studies, the No Observable Adverse Effect Level (NOAEL) for these studies was considered to be 1,200 mg/kg/day.

Bone neoplasms in F344 rats given teriparatide [rhPTH(1-34)] are dependent on duration of treatment and dose.

A long-term study was conducted in female F344 rats to determine the relative importance of dose, treatment duration, and age at initiation of treatment on the incidence of teriparatide [rhPTH[1-34)]-induced bone proliferative lesions. Treatment groups consisted of different combinations of dose (0, 5, or 30 microg/kg/d), treatment duration (6, 20, or 24 months) and age at initiation of treatment (2 or 6 months of age). The primary endpoints were the incidence of bone neoplasms and effects on bone mass and structure as evaluated by quantitative computed tomography and histomorphometery. Significant increases in the incidence of bone tumors (osteoma, osteoblastoma, and osteosarcoma) occurred in rats treated with 30 microg/kg for 20 or 24 months. No neoplasms were found when the 5 microg/kg treatment was initiated at 6 months of age and continued for either 6 or 20 months (up to 70% of life span). This treatment regimen defined a "no-effect" dose for neoplasm formation that nevertheless resulted in substantial increases in bone mass. These results demonstrate that treatment duration and administered dose are the most important factors in the teriparatide-induced bone tumors in rats.

An alternative perspective: a critical evaluation of the Waddell threshold extrapolation model in chemical carcinogenesis.

In a recent Perspective article (Toxicologic Pathology 31: 260-262, 2003) Waddell asserts that he has developed a log linear extrapolation model that can demonstrate a threshold and resolve for once and for all the uncertainies associated with low dose cancer risk extrapolation. However, his method essentially forces, rather than demonstrates, a threshold, and has many serious flaws that result in significant under-estimation of low dose risk. It would be a serious mistake for the scientific community to adopt Waddell's log linear extrapolation model for chemical carcinogenesis risk assessment.

Mortality, body weight, food and water consumption, and clinical signs in Slc: B6C3F1 (C57BL/6 x C3H) mice utilized in chronic toxicity and carcinogenicity studies.

In vivo historical control data for Slc: B6C3F1 mice, including mortality, body weight, food and water consumption, and clinical signs and which were obtained from long-term toxicity and carcinogenicity studies (11 male and 12 female studies) conducted at the Biosafety Research Center, Foods, Drugs and Pesticides, (An-Pyo Center) during the last five years are presented. Mean survival at 83 and 109 weeks of age was 96.4% (min: 94.0%, max: 100%) and 79.0% (min: 74.0%, max: 86.0%) in males and 98.7% (min: 96.0%, max: 100%) and 81.7% (min: 70.0%, max: 90.0%) in females, respectively. The maximum mean body weight of males and females was 45.1 +/- 3.1 g (mean +/- S.D.) and 39.2 +/- 4.1 g, respectively. Male mice attained their maximum body weight at 72.7 +/- 4.3 weeks of age and females at 76.2 +/- 5.9 weeks of age. Clinical symptoms increased with age, particularly after week 84, and included: wasting, piloerection and palpable abdominal masses. Hypothermia and auricular pallor were common findings in moribund animals from week 79 to 104 of the studies. The use of in-house, historical control data can prove invaluable in the evaluation and interpretation of experimental results, especially in long-term and life-time studies.

Historical control data of organ weight and gross findings in F344/DuCrj rats and B6C3F1 mice.

Organ weight and gross postmortem findings of control Fischer 344/DuCrj rats and B6C3F1 mice are presented from subchronic, chronic toxicity and carcinogenicity studies that were conducted over a 9 year period at our center (An-Pyo Center). The mean organ weight of the liver, kidney, spleen and the lung were increased associated with age in both species. The most common findings observed at 109 weeks in both species were thymic atrophy, enlargement of the spleen, dilation of the lumen of the uterus and ovarian cysts. Male and female Fischer 344/DuCrj rats commonly exhibited a granular surface of the kidneys, hypertrophy of and/or nodular pituitary glands and subcutaneous tissue masses. Multiple white patches and hypertrophy or atrophy of the testes, atrophy of the seminal vesicles and prostate and nodules in the pancreas were seen frequently in male rats. Malformative nodule in the liver was a common finding in female rats. The most common lesions seen in both male and female B6C3F1 mice were nodules in the liver and lungs and enlargement of the lymph nodes. Nodules on the preputial gland were commonly seen in the males. Sex differences were evident in the incidence of some of the above findings. These historical data will contribute to analyze the organ weight and gross findings at necropsy in long-term toxicity and carcinogenicity studies.

Establishment and characterization of a chicken mononuclear cell line.

A new chicken mononuclear cell line (MQ-NCSU) has been established. The starting material used to initiate this cell line was a transformed spleen from a female Dekalb XL chicken which had been experimentally challenged with the JM/102W strain of the Marek's disease virus. After homogenization, a single cell suspension of splenic cells was cultured using L.M. Hahn medium supplemented with 10 microM 2-mercaptoethanol. Under these culture conditions, a rapidly proliferating cell was observed and then expanded after performing limiting dilution cultures. These cells were moderately adherent and phagocytic for sheep red blood cells and Salmonella typhimurium. When tested against a panel of monoclonal antibodies (mAb) using the flow cytometry, MQ-NCSU cells stained readily with anti-chicken monocyte specific (K-1) mAb but did not stain with mAb detecting T-helper, T-cytotoxic/suppressor, and NK cells. MQ-NCSU cells expressed very high levels of Ia antigens and transferrin receptors. In addition, cell-free supernatant obtained from MQ-NCSU culture contained a factor which exhibited cytolytic activity against tumor cell targets. Based on their cultural, morphological, and functional characteristics and mAb reactivity profile, we conclude that MQ-NCSU cell line represents a malignantly-transformed cell which shares features characteristic of cells of the mononuclear phagocyte lineage.