Report of final results regarding brain and heart tumors in Sprague-Dawley rats exposed from prenatal life until natural death to mobile phone radiofrequency field representative of a 1.8 GHz GSM base station environmental emission.

BACKGROUND: In 2011, IARC classified radiofrequency radiation (RFR) as possible human carcinogen (Group 2B). According to IARC, animals studies, as well as epidemiological ones, showed limited evidence of carcinogenicity. In 2016, the NTP published the first results of its long-term bioassays on near field RFR, reporting increased incidence of malignant glial tumors of the brain and heart Schwannoma in rats exposed to GSM - and CDMA - modulated cell phone RFR. The tumors observed in the NTP study are of the type similar to the ones observed in some epidemiological studies of cell phone users. OBJECTIVES: The Ramazzini Institute (RI) performed a life-span carcinogenic study on Sprague-Dawley rats to evaluate the carcinogenic effects of RFR in the situation of far field, reproducing the environmental exposure to RFR generated by 1.8 GHz GSM antenna of the radio base stations of mobile phone. This is the largest long-term study ever performed in rats on the health effects of RFR, including 2448 animals. In this article, we reported the final results regarding brain and heart tumors. METHODS: Male and female Sprague-Dawley rats were exposed from prenatal life until natural death to a 1.8 GHz GSM far field of 0, 5, 25, 50 V/m with a whole-body exposure for 19 h/day. RESULTS: A statistically significant increase in the incidence of heart Schwannomas was observed in treated male rats at the highest dose (50 V/m). Furthermore, an increase in the incidence of heart Schwann cells hyperplasia was observed in treated male and female rats at the highest dose (50 V/m), although this was not statistically significant. An increase in the incidence of malignant glial tumors was observed in treated female rats at the highest dose (50 V/m), although not statistically significant. CONCLUSIONS: The RI findings on far field exposure to RFR are consistent with and reinforce the results of the NTP study on near field exposure, as both reported an increase in the incidence of tumors of the brain and heart in RFR-exposed Sprague-Dawley rats. These tumors are of the same histotype of those observed in some epidemiological studies on cell phone users. These experimental studies provide sufficient evidence to call for the re-evaluation of IARC conclusions regarding the carcinogenic potential of RFR in humans.

Results of lifespan exposure to continuous and intermittent extremely low frequency electromagnetic fields (ELFEMF) administered alone to Sprague Dawley rats.

BACKGROUND: Up to now, experimental studies on rodents have failed to provide definitive confirmation of the carcinogenicity of extremely low frequency electromagnetic fields (ELFEMF). Two recent studies performed in our laboratory on Sprague-Dawley rats reported a statistically significant increase in malignant tumors of different sites (mammary gland, C-cells carcinoma, hemolymphoreticular neoplasia, and malignant heart Schwannoma) when ELFEMF exposure was associated with exposure to formaldehyde (50 mg/l) or acute low dose of γ-radiation (0.1 Gy) (Soffritti et al., 2016a) (Soffritti et al., 2016b). The same doses of known carcinogenic agents (50 mg/l formaldehyde, or acute 0.1 Gy γ-radiation), when administered alone, previously failed to induce any statistically significant increase in the incidence of total and specific malignant tumors in rats of the same colony. OBJECTIVES: A lifespan whole-body exposure study was conducted to evaluate the possible carcinogenic effects of ELFEMF exposure administered alone to Sprague-Dawley rats, as part of the integrated project of the Ramazzini Institute (RI) for studying the effects on health of ELFEMF alone or in combination with other known carcinogens. METHODS: Male and female Sprague-Dawley rats were exposed 19 h/day to continuous sinusoidal-50 Hz magnetic fields (S-50 Hz MF) at flux densities of 0 (control group), 2, 20, 100 or 1000µT, and to intermittent (30 min on/30 min off) S-50 Hz MF at 1000 µT, from prenatal life until natural death. RESULTS: Survival and body weight trends in all groups of rats exposed to ELFEMF were comparable to those found in sex-matched controls. The incidence and number of malignant and benign tumors was similar in all groups. Magnetic field exposure did not significantly increase the incidence of neoplasias in any organ, including those sites that have been identified as possible targets in epidemiological studies (leukemia, breast cancer, and brain cancer). CONCLUSIONS: Life-span exposures to continuous and intermittent sinusoidal-50 Hz ELFEMFs, when administered alone, did not represent a significant risk factor for neoplastic development in our experimental rat model. In light of our previous results on the carcinogenic effects of ELFEMF in combination with formaldehyde and γ-radiation, further experiments are necessary to elucidate the possible role of ELFEMF as cancer enhancer in presence of other chemical and physical carcinogens.

Potential carcinogenic effects of world trade center dust after intratracheal instillation to Sprague-Dawley rats: first observation.

BACKGROUND: More than 10 years have passed since the terrorist attack on the New York City World Trade Center on September 11, 2001. It is well known that long-term carcinogenic bioassays on rodents can predict the potential carcinogenic effects of chemical and physical agents for humans. OBJECTIVE: A life-span carcinogenicity bioassay was conducted on Sprague-Dawley rats at the CMCRC of the Ramazzini Institute to test the potential carcinogenic effects of settled dust collected at the WTC immediately after the terrorist attack. METHODS: The WTC material tested is a complex mixture of coarse particles (95%) contain pulverized cement, glass fibres, asbestos, lead, polycyclic aromatic hydrocarbons (PAH(S) ), polychlorinated biphenyls (PCB(S) ) and polychlorinated furans, and dioxin. The test matter was suspended in sterile saline and administered by intratracheal instillation (IT) to 8-week-old Sprague-Dawley rats (100 animals/sex), 3-4 days/week for 4 weeks. A group of 200 male and female rats served as controls. The animals were kept under observation until natural death. RESULTS: Histopathological evaluation of the lungs (target organ) of instilled control and treated male and female rats, did not show any significant increased incidence of lung tumors. Two hemangiomas (one with endothelial atypia) and one hemangiosarcoma were found in the lungs of treated males. Moreover a modest increased incidence of terminal bronchiolar hyperplasia (TBH) and squamous metaplasia occurred in the lung of treated males and females compared to the controls. CONCLUSION: Hemangioma and hemangiosarcoma are extremely rare tumors in the lung of our colony and we believe they are caused by WTC dust.

The scientific and methodological bases of experimental studies for detecting and quantifying carcinogenic risks.

This paper outlines the aims and potential scope of experimental research for risk identification and assessment in industrial carcinogenesis (environmental and occupational). It then reviews the basic, general, and specific requisites of a rigorously scientific nature that are required to render experiments to be more appropriate and better geared to the information they seek. A range of experimental approaches to risk assessment are illustrated by results achieved in the Cancer Research Centre of the Ramazzini Foundation (CRC/RF). The paper ends with a call for closer relations and integration among experimental, epidemiologic, and biostatistical studies.

Mega-experiments to identify and assess diffuse carcinogenic risks.

Diffuse carcinogenic risks, that is, those of low potency involving large areas of population and sometimes all mankind, pose a serious public health problem. Controlling these risks might help to reduce the incidence of, and mortality from, cancer. Because of their low expected carcinogenic potential, these risks are difficult to expose or assess. Epidemiologic investigation is of limited use in this field and yields its data too late to be useful. Experimental studies offer the only possible approach for assessing such risks. To increase experimental sensitivity and consistency of results, mega-experiments must be designed. That is, experiments that use a large number of animals with a well-known basic tumorigram, that extend the exposure and the biophase for as long as possible, that carefully observe the effects, and that are performed with suitable standardized methods. In the last 15 years the Ramazzini Foundation, in its Cancer Research Center at Bentivoglio, has conducted or planned five mega-experiments. Initial results indicate the great potential of these methods for identifying and assessing diffuse risks.

p53 gene mutation pattern in rat liver tumors induced by vinyl chloride.

Vinyl chloride (VC) induces angiosarcomas of the liver (ASL) and hepatocellular carcinomas (HCCs) in humans and rodents. We examined the presence of p53 gene mutations in ASL and HCC induced by VC in Sprague Dawley rats; 25 ASL and eight HCCs were analyzed for point mutations in exons 5-8, using PCR amplification, single-strand conformation polymorphism analysis, and direct DNA sequencing. Mutations were found in 11 (44%) of the ASL and in 1 HCC. A 12-base pair deletion was found in one tumor; all others were base pair substitutions. Nine of the point mutations were observed at A:T base pairs (5 A:T --> T:A; 2 A:T --> G:C, and 2 A:T --> C:G), and of three G:C --> A:T transitions, only one was at a CpG site. In ASL, four mutations were found in exon 5, two in exon 6, and six in exon 7; the base pair substitution found in one HCC was in exon 8. One ASL exhibited two point mutations, including a silent one. Two ASL exhibited the same mutation in codon 203 and two other samples in codon 253. Codon 235 was found to be mutated in three ASL. These data show that p53 is often mutated in ASL induced by VC in rats and, as observed in ASL in humans exposed to VC, the majority of the missense mutations involved A:T base pairs. The characteristic patterns of mutations found suggest that a common mechanism operates in VC-induced p53 mutagenesis in both species, and these mutations are consistent with the formation of DNA etheno adducts by VC in the liver. The A:T --> T:A transversion observed in the first nucleotide of codon 253 in two rat ASL is equivalent to the A:T --> T:A transversion characterized previously in codon 255 in one human ASL associated with VC exposure.

Results of long-term experimental carcinogenicity studies of the effects of gasoline, correlated fuels, and major gasoline aromatics on rats.

Unleaded gasoline, with high aromatic content, leaded gasoline, gasoil (diesel), kerosene, toluene, xylenes, ethylbenzene, and 1,2,4-trimethyl-benzene were submitted to long-term experimental carcinogenicity bioassays. The mixtures and the compounds were administered by stomach tube, in olive oil, once daily, 4 days weekly, for 104 weeks, to male and female Sprague-Dawley rats. The animals were kept under control until the end of the experiments. With varying degrees of evidence, all the tested materials were found to increase the total number of malignant tumors and of some site-specific tumors. They must therefore be considered carcinogenic. On the basis of our results the rank of carcinogenic potency of the tested aromatic hydrocarbons increases in the following order: 1,2,4-trimethylbenzene, ethylbenzene, xylenes, toluene (benzene).

Results of long-term experimental studies on the carcinogenicity of methyl tert-butyl ether.

Methyl-tert-butyl ether (MTBE) was submitted to long-term carcinogenicity bioassays on Sprague-Dawley rats. The test compound was delivered in olive oil by stomach tube (gavage), at the doses of 1000, 250, and 0 mg/kg b.w. to groups of 60 males and 60 females, once daily, 4 times weekly, for 104 weeks. All animals were kept under control until spontaneous death. MTBE was found to cause in males an increased incidence of Leydig cell testicular tumors in the group treated with the higher dose, and in females a dose-related increase of leukemias, an increase of dysplastic proliferations of lymphoreticular tissues, and also an increase of uterine sarcomas at the lower tested dose. On the basis of the presented data, MTBE must be considered a potential carcinogen.

Results of long-term carcinogenicity studies of chlorine in rats.

Four groups, each of 50 male and 50 female Sprague-Dawley rats, of the colony used in the Cancer Research Center of Bentivoglio of the Ramazzini Foundation, 12 weeks old at the start of the study, received drinking water containing sodium hypochlorite, resulting in concentrations of active chlorine of 750, 500, and 100 mg/l (treated groups), and tap water (active chlorine < 0.2 mg/l) (control group), respectively, for 104 weeks. Among the female rats of the treated groups, an increased incidence of lymphomas and leukemias has been observed, although this is not clearly dose related. Moreover, sporadic cases of some tumors, the occurrence of which is extremely unusual among the untreated rats of the colony used (historical controls), were detected in chlorine-exposed animals. The results of this study confirm the results of the experiment of the United States National Toxicology Program (1991), which showed an increase of leukemia among female Fischer 344/N rats following the administration of chlorine (in the form of sodium hypochlorite and chloramine) in their drinking water. The data here presented call for further research aimed at quantifying the oncogenic risks related to the chlorination of drinking water, to be used as a basis for consequent public health measures.

Results of three life-span experimental carcinogenicity and anticarcinogenicity studies on tamoxifen in rats.

Tamoxifen was submitted to carcinogenicity bioassays on Sprague-Dawley rats (of the colony used at the Cancer Research Center in the Castle of Bentivoglio of the European Ramazzini Foundation of Oncology and Environmental Sciences) at the dose of 3.3 mg/kg b.w., by stomach tube, in three experiments. In the first experiment the drug was administered once daily, 6 days a week to male and female rats, 8 weeks old at start for their life span. In the second experiment, the drug was administered to female rats, 12 weeks old at start, once daily for 8 consecutive days every 8 weeks for their life span. In the third experiment the drug was administered to female rats, 56 weeks old at start, 6 times weekly for 40 weeks; and then the animals were kept alive for their life span. In the first experiment, a mild increase in hepatocarcinomas with low grading was detected. In the first and second experiments, a borderline increase in uterine malignancies was found. No carcinogenic effect was observed in the third experiment. In the three experiments, tamoxifen showed a strong, long-lasting chemopreventive effect on mammary benign tumors and cancers. The presented data also indicate that tamoxifen treatment reduces the incidence of other tumors: pituitary adenomas, adrenal pheochromocytomas, islet cell pancreatic tumors, Leydig cell testicular tumors, and polyps of the uterus.

Methyl-tertiary-butyl ether (MTBE)--a gasoline additive--causes testicular and lymphohaematopoietic cancers in rats.

In the framework of a series of experiments conducted to evaluate the carcinogenic effects of oxygenated gasoline additives, MTBE was analyzed in an oral lifetime carcinogenicity study using 8-week-old male and female Sprague-Dawley rats. These experiments were part of a large research project on gasoline carcinogenicity performed at the Bentivoglio (BT) Castle Cancer Research Center of the Ramazzini Foundation and of the Bologna Institute of Oncology, MTBE, dissolved in oil, was administered by stomach tube at the doses of 1000, 250, or 0 mg/kg b.w., once daily, four days weekly, for 104 weeks. The animals were maintained until natural death. The last animal died 166 weeks after the start of the experiment, i.e., at 174 weeks of age. Under the tested experimental conditions, MTBE was shown to cause an increase in Leydig interstitial cell tumors of the testes and a dose-related increase in lymphomas and leukemias in female rats.

Benzene, an experimental multipotential carcinogen: results of the long-term bioassays performed at the Bologna Institute of Oncology.

In 1976, a systematic and integrated project of long-term carcinogenicity bioassays began at the Bentivoglio Experimental Unit of the Bologna Institute of Oncology. The Bologna experiments proved for the first time that benzene is an experimental carcinogen. These experiments demonstrated that benzene is carcinogenic when administered by ingestion and by inhalation and that it cause tumors in the various tested animal models (Sprague-Dawley rats, Wistar rats, Swiss mice, and RF/J mice). They also showed that benzene is a multipotential carcinogen, as it produces a variety of neoplasias in one or more of the tested animal models, including Zymbal gland carcinomas, carcinomas of the oral cavity, nasal cavities, skin, forestomach, and mammary glands, as well as angiosarcomas of the liver, hemolymphoreticular neoplasias, tumors of the lung, and possibly hepatomas. The Bologna experiments also indicated a clear-cut dose-response relationship in benzene carcinogenesis. This report presents the up-to-date results of the Bologna project. The need for more experimental research aimed at assessing the carcinogenic effects of low doses of benzene, of chemical mixtures containing benzene, and of benzene substitutes is emphasized. Also recommended are more comprehensive epidemiological investigations, extended to all types of malignancies, with particular regard to lung carcinomas.

Experimental studies on benzene carcinogenicity at the Bologna Institute of Oncology: current results and ongoing research.

In 1977 Maltoni and Scarnato were the first to demonstrate that benzene is an experimental carcinogen in rats. With that and other experiments, Maltoni et al have shown that benzene administered by ingestion (stomach tube) or inhalation is a multipotential carcinogen in rats (of two different strains) and mice and produces a variety of tumors, namely: Zymbal gland carcinomas, oral and nasal cavity carcinomas, skin carcinomas, acanthomas, dysplasias and carcinomas of forestomach, mammary malignant tumors, hepatomas, liver angiosarcomas, hemolymphoreticular neoplasias, and pulmonary tumors. The incidence of Zymbal gland carcinomas and carcinomas of the oral and nasal cavities is affected by the length of treatment by inhalation and by the age of animals. However, the available epidemiological and experimental data at present do not provide precise information on the risk of doses around or below 10 ppm. Long-term carcinogenicity bioassays at 50, 25, 10, 5 and 1 ppm may be helpful for scientific risk assessment. In addition, these experiments have shown that toluene, xylene, and ethylbenzene, at high concentrations, cause an increase in the number of total malignant tumors.