Histopathological analysis of malignant tumors induced by intraperitoneal injection of carbon nanotubes

Biological effects of tailor-made multi-walled carbon nanotubes (MWCNTs) were investigated in vivo in a 2-year carcinogenicity study in a project funded by the German Federal Ministry of Education and Research (contract no. 03X0109A). Five male Wistar rats per group (total of 500 rats) were treated once by an intraperitoneal (i.p.) injection of a low (1x109 WHO fibers) and high (5x109 WHO fibers) dose each of MWCNTs (MWCNT 1, 2, 3 and 3a) suspended in artificial lung medium, also used as negative control. Amosite asbestos (0.1x109 WHO fibers) served as positive control. It is well known that only fibrous but not granular dusts can cause tumors in rats after i.p. application. Moribund rats were sacrificed and necropsy comprising all organs was performed. A histopathological classification of tumors and in addition, immunohistochemistry was conducted to compare the induced tumors with mesotheliomas occurring in humans. The treatments induced mesotheliomas in all dose groups, whereas incidence and time to tumor were different between the groups. Rats treated with MWCNTs 3 (L= 8.57 mm; D: 0.085 mm) and 3a (L= 9.3 mm; D= 0.062 mm) were killed moribund between 8 and 12 months after treatment and exhibited high tumor incidences in the low and high dose groups. The survival time of rats treated with MWCNTs 2 (L= 10.24 mm; D= 0.04 mm) was even longer but resulted also in high tumor incidences in both dose groups in relation to the positive control. For MWCNTs 1 (L= 7.9 mm; D= 0.037 mm) the survival time for the low dose was similar to the amosite asbestos (L= 13.95 mm; D= 0.39 mm) positive control. Overall, tumor incidences were lower than for MWCNTs 2, 3 and 3a. Most tumors were histologically and immunohistochemically classified as malignant mesotheliomas comparable to those in humans, revealing a predominantly superficial growth on the serosal surface of the peritoneum especially the diaphragm.