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Mechanistic research specifically on DEA indicates that, to the extent DEA can potentially induce tumours in mice, it does so by a mechanism that is not relevant to humans. Therefore, based on the available data, DEA is not considered carcinogenic for humans.

Additional information

Mechanistic studies regarding carcinogenicity

 

In vitro

The effect of DEA (0 - 500 µg/ml) and choline depletion on DNA synthesis and changes in expression of genes involved in cell growth pathways in primary cultures of mouse, rat, and human hepatocytes were examined. In mouse and rat hepatocytes DNA synthesis was increased following treatment with 10 µg/ml DEA and higher. DEA failed to increase DNA synthesis in human hepatocytes. Incubation of hepatocytes in medium containing reduced choline increased DNA synthesis. Choline depletion did not induce DNA synthesis in human hepatocytes. Mouse and rat hepatocytes incubated in medium supplemented with excess choline reduced DEA-induced DNA synthesis to control levels or below. Gene expression analysis of mouse and rat hepatocytes following DEA treatment showed increases in genes associated with cell growth, and decreases in expression of genes involved in apoptotic pathways. These results support that choline depletion is a key mode of action for the induction of rodent hepatic neoplasia by DEA (Kamendulis, 2002, 2006; Kamendulis et al., 2002, 2003; Kamendulis and Klaunig, 2005).

 

In addition the effect of DNA hypomethylation was examined.B6C3F1 mouse hepatocytes in primary culture were grown in the presence of either DEA, Phenobarbital (PB) or choline deficient medium (CD) for 48h. Average DNA methylation status was not affected. DEA, PB and CD medium treatment resulted in regions of altered methylation (RAMs), the majority was hypomethylations. A high proportion of RAMs were identical when DEA was compared to CD medium and similarly, 70% were identical between PB and CD medium. Altered patterns of methylation in GC-rich regions induced by DEA and PB resemble that of CD medium and suggest that altered DNA methylation is an epigenetic mechanism involved in the facilitation of mouse liver tumourigenesis (Bachman et al., 2005).

 

Gene alterations in hepatocellular neoplasms and hepatoblastomas in B6C3F1 mice, exposed dermally to DEA (0, 40, 80, and 160 mg/kg bw/day) for 2 years, were characterized immunohistochemically. There was a lack of H-ras mutations in hepatocellular neoplasms and hepatoblastomas as an indication that the signal transduction pathway is not involved in the development of liver tumours following DEA treatment (Hayashi et al., 2003).

 

Screening for effects of DEA on cell phospholipid biosynthesis and choline uptake in Syrian Golden Hamster (SHE) cells showed that DEA disrupted intracellular choline homeostasis by inhibiting choline uptake and altering phospholipid synthesis. Excess choline blocked these biochemical effects (Lehman-McKeeman and Gamsky, 2000).

 

In vivo

DEA was investigated in a series of studies in B6C3F1 mice for its effects on cell proliferation and apoptosis in the liver and kidneys after repeated dermal administration. The same strain of mice as in the carcinogenicity study was used and DEA was generally applied as ethanolic solution.

 

In the first study, DEA administered dermally to groups of 10 male B6C3F1 mice for a period of 1, 4 and 13 weeks at doses of 0 and 160 mg/kg bw/day caused sustained cell proliferation in the liver and kidneys as well as increased number of mitosis in the kidneys (Mellert and Bahnemann, 2001; Mellert et al., 2004; BASF AG, 2001).

 

In the second study, when DEA was topically applied to groups of 8 male B6C3F1 mice for a period of 1 week and 13 weeks at doses of 0, 10, 20, 40, 80, 160, 630 and 1,250 mg/kg bw/day, the treatment with 630 and 1,250 mg/kg bw/day resulted in severe skin lesions (e.g., ulcers) and therefore, the animals were necropsied prematurely. In all treated groups, liver weights were slightly increased after 1 as well as after 13 weeks of treatment. The kidney weights showed only a tendency for an increase in all dose groups after 1 week of treatment, while after 13 weeks all test groups exhibited significantly increased kidney weights. After 1 week of treatment, in the liver a clear and significant increase of cell proliferation was found in a dose - dependent manner for groups treated with 80 mg/kg and above, while after 13 weeks a significant increase of cell proliferation occurred, which was noted for the 160 and 80 mg/kg groups at least in zone 3 hepatocytes. Light microscopy of liver slides revealed cytoplasmic changes in portal hepatocytes (zone 1) of treated animals. In the kidneys, cell proliferation was significantly increased in all dose groups after 1 week of treatment except in the lowest dose of 10 mg/kg and after 13 weeks of treatment, in all dose groups a significant increase was visible but to a much lower extent with a highest value of a 2.4-fold increase in the OSOM of the 160 mg/kg group. Cell proliferation was increased in liver and/or kidneys of all treatment groups (Mellert et al., 2004; BASF AG, 2002b).

 

In the third study, the reversibility was investigated by dermal application of DEA to groups of 8 male and 8 female B6C3F1 mice for a period of 1 week at doses of 0 and 160 mg/kg bw/day, followed by a 3 week recovery period. There was a clearly increased cell proliferation rate after 1 week of treatment in the liver of both sexes without distinct differences. Cell proliferation was also increased in kidneys of males. The effects were reversible after a recovery-period of 3 weeks (BASF AG, 2002c).

 

In the forth study, the effect of dietary choline supplementation (20,000 ppm) on cell proliferation in liver and kidney was examined after dermal administration of DEA to groups of 8 male B6C3F1 mice for a period of 1 week and 4 weeks at doses of 0, 10 and 160 mg/kg bw/day. A treatment - related increase of cell proliferation was found in the liver as well as in the kidneys at 160 mg/kg after treatment periods of 1 week and 4 weeks. Choline supplementation had no inhibiting influence on the substance-induced increase of cell proliferation. Choline supplementation itself led to an increase of cell proliferation (BASF AG, 2003).

 

A subacute screening study on effects of dermally administered DEA on choline (Cho) and metabolites (phosphocholine (PCho); glycerophosphocholine (GCP); phosphatidylcholine (PC); s-adenosylmethionine (SAM); s-adenosylhomocysteine (SAH) was examined in groups of 6 – 8 male mice of two different strains. After dermal treatment of B6C3Fl mice with DEA at 0, 10, 20, 40, 80, 160 mg/kg bw/day for 4 weeks, PCho was most sensitive to DEA treatment, decreasing at ≥20 mg/kg bw/day. GPC, choline, and PC also decreased in a dose-dependent manner. At ≥80 mg/kg bw/day, SAM levels decreased, while SAH levels increased in liver. The no-observed effect level (NOEL) for DEA induced changes was 10 mg/kg bw/day. Choline metabolites, SAM and SAH returned to control levels in mice allowed a 2-week recovery period. No fatty change was observed in the liver of DEA treated mice in a manner similar to dietary choline deficiency. In C57BL/6 mice, DEA treatment also decreased PCho concentrations, without affecting hepatic SAM levels. Dermal application of 95% ethanol for 4 weeks alone decreased hepatic betaine levels, suggesting that the use of ethanol as a vehicle for dermal application of DEA may exacerbate or confound the biochemical actions of DEA (Lehman-McKeeman et al., 2002).

 

The possibility of nitrosation of DEA under the experimental condition of the NTP mouse bioassay was investigated via subacute application of 160 mg/kg bw/day DEA with or without supplemental sodium nitrite to male B6C3F1 mice. The application was dermal (with or without access to the application site) or via oral gavage, 7 days/week, for two weeks. No NDELA was observed in the urine, blood or gastric contents of any group of treated mice. Blood levels in orally dosed mice were higher than in dermally dosed mice with access to the application site, which were in-turn greater than dermally dosed mice without access. In addition the influence of DEA on metabolism of choline was investigated. Choline, phosphocholine and glycerophosphocholine were decreased ≥62-84% in an inverse relationship to blood DEA levels. After oral application of 160 mg/kg bw/day DEA by gavage, 5 days/week, for 4 weeks. Similar evidence of DEA-induced choline deficiency was obtained. These changes were not accompanied by biochemical evidence of liver damage; however, a statistically identified decrease in SAM levels and increases in diacylglycerol (DAG) were noted. An approximately 2-fold increase in the amount of PKC isoforms was also noted. Thus, there was a lack of NDELA formation at tumourigenic dosages of DEA but a pronounced depletion of choline-containing structures in male B6C3F1 mice (Stott et al., 2000).