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Conditions of marked and long-lasting hypothermia have been shown to increase the formation of micronucleated polychromatic erythrocyte (MNPCE) in mouse bone-marrow. Stimulation of erythropoiesis as a consequence of anoxic conditions associated with decreased body temperature has been suggested as a possible mechanism for hypothermia-induced micronucleus formation. We examined whether chemically induced hypothermic conditions that produced increased MNPCE formation were associated with stimulation of erythropoiesis by measuring erythropoietin (EPO) concentrations in blood. Marked and long-lasting hypothermia was induced in male mice by oral administration of the antipsychotic compounds E-5842 (200 mg/kg) or chlorpromazine (100 mg/kg). Maximum decreases from the basal temperature, achieved 8 h after treatment, were 14.8 and 12.8 degrees C, respectively. A statistically significant increase in bone-marrow MNPCE frequency was observed 48 h after administration of E-5842 (p<0.01) or chlorpromazine (p<0.05). Mice made anaemic by retro-orbital bleeding (0.5 ml), which acted as positive control for stimulation of erythropoiesis, showed no relevant variation in mean rectal temperature and a slight non-statistically significant increase in MNPCE frequency after 48 h. Blood samples for determination of EPO levels were obtained 4 (bleed-control animals only), 8, 16 and 24 h after treatment. In spite of the induced hypothermia, no significant variation in EPO blood levels was observed after administration of E-5842 or chlorpromazine. Bleed-induced anaemic mice showed a clear increase in EPO blood levels at all sampled time points, differences from baseline values being statistically significant (p<0.001) at the 8-h samplings and beyond. These results indicate that induction of MNPCE secondary to chemically induced hypothermia is not mediated by stimulation of erythropoiesis.

Three structurally related phenyltetrahydropyridinyl butylazole (PTHPB)-derived drug candidates with sigma receptor-binding properties were evaluated for genotoxic potential in the ICH standard battery of genetic toxicology assays. These comprised an Ames test, a mouse-lymphoma assay, and a mouse bone-marrow micronucleus test. The maximum test concentrations in the in vitro assays were determined by the solubility and/or the cytotoxicity of the compounds. In the mouse micronucleus assay, the compounds were administered orally at three levels up to the maximum tolerated dose (MTD). Negative results were obtained for all three drug candidates in the Ames test and in the mouse-lymphoma assay, both in the absence or presence of metabolic activation. In the mouse micronucleus test, there was no effect on the frequency of micronucleated polychromatic erythrocytes (MNPCE) in bone marrow after oral administration of any of the three test compounds, at any dose level or sampling time (24 and 48h). Administration of all three compounds at the MTD induced a clear decrease in mouse body-temperature of 3.1-4.8 degrees C below normal; the temperature returned to normal within 8h of dose administration. The produced mild hypothermia and absence of micronucleus induction was in contrast to the induction of MNPCE secondary to marked hypothermia reported for a structurally similar PTHPB-derived sigma-receptor ligand, the antipsychotic compound E-5842. The results obtained in the current series of studies suggest that exposure to the three tested PTHPB-derived drug candidates would not pose a genotoxic risk under clinical conditions.

The genotoxic potential of the natural neurotoxin Tetrodotoxin (TTX) was evaluated in a battery of in vitro and in vivo genotoxicity assays. These comprised a bacterial reverse-mutation assay (Ames test), an in vitro human lymphocyte chromosome-aberration assay, an in vivo mouse bone-marrow micronucleus assay and an in vivo rat-liver UDS assay. Maximum test concentrations in in vitro assays were determined by the TTX limit of solubility in the formulation vehicle (0.02% acetic acid solution). In the Ames test, TTX was tested at concentrations of up to 200 microg/plate. In the chromosome-aberration assay human lymphocytes were exposed to TTX at concentrations of up to 50 microg/ml for 3 and 20 h in the absence of S9, and for 3h in the presence of S9. For the in vivo assays, maximum tested dose levels were determined by the acute lethal toxicity of TTX after subcutaneous administration. In the mouse micronucleus assay TTX dose levels of 2, 4 and 8 microg/kg were administered to male and female animals, and bone-marrow samples taken 24 and 48 h (high-dose animals only) after administration. In the UDS assay, male rats were given TTX on two occasions with a 14-h interval at dose levels of 2.4 and 8 microg/kg, the last dose being administered 2h before liver perfusion and hepatocyte culturing. Relevant vehicle and positive control cultures and animals were included in all assays. TTX was clearly shown to lack in vitro or in vivo genotoxic activity in the assays conducted in this study. The results suggest that administration of TTX as a therapeutic analgesic agent would not pose a genotoxic risk to patients.

The genotoxic potential of E-5842, a sigma ligand compound being developed as an antipsychotic drug, was evaluated by means of an extensive battery of in vitro and in vivo assays. Negative results were obtained in an Ames test (up to 5000 μg/plate), a mouse lymphoma assay (up to 535.1 μg/ml (-S9) and 891.8 μg/ml (+S9)), an in vivo rat hepatocyte micronucleus assay (up to 100 mg/kg/day on 2 days), and a two-dose mouse micronucleus assay (up to 40 mg/kg/day on 2 days). In a single-dose mouse bone-marrow micronucleus assay (up to 400 mg/kg; 24, 48 and 72 h sampling) a slight and non-statistically significant increase in the frequency of micronucleated polychromatic erythrocytes (MNPCE) was observed 48 h after administration of a 200 mg/kg dose, in the absence of bone-marrow toxicity. This minor increase in MNPCE frequency was considered of questionable biological relevance, because it was observed under conditions of marked animal toxicity including mortality. In addition, it occurred in association with a strong hypothermic effect produced by administration of E-5842. A clear increase in the frequency of structural chromosomal aberrations was observed in human lymphocytes at concentrations ≥350.6 and 1685.4 μg/ml in the presence and absence of S9, respectively. Mitotic accumulation was observed at those concentrations at which clastogenic effects were observed, a condition that may have masked toxicity. Concentrations lacking clastogenic effects in this chromosome aberration assay (300.7 and 173.2 μg/ml in the presence and absence of S9, respectively) were well in excess of maximum human plasma concentrations attained in clinical studies at the maximum tolerated dose (19.1 ng/ml). A weight-of-evidence analysis, taking into consideration the results obtained in the different in vitro and in vivo assays and the conditions of clinical use, suggest that E-5842 would not pose a genotoxic risk under clinical conditions.

Oral administration of E-5842, a new sigma1 receptor ligand being developed as an antipsychotic drug, to male mice at single doses of 50, 100, 200 and 400 mg/kg produced marked and sustained decreases in rectal temperature. Both the intensity and the duration of the hypothermic effect increased with dose. Maximum decreases from the mean pre-administration temperature (36.2 degrees C) ranged from 7.5 to 12.9 degrees C for animals receiving 50 and 400 mg/kg doses, respectively. Examination of bone-marrow smears obtained 24, 48 and 72 h after administration revealed a slight but statistically significant (p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (MNPCE) at the 48 h sampling for animals receiving the 200 mg/kg dose. These animals showed decreases from pre-administration temperature of approximately 12 degrees C, with recovery being observed 24 h after administration. When the hypothermic effect of E-5842 administration was avoided by housing treated animals under conditions of increased environmental temperature (30 degrees C) for 24 h, MNPCE frequency reverted to vehicle control values. Further, in E-5842-treated animals with an increased MNPCE frequency there was a shift in the distribution of the relative areas of micronuclei in MNPCE towards higher values. In addition, there was a statistically significant increase (p < 0.001) in the number of relatively large micronuclei (micronucleus diameter > or = 1/4 cytoplasm diameter) similar to that produced by administration of the mitotic spindle inhibitor colchicine (1 mg/kg), suggesting disturbance of mitotic apparatus as the possible underlying mechanism. The results suggest that the slight increase in MNPCE frequency observed 48 h after administration of a 200 mg/kg dose of E-5842 is due to a hypothermic effect and not to a direct effect of E-5842 on DNA.