Evaluation of the genetic and embryotoxic effects of bis(tri- n-butyltin)oxide (TBTO), a broad-spectrum pesticide, in multiple in vivo and in vitro short-term tests

Abstract

The genetic and embryotoxic effects of bis(tri- n-butyltin)oxide (TBTO) were evaluated in multiple in vivo and in vitro short-term tests preparatory to its potential wide use as a molluscicide in control of schistosomiasis. When tested in the rec assay in Bacillus subtilis, TBTO was not mutagenic and it did not induce reverse mutations in Klebsiella pneumoniae. Neither in the presence nor in the absence of rat liver activation system did TBTO produce point mutations in Salmonella typhimurium strains TA1530, TA1535, TA1538, TA97, TA98 or TA100. TBTO was mutagenic in strain TA100 in a fluctuation test, but only in the presence of rat liver S9 (Aroclor-induced). TBTO did not induce gene mutations in the yeast Schizosaccharomyces pombe, mitotic gene conversions in the yeast Saccharomyces cerevisiae, nor sister-chromatid exchange in Chinese hamster ovary cells in the presence or absence of rat or mouse liver S9. In the latter cells, structural chromosomal aberrations, endoreduplicated and polyploid cells were induced. TBTO did not induce gene mutations in V79 Chinese hamster cells (to 8-azaguanine-, ouabain- or 6-thioguanine-resistance) in the presence of a rat liver postmitochondrial fraction or in cell (hamster embryo cells and human and mouse epidermal keratinocyte)-mediated assays. In mouse lymphoma cells, TBTO did not induce 6-thioguanine- or BUdR-resistant mutations. As many tumour promoters inhibit metabolic cooperation between V79 Chinese hamster 6-thioguanine-resistant/-sensitive cells, TBTO was tested but showed no such activity. TBTO was examined for the induction of recessive lethal mutations in adult Berlin K male Drosophila melanogaster, either by feeding or by injection. Doses of 0.37 or 0.74 mM did not increase the number of X-linked recessive lethal mutations. An increased number of micronuclei was observed in the polychromatic erythrocytes of male BALB/c mice 48 h after a single oral dose of TBTO (60 mg/kg bw), while a lower dose (30 mg/kg bw) was ineffective. Neither of the two doses had induced micronuclei 30 h after treatment. The reproductive toxicity of TBTO was studied in NMRI mice. In a 10-day toxicity study, the LD 50 and LD 10 were 74 and 34 mg/kg bw, respectively. An increased frequency of cleft palates was seen in the fetuses of mice (compared with controls, 0.7%) treated orally during pregnancy with 11.7 mg/kg TBTO (7%), 23.4 mg/kg (24%) or 35 mg/kg (48%). The two highest doses of the drug also increased the frequencies of irregular ossification centres of sternebrae and of minor abnormalities, such as fusion of the bases of the os occipitalis. Doses of 6 mg of TBTO/kg bw do not produce teratogenic effects in mice. Teratogenic effects were only seen at TBTO doses that were toxic to the maternal organism. Electron microscopy 26 and 48 h after TBTO treatment showed no evidence that embryos were damaged. In contrast, the maternal liver was greatly affected. The tin contents of different organs of the maternal organism and of the whole fetus were determined after TBTO treatment. After a single oral treatment with 117 mg TBTO/kg bw, the total tin content in 11.5-day-old embryos 12 h after TBTO administration was 9.5 nmoles Sn/g wet weight — 70% greater than the normal value. The total concentration of tin in the maternal liver was increased to a similar extent (by approximately 60%) over the normal level between 6 and 24 h after treatment. No other organ studied showed similarly high increases in tin content. These results show that TBTO gives negative results in short-term tests using various genetic endpoints. However, at cytotoxic concentrations, it was mutagenic in one bacterial strain, clastogenic in CHO cells in vitro and produced micronuclei in mouse bone marrow cells in vivo. In view of these adverse biological effects, and its high capacity to interfere with morphogenetic differentiation processes in vitro careful evaluations must be made before TBTO is released in large quantities into the aquatic environment, and levels to which humans are exposed must be strictly controlled when it is used as a molluscicide.