发酵法L-丙氨酸对小鼠的急性毒性和遗传毒性研究

Acute toxicity and genotoxicity among mice caused by L-alanine from fermentation broth:acute oral test

目的评价发酵法L-丙氨酸的急性毒性与遗传毒性。方法选择健康成年清洁级ICR雌雄小鼠各10只,以最大耐受量(MTD,15.0 g/kg)经口灌胃进行急性经口毒性试验。分别设8、40、200、1 000、5 000μg/皿发酵法L-丙氨酸染毒组,同时设空白对照组(自发回变)、溶剂对照组(纯净水)和阳性对照组(TA97a-S9、TA98-S9、TA102-S9均采用50.0μg/皿敌克松,TA97a+S9、TA98+S9、TA100+S9均采用10.0μg/皿的2-氨基芴,TA100-S9采用1.5μg/皿叠氮钠,TA102+S9采用50.0μg/皿的1,8-二羟蒽醌),采用平板掺入法进行Ames试验。将50只健康成年清洁级小鼠随机分为5组,分别为阴性对照组(0.5%羧甲基纤维素钠水溶液)和1.25、2.50、5.00 g/kg发酵法L-丙氨酸染毒组及阳性对照组(0.04 g/kg环磷酰胺),每组10只,雌雄各半;采用间隔24 h两次经口灌胃法进行小鼠骨髓细胞微核试验。将25只健康成年清洁级雄性小鼠随机分为5组,分别为阴性对照组(0.5%羧甲基纤维素钠水溶液)和1.25、2.50、5.00 g/kg发酵法L-丙氨酸染毒组及阳性对照组(2 mg/kg丝裂霉素C),每组5只。采用连续5 d灌胃方式进行小鼠睾丸染色体畸变试验。结果发酵法L-丙氨酸对雌、雄性小鼠的MTD均大于15.0 g/kg。在加或不加S9的情况下,各剂量发酵法L-丙氨酸对TA97a、TA98、TA100、TA102菌株均无致突变作用。各剂量发酵法L-丙氨酸染毒组小鼠骨髓嗜多染红细胞微核率与阴性对照组比较,差异均无统计学意义(P>0.05);PCE/NCE值均在正常范围内。各剂量发酵法L-丙氨酸染毒组小鼠睾丸初级精母细胞常染色体单价体率、性染色体单价体率及畸变细胞率与阴性对照组比较,差异均无统计学意义(P>0.05)。结论发酵法L-丙氨酸急性毒性属无毒级,未见其明显致突变作用或遗传毒性。

Objective To evaluate acute toxicity,genotoxicity and mutagenicity of L-alanine from fermentation broth. Methods L-alanine from fermentation broth was administered by gavage to 20 cleaning grade ICR mice（male：female=1∶1） at a dose of 15.0 g/kg body weight using maximal tolerance dose（MTD）; Ames test was conducted with the concentrations of8,40,200,1 000 and 5 000 μg/plate test article,while blank（spontaneous revertants）,solvent（purified water） and positive controls（50.0 μg/plate fenaminosulf for strain TA97a-S9,TA98-S9,TA102-S9;10.0 μg/plate 2-aminofluorene for strain TA97 a ＋S9,TA98 ＋S9,TA100 ＋S9;1.5 μg/plate sodium azide for strain TA100-S9;50.0 μg/plate 1,8-dihydroxyanthraquinone for strain TA102 ＋S9） according to plate incorporation assay. A total of 50 cleaning grade ICR mice of both sexes were randomly assigned into five groups,10 in each（male ∶female =1 ∶1）,negative control（0.5% sodium carboxymethyl cellulose solution）,positive control（0.04 g/kg cyclophosphamide） and three test groups（1.25,2.50,5.00 g/kg body weight test substance）,test substance was administered by gavage for twice at 24-hour intervals. Chromosome aberration test was carried out with 25 cleaning grade ICR male mice,five groups,five males in each,test article was given through gavage at the doses of 1.25,2.50 and 5.00 g/kg body weight respectively for five consecutive days at 24-hour intervals. The mice in the positive group were given mitocycin-C at the dose of 2 mg/kg body weight only once on the last test article administration day,0.5% sodium carboxymethyl cellulose solution was taken as negative control with the same time procedure and route as the test article.Results The oral MTD of L-alanine was higher than 15.0 g/kg body weight. None of the bacterial strains tested（TA97a,TA98,TA100 and TA102） showed an increase in revertants with or without S9 mixture at any dose. There were no significant differences in the frequency of micronucleated PCE cells observed in the bone marrow of mice between the treated groups and the negative control group（P〉0.05）,and the average ratio of the PCE/NCE in the treated groups was in the normal reference range. No significant differences were seen in the rates（%） of autosome univalent,sex chromosome univalent and aberration cells of mice testicle primary spermatocytes between the treated groups the negative control group. Conclusion These results confirm that L-alanine from fermentation broth belongs to the non-toxic class by acute oral toxicity test and no significant genotoxicity and mutagenicity are observed in the present study.