Studies of the toxicological potential of tripeptides (L-valyl-L-prolyl-L-proline and L-isoleucyl-L-prolyl-L-proline): III. Single- and/or repeated-dose toxicity of tripeptides-containing Lactobacillus helveticus-fermented milk powder and casein hydrolysate in rats.

The objective of these studies was to assess the toxicological potential of orally administered tripeptides in rats. The studies employed powdered L-valyl-L-prolyl-L-proline (VPP)- and L-isoleucyl-L-prolyl-L-proline (IPP)-containing test articles, including (1) powdered Lactobacillus helveticus-fermented milk (FM), (2) pasteurized casein hydrolysate (CH) generated by Aspergillus oryzae protease, and (3) synthesized VPP. All test articles were administered by oral gavage to male and female Sprague-Dawley rats. Specific goals of the single-dose and repeated-dose studies were to (1) identify doses that produce evidence of systemic and/or local (i.e., gastrointestinal) toxicity (e.g., lowest-observable-effect level [LOEL]); (2) estimate the maximally tolerated oral dose (MTD); and (3) identify specific target organs for toxicity of these tripeptides. Single doses of CH (2000 mg/kg), powdered FM (2000 or 4000 mg/kg), or VPP (40, 200, or 400 mg/kg) were administered 14 days prior to study termination. No treatment regimen caused either antemortem (gross observations, body weight, and food consumption parameters) or postmortem (necropsy) evidence of either systemic or local toxicity. In the repeated-dose study, powdered FM (0, 500, 1000, or 2000 mg/kg body weight [BW]/day) was administered by gastric gavage to male and female rats for 28 consecutive days. Antemortem evaluative parameters included gross observations, ophthalmic examinations, and clinical pathology (clinical chemistry, hematology, and urinalysis). Post mortem parameters included necropsy, determination of organ weights, and microscopic examination of major organs. There was neither in-life nor postmortem evidence that powdered FM administration caused physiological or toxicological changes. Under the conditions of these experiments, the single-dose LOEL of powdered FM, CH, and VPP were found to be greater than 4000, 2000, and 400 mg/kg, respectively. The results of the repeated-dose study do not support identification of a target organ for powdered FM toxicity. Similarly, there was no evidence to support establishment of either the LOEL or MTD; both being greater than 2000 mg/kg/day for up to 28 consecutive days.

Studies of the toxicological potential of tripeptides (L-valyl-L-prolyl-L-proline and L-isoleucyl-L-prolyl-L-proline): VI. Effects of Lactobacillus helveticus-fermented milk powder on fertility and reproductive performance of rats.

The objective of these studies was to assess the effects of the tripeptides, L-valyl-L-prolyl-L-proline (VPP) and L-isoleucyl-L-prolyl-L-proline (IPP), on reproductive capabilities of male and female rats. The specific goals of the experiments were (1) to determine the effects of orally administered tripeptides on (a) fertility and reproductive behavior in both sexes of rats, (b) embryo-fetal development in pregnant rats, and (c) pre- and postnatal development of rats exposed to tripeptides in utero and during lactation; and (2) to estimate the no-observable-adverse-effect doses of tripeptides in maternal and fetal rats. During the conduct of these classical segment I, II, and III studies, the test material was powdered Lactobacillus helveticus-fermented milk (FM), which contains the tripeptides, VPP and IPP. FM (0, 500, 1000 or 2000 mg/kg body weight [BW]/day--equivalent to 0, 0.8, 1.6, or 3.3 mg/kg BW/day of VPP plus IPP) was administered to males by oral gavage from 4 weeks prior to mating until sacrifice, and to females from 2 weeks prior to mating through day 20 of lactation. Evaluative parameters included monitoring grossly observable clinical signs; food consumption and body weight gains; mating behavior and fertility indices of both sexes; implantation and maintenance of embryos; sex ratio of live pups; fetal viability; incidences of external, visceral or skeletal variations; growth and behavioral development; as well as reproductive capabilities of F1 offspring exposed to FM during gestation and lactation. All animals were subjected to macroscopic examination at termination of their segment of the studies. Clinical signs, body weights, and food consumption were unaffected by administration of FM. During segment I, the test agent had no effect on estrus cycle, mating behavior, fertility index, or reproductive competence of either males or females. The results of segment II experiments revealed no effects of FM on postimplantation survival-loss, sex ratio or birth weights of live fetuses, and there was no evidence of treatment-associated developmental or teratological effects. During segment III, FM was without effect on pup viability, behavioral and sexual maturation, and reproductive capability of the F1 generation. Under the conditions of these experiments, the no-observable-adverse-effect level (NOAEL) of FM on reproductive performance in male and female rats is greater than 2000 mg/kg BW/day, the equivalent of 3.3 mg/kg BW/day of VPP plus IPP.

Studies of the toxicological potential of tripeptides (L-valyl-L-prolyl-L-proline and L-isoleucyl-L-prolyl-L-proline): VII. Micronucleus test of tripeptides-containing casein hydrolysate and Lactobacillus helveticus-fermented milk powders in rats and mice.

The objective of these in vivo experiments was to assess the mutagenic potential of tripeptides as reflected by the ability of the test compound to induce the formation of micronuclei in mouse polychromatic erythrocytes. The test agents used in these experiments were (1) powdered Aspergillus oryzae protease casein hydrolysate (CH) and (2) powdered Lactobacillus helveticus-fermented milk (FM). Both test agents contain two tripeptides, L-valyl-L-prolyl-L-proline (VPP) and L-isoleucyl-L-prolyl-L-proline (IPP). Male Sprague-Dawley rats (five per group) were administered doses of 0, 500, 1000, or 2000 mg (0, 3, 6, or 12 mg VPP plus IPP)/kg body weight (BW)/day CH by oral gavage for 2 days. Male CD-1 mice (six per group) received a single oral gavage dose of 0, 500, 1000, or 2000 mg (0, 0.8, 1.6 or 3.3 mg VPP plus IPP)/kg BW of FM. Positive-control agents were cyclophosphamide (10 mg/kg, intraperitoneal [i.p.]) in rats and mitocycin C (2 mg/kg, i.p.) in mice. Twenty-four hours after the second dose of CH, or the dose of cyclophosphamide to rats, or FM or mitocycin C to mice, bone marrow cells were fixed and examined for the presence of polychromatic erythrocytes (PCEs) and the presence or absence of micronucleated PCEs (MNPCEs). Administration of CH to rats and FM to mice produced neither changes in body weights nor signs of systemic toxicity. Similarly, neither CH nor FM caused statistically significant variations in the incidences of either PCEs or MNPCEs. Both positive-control agents caused unequivocal increases in the incidence of MNPCEs and cyclophosphamide significantly reduced the percent of rat erythrocytes appearing as PCEs. The results of these micronucleus assays conducted with either powdered CH or FM in rats and mice, respectively, show that neither form of the tripeptides possesses the potential to induce micronuclei formation in these rodent species.

Studies of the toxicological potential of tripeptides (L-valyl-L-prolyl-L-proline and L-isoleucyl-L-prolyl-L-proline): VIII. Assessment of cytotoxicity and clastogenicity of tripeptides-containing casein hydrolysate and Lactobacillus helveticus-fermented milk powders in Chinese hamster lung cells.

The objective of this chromosomal aberration test was to assess the mutagenic potential of tripeptides by determining their ability to induce chromosomal aberrations in cultured Chinese hamster lung (CHL) cells. The test agents used in these experiments were (1) powdered casein hydrolysate (CH) and (2) powdered Lactobacillus helveticus-fermented milk (FM). Both test agents contain two tripeptides, L-valyl-L-prolyl-L-proline (VPP) and L-isoleucyl-L-prolyl-L-proline (IPP). CHL cells were cultured and exposed in the presence or absence of a rat hepatic metabolizing system (S9); CH or FM (1250, 2500, 5000 microg/ml of incubation medium); or positive-control agents, mitomycin C (0.1 or 0.05 microg/ml) or benzo(a)pyrene (20 microg/ml). In experiments with CH, cells were incubated for 6 h (either in the presence or absence of S9) or for 24 h (without S9). In experiments with FM, the cells were incubated for 6 h (either in the presence or absence of S9) or for 24 or 48 h (without S9). Neither short-term nor continuous exposure to either CH or FM caused the induction of significant changes in cell growth indices, incidences of chromosomal aberrations or the incidence of polyploids. Exposures of cells to mitomycin C and benzo(a)pyrene consistently resulted in the induction of the anticipated aberrant cells after either short-term or continuous exposures. The results of these assays support the conclusions that, under the conditions of these experiments, neither CH nor FM possesses demonstrable potential for the induction of cytotoxicity or clastogenesis.