A review of mammalian carcinogenicity study design and potential effects of alternate test procedures on the safety evaluation of food ingredients.

Extensive experience in conducting long term cancer bioassays has been gained over the past 50 years of animal testing on drugs, pesticides, industrial chemicals, food additives and consumer products. Testing protocols for the conduct of carcinogenicity studies in rodents have been developed in Guidelines promulgated by regulatory agencies, including the US EPA (Environmental Protection Agency), the US FDA (Food and Drug Administration), the OECD (Organization for Economic Co-operation and Development) for the EU member states and the MAFF (Ministries of Agriculture, Forestries and Fisheries) and MHW (Ministry of Health and Welfare) in Japan. The basis of critical elements of the study design that lead to an accepted identification of the carcinogenic hazard of substances in food and beverages is the focus of this review. The approaches used by entities well-known for carcinogenicity testing and/or guideline development are discussed. Particular focus is placed on comparison of testing programs used by the US National Toxicology Program (NTP) and advocated in OECD guidelines to the testing programs of the European Ramazzini Foundation (ERF), an organization with numerous published carcinogenicity studies. This focus allows for a good comparison of differences in approaches to carcinogenicity testing and allows for a critical consideration of elements important to appropriate carcinogenicity study designs and practices. OECD protocols serve as good standard models for carcinogenicity testing protocol design. Additionally, the detailed design of any protocol should include attention to the rationale for inclusion of particular elements, including the impact of those elements on study interpretations. Appropriate interpretation of study results is dependent on rigorous evaluation of the study design and conduct, including differences from standard practices. Important considerations are differences in the strain of animal used, diet and housing practices, rigorousness of test procedures, dose selection, histopathology procedures, application of historical control data, statistical evaluations and whether statistical extrapolations are supported by, or are beyond the limits of, the data generated. Without due consideration, there can be result conflicting data interpretations and uncertainty about the relevance of a study's results to human risk. This paper discusses the critical elements of rodent (rat) carcinogenicity studies, particularly with respect to the study of food ingredients. It also highlights study practices and procedures that can detract from the appropriate evaluation of human relevance of results, indicating the importance of adherence to international consensus protocols, such as those detailed by OECD.

Developmental toxicity study of vegetable oil-derived stanol fatty acid esters.

In a standard developmental toxicity study, a mixture of vegetable oil-derived stanol fatty acid esters was administered in the diet to groups of 28 mated female HsdCpb:WU Wistar rats at concentrations that provided 0, 1, 2.5, and 5% total stanols (equivalent to 0, 1.75, 4.38, and 8.76% plant stanol esters). Test diets were adjusted with rapeseed oil to maintain an equivalent caloric content of fatty acids at each of the treatment levels. The treatment period extended from day 0 to 21 of gestation. No compound-related toxicity or clinical effects were seen in any of the treated groups. No statistically significant differences were seen in body weights or body weight gain in the low- or mid-dose groups, although slight but statistically significant decreases in mean body weight relative to controls were seen at gestation days 7 and 14 in the high-dose group. The decreases in body weight in the high-dose group may be attributable to the virtual lack of absorption of the dietary stanols. Body weight gains were equivalent to controls throughout the study except for a statistically significant decrease seen only in the 0- to 7-day gestation period in the high-dose group. No significant effects were seen on food consumption in terms of g/rat/day, but a slight, statistically significant increase was seen in the mid-dose group during gestation days 7-14. A significant increase was seen in the high-dose group during the 7- to 21-day period of gestation. Reproductive performance was not affected by the treatment. There were no statistically significant differences in uterine weight, placental weight, fetal weight, number of fetuses, number of implantation sites, number of corpora lutea, and early/late resorptions between the treated and control groups. In addition, there was no biologically meaningful effect on fetal sex ratio. Visceral and skeletal examinations did not show any significant increases in the incidence of malformations, anomalies, or variations that were considered to be treatment related. Dietary plant (8.76% plant stanol esters) stanol esters at concentrations up to 5% total stanols were concluded to have no adverse effects on reproduction or development.

An evaluation of the roles of mammalian cell mutation assays in the testing of chemical genotoxicity.

The present status of the applicability of mammalian cell gene mutation assays in the safety evaluation of industrial chemicals is evaluated from the industrial and regulatory point of view, with emphasis being placed on the CHO/HGPRT and mouse lymphoma tk +/- assays. The CHO/HGPRT assay was concluded to be a highly specific assay, but it might be less sensitive to mutagens that mainly induced large deletions. The mouse lymphoma assay was concluded to be sensitive, but it might have a lower specificity due to experimental artifacts such as pH and osmolality changes. Mammalian gene mutation assays, when conducted within their limitations, are concluded to be valuable in safety evaluation, providing results complementary to the Ames test and cytogenetic assays.

In vitro and in vivo evaluation of the genotoxic potential of 2-ethyl-1,3-hexanediol.

2-Ethyl-1,3-hexanediol (EHD) has intentional human exposure because of its application to skin as an insect repellent and its use in various skin care products. Genotoxicity studies on EHD were conducted to determine mutagenic and clastogenic potential using in vitro and in vivo test systems. In vitro tests were conducted both with and without an Aroclor-induced, rat-liver S9 metabolic activation system and within a range of cytotoxic to non-cytotoxic doses. EHD did not produce dose-related positive increases in gene mutations in the Salmonella (Ames) test or in the CHO/HGPRT forward mutation test. No statistically significant or dose-related increases in sister chromatid exchanges indicative of DNA damage were produced by EHD in CHO cells. Small but statistically significant increases in chromosome aberrations were produced in CHO cells only in tests with S9 activation. However, no evidence of clastogenicity of EHD was obtained in vivo in a mouse peripheral blood micronucleus test or in 2 rat bone marrow chromosome aberration studies using single or repeated dosing procedures. The overall negative pattern of mutagenic and clastogenic results in the majority of tests conducted suggests that EHD is unlikely to pose significant hazard as a genotoxic agent or to possess carcinogenic initiating activity in animals.

Mutagenicity studies on ketone solvents: methyl ethyl ketone, methyl isobutyl ketone, and isophorone.

3 ketone solvents (methyl ethyl ketone (MEK), methyl isobutyl ketone (MiBK), and isophorone) were tested for potential genotoxicity. The assays of MEK and MiBK included the Salmonella/microsome (Ames) assay, L5178Y/TK+/- mouse lymphoma (ML) assay, BALB/3T3 cell transformation (CT) assay, unscheduled DNA synthesis (UDS) assay, and micronucleus (MN) assay. Only the ML, UDS, and MN assays were conducted on samples of isophorone. No genotoxicity was found for MEK or isophorone. The presence of a marginal response only at the highest, cytotoxic concentration tested in the ML assay, the lack of reproducibility in the CT assay, and clearly negative results in the Ames assay, UDS and MN assays, suggest that MiBK is unlikely to be genotoxic in mammalian systems.

The acute toxicity and mutagenic potential of 3-methyl-2-benzothiazolinone hydrazone.

3-Methyl-2-benzothiazolinone hydrazone (MBTH), widely used in analytical laboratories, was investigated for potential handling hazards. Tested as the hydrochloride, it was found to be of moderately high acute peroral toxicity with LD50 values in rabbits of 177 mg/kg (males) and 268 mg/kg (females), and in the rat 308 mg/kg (males) and 149 mg/kg (females). The major signs of toxicity, seen at peroral doses of 125 mg/kg and above, were convulsions. Although of low acute lethal percutaneous toxicity in rats (LD50 greater than 16 g/kg), rabbits were more sensitive with one of five males dying at an applied dose of 16 g/kg, and females having an LD50 of 12.3 g/kg; convulsions were seen in rabbits having applied cutaneous doses of 4 g/kg and above. There was no evidence for cutaneous inflammation after a 4 hour occluded contact with MBTH in rabbits, although following 24 hour occlusive contact in the acute percutaneous toxicity study there was erythema, edema, desquamation and, in a few animals, local necrosis. Ocular studies in rabbits indicated that, depending on the degree of contamination, MBTH produced mild to moderate eye irritation. In keeping with its low vapor pressure, there were no adverse effects from a 6 hour exposure of rats to an atmosphere saturated with any vapor produced from solid MBTH at ambient temperature. MBTH was positive in an Ames bacterial mutagenicity assay, particularly in the absence of metabolic activation. These studies indicate MBTH to be of moderately high acute peroral toxicity, of moderate percutaneous toxicity, a mild primary skin irritant, a mild to moderate eye irritant, and produced mutations in Salmonella. There is a need for skin and eye protection, and avoidance of swallowing, when handling MBTH.

Studies on the acute toxicity primary irritancy and genotoxic potential of 1,3,5-triacryloylhexahydro-s-triazine (TAHT).

TAHT (1,3,5-triacryloylhexahydro-s-triazine), a reactive chemical coupling agent, was highly toxic following a single peroral dose of an aqueous suspension (10% w/v) to Wistar rats, or following application of TAHT in dichloromethane (DCM) solution (10% w/v) to covered skin of New Zealand rabbits. It was moderately toxic when applied dermally as an aqueous paste. Ocular contact with 25 mg of TAHT in a 5% aqueous suspension, or of 0.5 mg of TAHT in a 10% (w/v) solution in DCM, produced severe corneal damage, iritis and blepharo-conjunctivitis. A 30-min exposure of uncovered rabbit skin to 1 mg of TAHT in a 10% (w/v) aqueous suspension produced only slight skin irritation. However, 24-h exposures to TAHT on covered skin produced erythema, edema, ecchymoses, scabs, and death depending upon dosage and vehicle. In vitro genotoxicity studies revealed no positive effects upon gene mutations (HGPRT locus) or on sister chromatid exchanges (SCEs) of CHO cells exposed to TAHT with and without a rat-liver S9 metabolic activation system. TAHT did not increase the levels of [3H]thymidine incorporation in a test for unscheduled DNA synthesis with primary rat hepatocytes. In contrast, substantial increases in the number of chromosome breaks and rearrangements were observed in chromosome preparations used for the SCE analyses. The clastogenic activity of TAHT was confirmed in an in vitro chromosome aberration test with CHO cells. Treatment-related increases in chromosome breakage were observed at two independent sampling times and positive effects did not depend upon the presence or absence of a metabolic activation system. Clastogenic activity of TAHT was also demonstrated in vivo in a micronucleus test using mouse peripheral polychromatic erythrocytes. Significant, treatment-related increases in micronucleated polychromatic erythrocytes were obtained at two of three sampling times. The high degree of mammalian toxicity, severe eye irritancy and the in vitro and in vivo clastogenicity indicate that TAHT should be handled as a hazardous material using suitable caution and protective equipment.

Assessment of genotoxic potential of ethylenediamine: in vitro and in vivo studies.

Ethylenediamine (EDA) was evaluated for potential genotoxic activity using a battery in vitro and in vivo mammalian tests. The tests employed were the Chinese hamster ovary (CHO) gene mutation assay, the sister-chromatid exchange (SCE) test with CHO cells, unscheduled DNA synthesis (UDS) assays with primary rat hepatocytes and a dominant lethal study with Fischer 344 rats. EDA did not produce a positive, dose-related, mutagenic effect in either the CHO mutation assay or in the SCE test when evaluated both with and without the addition of a rat-liver S9 activation system. With hepatocytes, no positive effects of EDA upon UDS values were noted in 2 separate studies using either a scintillation counting procedure or an autoradiographic method to determine UDS activity. In a dominant lethal study, male rats fed for 23 weeks with dietary levels of EDA X 2HCl of 0, 0.05, 0.15 or 0.50 g/kg/day, and mated with 1 virgin female/week for 3 consecutive weeks, showed no dose-related or statistically significant effects upon fertility, total number of implantations/female, or the number of living and dead implants per female; marked effects upon the incidence of dominant lethal mutations were noted in the positive control group injected intraperitoneally with one dose of 0.25 mg/kg triethylenemelamine. We conclude that EDA was not genotoxic in the in vitro and in vivo mammalian test systems employed.

Mutagenicity evaluation of glutaraldehyde in a battery of in vitro bacterial and mammalian test systems.

Glutaraldehyde was evaluated for genotoxicity using a battery of four in vitro test systems: the Salmonella/microsome assay, the Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyltransferase (CHO/HGPRT) gene mutation system, the sister-chromatid exchange test with Chinese hamster ovary cells, and measurements of unscheduled DNA synthesis in primary rat-hepatocyte cultures. No significant, dose-related increases in the various end-points were produced by glutaraldehyde in tests with or without the addition of a rat-liver metabolic activation system (S-9 mix) or with the cell-mediated activation of the hepatocyte test system. A range of concentrations which spanned cytotoxic to non-cytotoxic doses was evaluated in each test system and marked cytotoxicity was typically noted at micromolar concentrations. Within a range of biologically active concentrations, glutaraldehyde did not produce significant genotoxic effects with the assays and conditions used for these studies.

Variation in melanosome numbers in cultured B-16 melanoma cells.

Melanosomes from B-16 mouse melanoma cells in culture were isolated by treatment of pigmented cells with 2% SDS, sonication, and heating at 100 degrees C. The total number of melanosomes in cultures of B-16 mouse melanoma cells increased exponentially during the rapid phase of sigmoid growth. The numbers of melanosomes per cell decreased during rapid phase of growth, and repigmentation was observed only when the cultures attained the stationary growth phase. BUdr at a minimum concentration of 0.5 mug/ml decreased both cell growth and numbers of melanosomes per cell, and completely inhibited repigmentation following a period of active growth. Cells cultured in 0.1 mug/ml BUdr grew at the same rate as untreated cells but contained fewer melanosomes/cell and lower total numbers of melanosomes during the late stages of the growth cycle.

Chemical mutagenesis at the phosphoribosyltransferase locus in cultured human lymphoblasts.

The presence of selectable genetic markers in long-term human lymphoblast cultures would facilitate cell hybridization experiments on the biosynthesis of immunoglobulins, as well as other studies. This work reports the induction with ethylmethane sulfonate of 6-thioguanine - resistant, phosphoribosyltransferase - deficient mutants in a lymphoblast line from a patient with infectious mononucleosis. These cells were unusually sensitive, with a D(0) value of 28 mug of ethylmethane sulfonate per ml; the sensitivity curve followed a biphasic pattern suggesting the presence of 3% resistant cells. Ethylmethane sulfonate increased the frequency of mutants resistant to 6-thioguanine over 100-fold, to about 2 x 10(-4); nitrosoguanidine was less effective. Almost all the mutants contained considerably less than 1% of the hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) activity of wild-type cells. The mutation did not appear to result from loss of an X chromosome.