Doxylamine succinate (DA), a compound which was formerly used as an antinauseant during pregnancy, showed no substantial mutagenicity in mouse embryos following transplacental exposure. A small dose-dependent induction of chromosomal aberrations was found in mouse embryos on day 11 of gestation. No induction of sister chromatid exchanges (SCE) was found in embryos on day 11 of gestation. A micronucleus test with fetal blood on day 17 of gestation was negative. Additionally, DA was negative in Chinese hamster bone marrow in vivo (micronuclei) and in human lymphocyte cultures in vitro (SCE).

Sensitivity to the hypnotic effects of ethanol was increased selectively by central administration of muscarinic agonists. Carbachol or oxotremorine, but not nicotine, i.c.v., enhanced hypnotic sensitivity to ethanol markedly, as measured by blood ethanol concentration at loss or righting response, in short-sleep (SS) but not long-sleep (LS) mice. Likewise, the acetylcholinesterase inhibitor, neostigmine, i.c.v., differentially enhanced hypnotic sensitivity to ethanol in these mouse lines. LS and SS mice were equally sensitive to the hypothermic effects of carbachol, neostigmine or oxotremorine i.c.v. The muscarinic antagonists, atropine or pirenzepine, i.c.v., were without effect on ethanol sensitivity, but these compounds antagonized muscarinic agonist-enhanced ethanol sensitivity in SS mice effectively. Pirenzepine, and M1 selective antagonist, produced a parallel shift in the oxotremorine dose-response curve, indicating that the enhanced hypnotic sensitivity to ethanol may be due to interaction of oxotremorine with M1 muscarinic receptors. This possibility was supported by the finding that atropine and pirenzepine which are known to have comparable affinities for M1 but not M2 receptors, had comparable potencies in antagonizing the action of oxotremorine or neostigmine. The results suggest that LS and SS mice differ genetically in neuronal processes activated by specific muscarinic agonists and are consistent with the hypotheses that ethanol acts in part via membrane receptor coupling to intracellular processes known to mobilize intracellular Ca++.

The hypothesis that some of ethanol's acute effects are mediated via neurotensinergic systems was investigated by characterizing neurotensin (NT) receptors in mice (LS and SS) selectively bred for differences in sensitivity to ethanol. [3H]Neurotensin binding in brain membranes from both mouse lines was specific, saturable, reversible, and linear with protein concentrations. Subcellular localization studies showed specific NT binding to be concentrated in the microsomal/synaptosomal fractions. Scatchard analyses of [3H] NT binding indicated similar KD values for membranes from various brain regions of LS and SS mice. However, Bmax values in frontal cortex, cerebellum, and striatum were greater in SS than in LS mice. In competitive binding studies IC50 values were lower for NT8–13 than for NT1–13, and IC50 values for NT1–8, NT1–11, D-Trp11-NT, and D-Tyr11-NT were greater than 1000 nM. Association and dissociation rate constants for [3H]NT and resulting KD values (0.8 nM) were similar for LS and SS brain membranes. Ethanol, in vitro, had no effect on NT binding characteristics, but as expected various cations markedly increased KD values.

Neurotensin (NT) differentially altered ethanol-induced anesthesia as measured by duration of loss of righting response or by blood ethanol levels producing loss of righting response in mice (LS and SS) which were selectively bred for differences in response to ethanol. At dose of 5–500 ng i.c.v., NT increased ethanol sensitivity in SS mice, but not in LS mice, as measured by blood ethanol concentrations at loss of righting response. At higher doses, 0.5–10 μg i.c.v., NT enhanced the sensitivity of both SS and LS mice to ethanol-induced anesthesia. The hypothermic effect of ethanol determined at loss of righting response was not altered in either LS or SS mice at low doses of NT, but at higher doses NT enhanced ethanol-induced hypothermia in both lines of mice. The altered anesthetic sensitivity was specific for ethanol in that NT did not alter pentobarbital-induced sleep time in either LS or SS mice and halothane anesthesia was altered slightly on ly in LS mice. NT analogues, N-acetyl-NT8−13, and [d-Trp11]-NT but not NT1−8 enhanced the anesthetic action of ethanol in SS mice. Bombesin, cholecystokinin sulfate, substance P, [d-Trp8,d-Cys14]-somatostatin and corticotropin releasing hormone (CRF) were not effective in enhancing ethanol-induced anesthesia in LS or SS mice. CRF appeared to decrease ethanol sensitivity in LS but not in SS mice. β-Endorphin (β-END) markedly increased the ethanol sensitivity of SS and to a lesser extent of LS mice at relatively high doses, e.g. 0.5–1.0 μg i.c.v. The results of the present study indicate that differences in brain sensitivity of LS and SS mice to ethanol may be mediated by genetic differences in NT systems. Likewise, NT, and probably β-endorphin, may interact with other neurochemical processes that are involved in the mechanism of ethanol-induced anesthesia and that differ genetically in LS and SS mice.

Quality and quantity of mutagenicity testing were analyzed for drugs with new active compounds which were submitted for registration in the Federal Republic of Germany from mid 1982 to mid 1986. A large variety of deficiencies was found, applying to selection and number of mutagenicity tests as well as to test performances. Only 65 out of the 144 drugs submitted for registration were tested sufficiently in the initial phase of registration. From 1982 to 1986 this situation has not been changed markedly. Inadequate test performance still remains the main reason for insufficient testing, leading in some cases to artificially positive results. For in vivo tests the selection of test species was mainly motivated by technical reasons and not by characteristics of the test compound. Most of the insufficiencies were eliminated during the second phase of registration. In some cases insufficient mutagenicity testing led to consequences concerning risk-benefit assessment of the drug and its regulation.

Three animal species used in in vivo mutagenicity testing--rats, mice and Chinese hamsters--were compared with respect to their mutagenic response to the mycotoxin aflatoxin B1 (AFB1). The micronucleus test and the SCE test with bone marrow cells were chosen as test methods, employing similar protocols for all species. The mutagenic potential of AFB1 was detected with rats and mice but not with Chinese hamsters. Rats were more susceptible to the mutagenic action of AFB1 than mice with regard to the effective dose. A difference in sensitivity between males and females was evident in rats and mice: male animals exhibited higher induced micronucleus frequencies than females, and a clear SCE-inducing effect was only detectable in male animals. These results are in agreement with those of in vitro and carcinogenicity studies. They may be due to metabolic differences between the species and sexes, predominantly differences in glutathione conjugation of the reactive AFB1 epoxide and in the formation of the metabolite aflatoxicol. Furthermore, it could be demonstrated that AFB1 seems to be a more potent inducer of micronuclei than of SCE. Since our results obtained with rats and mice were clearly positive, but with the Chinese hamster the mutagenic potential of AFB1 was not detectable with the test systems used, it can be concluded that the choice of an "inappropriate" test species may lead to a false negative judgment on the genotoxic potential of a test compound.

This manuscript reports on the limb malformations and axial skeleton alterations found in legless fetuses and their heterozygote and wild-type littermates transplacentally exposed to all-trans-retinoic acid via a single intraperitoneal injection on Day 7, 8, 9, 9.5, 10, 10.5, or 11 of gestation. The most surprising aspect of the results was the temporal sensitivity of the legless mouse limb to exogenous retinoic acid. On Day 11, when both fore- and hindlimbs of nonmutant embryos can be made abnormal by retinoic acid and other teratogens, retinoic acid did not increase the frequency or severity of legless hindlimb defects and forelimb malformations were only slightly enhanced. On the other hand, retinoic acid administration on Day 7 exacerbated forelimb malformations in legless fetuses at a time when visible emergence of the affected structure is still 48 hr away. Heterozygote and wild-type fetuses had no limb malformations at this time point. A similar phenomenon was observed with hindlimb malformations after retinoic acid exposure on Day 8 except for a few mild limb malformations in heterozygotes at a high dose of retinoic acid. This early hypersensitivity of fore- and hindlimbs was followed by a period of reduced sensitivity (Day 8 forelimb; Day 9 hindlimb) when even very high doses (50 mg/kg) induced minimal changes in the typical legless malformation pattern. Subsequent]y, at the time of visible limb bud emergence (Day 9 forelimbs; Day 10 hindlimbs), sensitivity to exogenous retinoic acid was again detected. Surprisingly, the altered malformation patterns induced by retinoic acid in lgl mutants were nearly identical to those from earlier, preemergence exposure. A number of axial skeleton alterations were induced in legless fetuses by retinoic acid, especially after exposure on Days 7, 8, or 9. Posterior truncations were particularly noteworthy, showing a graded response in which frequency and severity of truncation were worst in lgl/lgl fetuses; heterozygotes gave an intermediate response, and wild-type fetuses were least affected. This exacerbation of the legless phenotype by exogenous retinoic acid coupled with the similarity between legless and retinoid malformations suggest that the legless mutation has altered endogenous retinoid homeostasis or a downstream retinoid-responsive gene.

Comparative investigations of sister-chromatid exchange (SCE) and micronucleus induction in the bone marrow of rats, mice and Chinese hamsters with the cytostatic alkylating mutagen cyclophosphamide (CP) revealed remarkable species differences in their mutagenic responses. With both test systems the sensitivities of the three species can be ranked into the order rat greater than mouse greater than Chinese hamster. More explicit results were obtained with the SCE test than with the micronucleus test within the same dose range. This may be due to the influence of species-related differences in the cytotoxic response to CP in the micronucleus test. These results show that clearly different mutagenic responses in different test species may be obtained in standard assays even with a compound which is metabolized in a very similar manner in all animal species.

Embryotoxicity: Aflatoxin B1 (AFB1), G1 (AFG1), and Patulin (PA) were investigated in NMRI mice for embryotoxic and teratogenic activity. These three mycotoxins were injected intraperitoneally or given orally on day 12 and 13 of pregnancy. AFB1 (15, 45, and 90 mg/kg ip or 45 mg/kg po) produced moderate retardation in fetal development and a dose-related increase of cleft palates, wavy ribs, and diaphragm changes. The effects after injection of AFG1 (45 to 90 mg/kg ip) were reduction of fetal weights, increase of diaphragm changes, and malformations of kidneys. PA (1, 25, 2, 5, and 3.75 mg/kg ip or 3.75 mg/kg po) was found to elevate the rate of cleft palates after 3.75 mg/kg. Dominant lethal assay: Neither PA (2, 5, and 5 mg/kg ip) nor AFB1 (15 and 45 mg/kg ip) increased the frequency of the dominant lethal mutations. Both mycotoxins showed no mutagenic activity in this test system. Cytogenetic studies: The capability of the three mycotoxins AFB1, AFG1, and PA to induce chromosome damages in vivo has been tested in the Chinese hamster by examination of bone marrow cells. The substances were tested in each of two oral doses (AFB1: 12, 5, and 25 mg/kg; 25 and 50 mg/kg; PA: 10 and 20 mg/kg). The present data show that the three mycotoxins induce chromosome aberrations in the following order of activity: PA greater than AFB1 greater than AFG1.

A series of publications of the results of National Toxicology Program (NTP) studies (Tennant et al. (1987) Science, 236, 933–941; Haseman et al. (1990) J. Am. Stat. Assoc., 85, 964–971; Shelby et al. (1993) Environ. Mol. Mutagen., 21, 160–179) show that the commonly used short-term genotoxicity tests are less predictive of rodent carcinogenicity than once thought. These results have fueled a great deal of debate in the field of genetic toxicology regarding appropriate strategies for assessing the potential carcinogenicity of chemicals. The debate has continued in the recent discussion of harmonized genotoxicity test strategies (Ashby (1993) Mutation Res., 298, 291–295 and Ashby (1994) 308, 113–114; Madle (1993) Mutation Res., 300, 73–76 and Madle (1994) 308, 111–112; Zeiger (1994) Mutation Res., 304, 309–314) since the underlying problem still has not been resolved. The underlying problem is the fact that the current short-term genotoxicity tests in any combination do not provide both the necessary high sensitivity and high specificity needed for accurate rodent carcinogen detection. In this discussion, we describe the utility of the newly revised Syrian hamster embryo (SHE) cell transformation assay alone and in combination with the Salmonella mutation assay for improved accuracy of screening of rodent carcinogens relative to standard short-term genotoxicity tests. The accompanying papers provide details of improved methodologies for the conduct of the SHE cell transformation assay and an extensive review of the databases which support our conclusion that the SHE cell transformation assay provides an improved prediction of rodent bioassay results relative to other in vitro genotoxicity test batteries.

Various cigarette smoke condensates (CSC) were analyzed with respect to the induction of sister-chromatid exchanges (SCE) in human lymphocytes in vitro. CSC from a reference cigarette, from three different tobaccos of the reference cigarette, and from a British cigarette induced similar SCE frequencies. CSC from the reference cigarette did not induce SCE in Chinese hamster bone marrow cells in vivo.

Chinese hamsters (Cricetulus griseus) were treated with ethanol, with cigarette smoke and with both. During the experimental period of 12 weeks a control group of animals (C) received water ad libitum, another water drinking group received a cigarette smoke treatment during the last 4 weeks (S). Another group received 20% (v/v) ethanol during the whole experimental period as the only liquid supply (E), and one group with the same ethanol treatment was simultaneously treated with cigarette smoke during the last 4 weeks of the experiment (ES). The investigation of bone marrow cells after 12 weeks with regard to chromosomal aberrations and sister chromatic exchanges revealed no effects. A high mitotic activity was found in the smoke treated groups.

Chinese hamsters received 10% (v/v) ethanol as their only liquid supply during 46 weeks. At the end of the drinking period the rate of chromosomal aberrations was determined in cultured lymphocytes. The cultures were set up with the blood from the retro-orbital plexus. Bone-marrow metaphases of the same animals were analysed with regard to sister-chromatid exchanges (SCE) after implantation of BrdU tablets in vivo. No cytogenetic effects were found in either test system.

Mycotoxins are ubiquitously occurring metabolites of moulds that grow on foodstuffs. They are able to cause toxic diseases in man and animals. Aflatoxin B1 (AFB1), aflatoxin G1 (AFG1), and platulin (PA) induce chromosomal damage in Chinese hamster bone-marrow cells. With respect to the number of induced aberrant mitoses the 3 mycotoxins can be ranked in the order PA > AFB1 > AFG1.

Chromosomes were investigated from the bone marrow of Chinese hamsters which received 10% (v/v) ethanol as the only liquid supply for a period of 9 weeks. At the end of the ethanol drinking period 1 group of animals received 2 oral doses of 80 mg/kg cyclophosphamide (CP), a second group received 2 oral doses of 25 mg/kg aflatoxin B1 (AFB1), a third group 2 oral doses of 20 mg/kg patulin (PA). The 2 applications were separated by 24 h. The intake of ethanol had no effect on the bone marrow chromosomes, and had no potentiating effect on CP induced aberrations. 9 weeks consumption of 10% (v/v) ethanol revealed likewise no influence on the frequencies of chromosomal aberrations induced by the indirect mutagen AFB1. However, the rate of chromosomal aberrations induced by the direct mutagen PA was clearly suppressed in ethanol drinking animals.