Genotoxicity studies of a desealant solvent mixture, SR-51.

The Royal Australian Air Force (RAAF) has reported that personnel involved in F-111 fuel tank maintenance were concerned that exposure to a range of chemicals during the period 1977 to mid-1990s was the cause of health problems, including cancer. Particular concern was directed at SR-51, a desealant chemical mixture containing the following four solvents: aromatic 150 solvent (Aro150), dimethylacetamide, thiophenol (TP), and triethylphosphate. The present study examined the mutagenic potential of SR-51 using a range of well-known mutagen and genotoxin assays. The tests used were i) a modified version of the Ames test, ii) the mouse lymphoma assay, iii) the comet assay (a single-cell gel electrophoresis assay), and iv) a mouse micronucleus test. The modified Ames test used mixed bacterial strains in liquid suspension media. The Ames test results showed that SR-51 (tested up to the cytotoxic concentration of 36 microg/ml, 30 min incubation) in the presence and absence of S9 metabolic activation was not mutagenic. The mouse lymphoma assay used cultured mouse lymphoma cells in a microwell suspension method. The mouse lymphoma assay was also negative with SR-51 (tested up to the cytotoxic concentration of 22.5 microg/ml, 3 h incubation) in the presence and absence of S9 metabolic activation. The Comet assay, using cultured mouse lymphoma cells, showed no evidence of DNA damage in cells exposed up to the cytotoxic concentration of SR-51 at 11.25 microg/ml. The in-vivo mouse micronucleus test was undertaken in wild-type C57Bl6J male mice dosed orally with SR-51for 14 days with a single daily dose up to 360 mg/kg/day (the maximum-tolerated dose). No increases were observed in micronuclei (MN) frequency in bone marrow collected (24 h after final dose) from SR-51-treated mice compared to the number of MN observed in bone marrow collected from untreated mice. Tissues collected from treated mice at necropsy demonstrated a significant increase in spleen weights in the high dose mice. Gas chromatography analysis of SR-51 identified more than 40 individual components and an oxidation product, diphenyldisulfide derived from TP under conditions of mild heating. In conclusion, there was no evidence that SR-51 is mutagenic.

Replacement of ether with alternate volatile anesthetics for collection of rat serum used in embryo culture.

BACKGROUND: The traditional anesthetic used for collection of the serum culture medium for whole rat embryo culture studies has been ether. However ethical concerns have been raised due to the irritant nature of the vapour and safety concerns due to the risk of fire. METHODS: Growth and development of gestation day 9.5 rat embryos cultured for 48 h in serum collected from rats anesthetised with either ether, isoflurane or halothane were compared. RESULTS: There were no differences in any of the parameters used to assess embryonic development when embryos were grown in serum collected using either ether or isoflurane anesthetics. However, when embryos grown in serum collected using ether or halothane were compared, embryonic development was similar in all respects, except for a reduced number of embryos turned to become fully dorsally convex in the halothane group (p <0.05). CONCLUSIONS: The data indicate that isoflurane is an appropriate alternative to ether for collection of the serum culture medium for whole rat embryo culture, while halothane may cause some delay of embryonic development.

Testicular changes induced by chronic exposure to the herbicide formulation, Tordon 75D (2,4-dichlorophenoxyacetic acid and picloram) in rats.

The second most used herbicide in the Vietnam war was Agent White, which contained the active components 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-amino-3,5,6-trichloropicolinic acid (picloram). The herbicide formulation Tordon 75D is similar in terms of its active components to Agent White and is currently used by the agricultural industry in Australia. As part of an investigation into the possible adverse effects of this herbicide on male reproductive performance, groups of five male rats were gavaged 5 days a week for 9 weeks with either 0.125 ml/kg (low dose), 0.25 ml/kg (middle dose), or 0.5 ml/kg (high dose) Tordon 75D or water (controls). The high dose corresponded to 150 mg/kg body weight 2,4-D and 37.5 mg/kg picloram acid equivalents. At the end of the treatment period, the testes were collected, weighed, and examined histologically and blood samples were taken to determine serum testosterone. Groups of high dose animals were also examined after 1, 2, and 4 weeks treatment. The 9 weeks treatment with Tordon 75D caused severe reduction in testicular weight in some high dose animals. Histologically, the small testes showed shrunken tubules with germ cell depletion. This damage was still evident in some rats following a 21 weeks recovery period suggesting that the testicular damage was permanent. Testicular damage was not due to endocrine disruption as there were no significant differences in the serum concentration of testosterone in control animals compared to Tordon 75D-treated animals. Blood levels associated with the high dose were determined in a separate study and were much higher than those likely to be obtained by occupational exposure to this herbicide.

A study of the potential for a herbicide formulation containing 2,4-d and picloram to cause male-mediated developmental toxicity in rats.

Male Vietnam veterans have repeatedly expressed concern that exposure to herbicides in Vietnam may have caused birth defects in their offspring. The second most used herbicide was a mixture of 2,4-D and picloram called Agent White. This study is an investigation into the possible male-mediated reproductive toxicology of this herbicide. Male rats were gavaged for 5 days per week for 9 weeks with a mixture of 2,4-D and picloram called Tordon 75D(R) (the Australian derivative of Agent White). Three doses were tested; the high dose was considered the maximum tolerated dose. Each male was mated with two untreated females during weeks 2 and 3, 4 and 5, and 8 and 9 of treatment, and with four untreated females after an 11-week recovery period. Negative controls were males dosed with distilled water, and positive controls were males dosed with cyclophosphamide at 5.1 mg/kg/day. All mated females were killed on day 20 of gestation, and the fetuses were weighed and examined for either structural malformations or skeletal development. Litter size, fetal weight, and malformation rate were all unaffected by treatment. The cyclophosphamide positive controls showed the expected large increase in postimplantation loss. In general, within the limitations of the power of the study, the results did not show any evidence that exposure to a herbicide formulation containing 2,4-D and picloram is likely to cause male-mediated birth defects or other adverse reproductive outcomes.

Model predicting the teratogenic potential of retinyl palmitate, using a combined in vivo/in vitro approach.

Retinyl palmitate (RP) is a known laboratory animal teratogen inducing abnormalities of the second visceral arch when administered on day 9 of gestation in the rat. However, there are significant problems when attempting to extrapolate this result to the human. A combined in vivo/in vitro model was developed to assist in human risk assessment. The in vitro teratogenic threshold concentration of a number of retinyl palmitate metabolites was established. Serum concentrations of retinyl palmitate metabolites following a single teratogenic dose of RP in the pregnant rat were also measured. These dosed sera were also used to culture rat embryos. Our hypothesis was that malformations would only be induced by the dosed sera in vitro if the threshold concentration(s) of one or more metabolites was exceeded. Using this approach, it was determined that the teratogenicity of the sera were best predicted by serum retinol levels, with some indication that all-trans-retinoic acid and 4-oxo-all-trans-retinoic acid could be involved in some cases. The available human data suggest that threshold concentrations of these retinoids were unlikely to be exceeded following vitamin A supplements of 25,000 IU/day. While the proposed model does not take into account species differences, protein binding, and transfer to the embryo, it does have potential for human risk assessment.

A review of the contribution of whole embryo culture to the determination of hazard and risk in teratogenicity testing.

Whole embryo culture appears to be an excellent method to screen chemicals for teratogenic hazard. Compared to in vivo testing it is cheap and rapid and does not involve experimentation on live adult animals. Also in the important area of risk estimation whole embryo culture offers distinct advantages over in vivo teratogenicity testing. Adverse embryonic outcomes (malformations or embryotoxicity) are directly related to the serum concentration of the compound being tested and can be compared to the serum concentration in the human. A similar comparison is not possible after in vivo testing because for most compounds there are major pharmacokinetic differences between humans and experimental animals. In vivo testing is also limited by the possibility that metabolites that occur in the human do not occur in the test animal. This problem can be overcome in the in vitro system by adding the metabolite directly at the desired concentration either with or without the parent compound. There is only one major disadvantage to in vitro testing and that is the limited period of embryogenesis that is undertaken in the commonly used culture system. This restricts the range of malformations that can be induced and may render the testing system unsuitable for compounds that are likely to exert their major toxicological effect late in gestation. Any evaluation of whole embryo culture for hazard and risk assessment in teratology must take into account the limited value of currently used in vivo methods. Over 2000 chemicals have been reported to be teratogenic in experimental animals exposed in vivo (Shepard, Catalog of Teratogenic Agents, 1989). In comparison only about 20 chemicals are known to cause birth defects in the human. This large number of in vivo false-positive cannot easily be distinguished from true-positives. In this respect in vivo testing is severely deficient. The embryo culture testing system would also be expected to produce many false-positives; but by comparing effective drug concentrations with human therapeutic concentrations they can be differentiated from true-positives. The most serious deficiency for an in vivo or in vitro teratogenicity testing system would be false-negatives. This has not been a problem in the validation of in vitro testing so far (except perhaps procarbazine), but difficult drugs such as thalidomide were not included. Thalidomide remains an important index chemical because it is not teratogenic in rats or mice but is teratogenic in the rabbit and human. It is likely that these species differences are due to metabolic differences between species and it is possible that if the proximate teratogen/s of thalidomide were identified they would be teratogenic in rat embryo culture. Whole embryo culture remains a very powerful technique that should continue to contribute to the determination of the safety of drugs and other chemicals during pregnancy.

Fragilitas ossium (fro/fro) in the mouse: a model for a recessively inherited type of osteogenesis imperfecta.

The fragilitas ossium (fro/fro) mutation in the mouse has been demonstrated to have clinical, radiographic and morphologic manifestations similar to those which arise in autosomal recessive forms of osteogenesis imperfecta (OI) occurring in humans. Approximately 90% of mutant offspring in the mouse were perinatally lethal with clinical and roentgenographic findings similar to those of OI type II subgroup A in humans. The 10% of mutant mice surviving follow a course very similar to severe progressively deforming OI type III. In surviving mice, there is progressive fore-limb and hind-limb bowing in the absence of a high fracture frequency.

Fetal brain damage in the rat following prenatal exposure to cocaine.

The aim of this study was to identify fetal brain damage induced by 1) prenatal cocaine exposure or 2) physical procedures causing temporary constriction or occlusion of the uterine vessels in pregnant rats. Brains were examined from rat fetuses killed 48 hours after the dam was given one or more intraperitoneal doses of cocaine (50-70 mg/kg) on day 16 of gestation. Only brains from fetuses with hemorrhage in the extremities were examined, as this indicated they had undergone a circulatory disturbance. Four of the 10 brains examined showed bilateral necrosis and cavitation in the cerebral cortex. There were also hemorrhage and ectopic outgrowths in the corpus striatum, bilateral cavitation in the brainstem and vacuolization in the lens of the eye. A similar type and distribution of damage was seen in rat fetal brains from dams treated by temporary occlusion of the uterine vessels or direct handling of the pregnant uterus on day 16 of gestation and examined 48 hours later. It is proposed that the procedures act through the common mechanism of constriction/occlusion of the uterine vessels. The damage to the fetuses appears to be due to hemorrhage from the fetal vessels and ischemia. These findings are discussed in relation to cocaine use during human pregnancy.

Teratogenic effects of alcohol and isotretinoin on craniofacial development: an analysis of animal models.

The teratogens alcohol and isotretinoin cause different patterns of facial dysmorphogenesis in the human. For isotretinoin the pattern is consistent with interference with the normal development of the cranial neural crest, particularly that destined for the second visceral arch. In vitro studies in the rat indicate that, at threshold levels of exposure to isotretinoin, the development of the second arch crest represents the most sensitive process of organogenic development. For alcohol, the facial abnormalities result from exposure very early in development, during the gastrulation process. There is no evidence that this is a peculiarly sensitive stage of development with respect to alcohol; animal studies indicate that other processes in the organogenic period are equally or more vulnerable. The emphasis given to the abnormal facial features in the fetal alcohol syndrome is considered a phenomenon associated with the exclusivity of syndromes.

Embryotoxicity of xylene and toluene: an in vitro study.

Exposure to aromatic hydrocarbon solvents during pregnancy has been reported to adversely affect human embryonic development. This exposure may be due to deliberate abuse or may occur in the workplace. Xylene and toluene are the most common solvents encountered in the workplace and toluene is a constituent of commonly abused substances. This study was performed in an endeavour to fulfil two requirements for proof of teratogenicity of a substance, namely development of an animal model and demonstration of a dose-response relationship of teratogenicity. To fulfil these aims, the possible teratogenic and embryotoxic effects of xylene and toluene on rat embryos during the organogenic period was investigated in vitro. Rat embryos were explanted on day 9.5 of gestation and cultured in heat-inactivated rat serum to which xylene or toluene (0.1, 0.5 or 1.0 microL/mL) had been added, dispersed in 0.1% DMSO. The amount of solvent in the culture medium was quantitated using gas chromatography. Neither xylene nor toluene had any observable teratogenic effect on the embryos in terms of increased malformations. However, both solvents were embryotoxic and caused a dose-dependent retardation of growth and development. A no-effect level was not established for either xylene or toluene, however, the lowest levels used for each of these compounds caused only a slight retardation of growth. Although there was no indication that exposure to these solvents caused a teratogenic effect, there was clear evidence of embryotoxicity. The embryotoxic levels of these solvents needed in culture were higher than blood levels likely to occur in the human following industrial exposure or recreational abuse.

Animal model: skeletal anomalies in mice with cleidocranial dysplasia.

Cleidocranial dysplasia in mice, a radiation-induced skeletal mutation, showed striking homology with cleidocranial dysplasia in humans. Genetic studies indicated that the condition in mice is inherited as an autosomal dominant trait with variable expressivity and almost complete penetrance. The homozygous condition was lethal in utero. Radiographic and alcian blue/alizarin red S-stained whole-skeletal preparation studies were used to determine the extent, pattern, incidence, and distribution of skeletal abnormalities in heterozygous mice. Cleidocranial dysplasia in mice was characterized by variable clavicular hypoplasia, delayed closure of cranial fontanelles and sutures, and variable hypoplasia of pelvic bones, in particular ischiopubic rami. The gene symbol Ccd is proposed for the cleidocranial dysplasia mutation in mice and humans.

Treatment of guttural pouch mycosis.

Seventeen cases of guttural pouch mycosis (including two bilaterally affected cases) were diagnosed in a three year period. The presenting signs were, in order of frequency, epistaxis at rest, nasal catarrh, pharyngeal paralysis, ipsilateral laryngeal hemiplegia, swelling of the submandibular/parotid region, extension of the head and neck and dyspnoea. Ligation of the origin of the internal carotid and occipital arteries was attempted in 10 of the cases exhibiting epistaxis. Bilateral ligation was performed on one animal with an untoward sequelae. Where surgery was successfully completed further haemorrhage was prevented in eight out of nine affected pouches (89 per cent). Medical treatment involving local administration of various antifungal preparations via a specially designed catheter and/or the oral administration of benzimidazole drugs was successful in eliminating the mycotic plaque in most cases. Cases which presented with pharyngeal paralysis were all fatal.