Ludwig Machemer

Standard protocol for the dominant lethal test on male mice set up by the work group “dominant lethal mutations of the ad hoc Committee Chemogenetics”

AbstractThe members of the work group “Dominant Lethal Mutations of the ad hoc Committee Chemogenetics” jointly carried out experimental studies in the period from November 1972 until February 1976. On the basis of the results obtained and the experience gained, they worked out on February 27, 1976, a standard protocol for the dominant lethal test (DLT) on male mice. The recommendations are:1.The mating period should preferentially be as short as possible to obtain information about germ cell-stage specific actions of chemical mutagens. Its selection should be based on the objectives of individual investigators. For screening purposes, and with respect to a high fertilization rate, a 4-day mating period is recommended.2.The total test period should cover at least 12 mating periods, i.e. the whole spermatogenic cycle. A limitation of the DLT to certain “critical” mating periods of high sensitivity is only permissible in repeat tests, or if the parts of gametogenesis concerned, e.g. spermatogoniogenesis or spermatocytogenesis, are studied by cytogenetical tests.3.The mode of mating should preferentially be 1∶1.4.The number of test animals to be used depends on various biological and statistical factors; no generally valid statement can be made concerning the number of test animals. By rule of thumb in the order of 50 fertilized females per group per mating interval is recommended.5.The individual dosing of the substance in terms of mg/kg body weight is to be preferred. Otherwise, the weights of the animals should not deviate more than 5% from the mean value.6.Results obtained from ill animals or those that died in the course of the trial should not be included in the evaluation.7.The sensitivity of the mouse strain used against a standard dose of a known mutagen should be regularly checked; this evidence should be presented in publications.8.For the following test conditions each investigator should make an optimal choice: animal strain, animal age and housing conditions. The investigator has also to decide whether a preliminary mating, in order to check the fertility of the animals to be used, and vaginal-plug evidence are useful in a given case.9.The autopsy of the dams is best carried out a fortnight from the middle of the mating interval.10.The results should be documented as completely as possible in the investigational report. The following data were also considered as essential, serving a direct comparison of the test groups: number of mated females (absolute), number of females with implantation (absolute and in %), number of implantations and of live and dead implants (absolute and per female). If the corpora lutea graviditates were counted, their number as well as the pre-implantation loss (absolute and per female) are to be stated. These data are also desirable for publications.11.For the biological evaluation the following formula can be used for the calculation of dominant lethal factors: FL% = [1-live implants per female of the test group/live implants per female of the control group]×10012.Statistical evaluation is an essential part of the DLT and various methods are known. For the statistical evaluation it is decisive which biological model and which statistical criteria are most important for the investigator. The DLT must be carried out according to the requirement of the model chosen.

Mutagenicity studies with Praziquantel, a new anthelmintic drug, in mammalian systems

Praziquantel, a new anthelmintic drug with antischistosomal and anticestodal properties, was tested in comparison with a placebo control and a ‘positive control’ with cyclophosphamide in mammalian test systems in vivo for potential mutagenic effects. The test systems used and the tested doses of Praziquantel were: (1) Dominant lethal test on male NMRI mice, 12 mating periods of 4 days each, 1×1200 mg/kg BW by mouth; (2) Dominant lethal test on female NMRI mice, treatment during pre-estrus, 1×1200 mg/kg BW by mouth; (3) Micronucleus test on male and female NMRI mice, two doses with a 24-h interval and preparation of the femoral marrow 6 h after the second dose, 2 ×300 mg/kg and 2×600 mg/kg BW by mouth; (4) Spermatogonial test on the Chinese hamster, two doses with a 24-h interval and preparation of the spermatogonia 48 h after the second dose, 2×600 mg/kg BW by mouth. The 1200 mg/kg BW dose corresponded to approximately 1/2 of the LD50 after oral application in the mouse and about 40 times the therapeutic dose (1×30 mg/kg BW). The cyclophosphamide doses in the test systems were 1×200 mg/kg or 2 ×200 mg/kg BW by mouth.No indication was found of any mutagenic potency of Praziquantel. This agrees with the results of point-mutation tests done by other authors.ZusammenfassungPraziquantel, ein neues Anthelmintikum mit Antischistosomen- und Anticestodenwirkung, wurde im Vergleich zu einer Placebo-Kontrolle und zu einer ‚Positiv-Kontrolle’ mit Cyclophosphamid in verschiedenen Säuger-Testsystemen in vivo auf mögliche mutagene Wirkungen untersucht. Die Testsysteme und untersuchten Praziquantel-Dosen waren: (1) Dominant-Letal-Test an der männlichen NMRI-Maus, 12 Paarungsperioden zu je 4 Tagen, 1× 1200 mg/kg KG per os; (2) Dominant-Letal-Test an der weiblichen NMRI-Maus, Applikation im Pro-Oestrus, 1×1200 mg/kg KG per os; (3) Mikronucleus-Test an männlichen und weiblichen NMRI-Mäusen, zweimalige Gabe im Abstand von 24 Std und Aufarbeitung des Femurmarks 6 Std nach der zweiten Applikation, 2×300 mg/kg und 2×600 mg/kg KG per os; (4) Spermatogomen-Test am Chinesischen Hamster, zweimalige Gabe im Abstand von 24 Std und Aufarbeitung der Spermatogonien 48 Std nach der zweiten Applikation, 2 × 600 mg/kg KG per os. Die Dosis 1200 mg/kg KG per os entsprach etwa der halben LD50 nach oraler Gabe an der Maus und ca. dem 40fachen der therapeutischen Dosis (1×30 mg/kg KG per os). Die Cyclophosphamid-Dosen waren in den entsprechenden Testsystemen 1×200 mg/kg bzw. 2×200 mg/kg KG per os.Es fand sich kein Hinweis auf eine mutagene Potenz von Praziquantel. Dies stand im Einklang mit den Ergebnissen von Punktmutations-Tests, die von anderen Autoren durchgeführt worden sind.

Studies with N,N-dimethylformamide for embryotoxic and teratogenic effects on rats after dynamic inhalation

SummaryThe purpose of this study, which was conducted for industrial toxicological reasons, was to investigate the possible embryotoxic and teratogenic effects after inhalation of dimethylformamide in rats. In a dynamic inhalation apparatus groups of 22–23 rats were subjected to approx. 18 ppm (maximum allowable concentration in the working area, MAC=20 ppm) and 172 ppm in the air for 6 hrs/day on 10 consecutive days, i.e. from the 6th to 15th day of gestation. The dimethylformamide inhalation did not cause any visible impairment to female rats as regards general behaviour, appearance or weight development during the treatment or the entire gestation period. The gestation rate was unchanged. The development of the fetuses was not influenced in any way by the exposure of the pregnant animals to approx. 18 ppm. In contrast the fetuses taken by caesarean section from the dams exposed to 172 ppm weighed significantly less than the fetuses of the control animals. Skeletal development of these fetuses, however, was normal.All other reproduction parameters were within the normal range for this strain.The study showed that the inhalation of dimethylformamide in concentration up to approx. 10 times the MAC had no teratogenic effect on rats.

Mutagenicity studies with 2,4,5‐T on bacteria and mammalian germ cells

The herbicide 2,4,5-T (2,4,5-trichlorophenoxyacetic acid) was evaluated for potential mutagenicity by a Salmonella/mammalian-microsome test, a dominant lethal test on female rats, and by a cytogenetic assay on spermatogonia of Chinese hamster. In the Salmonella/mammalian-microsome test on four Salmonella typhimurium strains (TA 1535, TA 100, TA 1537, and TA 98), doses of up to and including 2500 micrograms/plate did not cause any mutagenic effects. In a dominant lethal test on female rats, 8-week dietary administration of 2,4,5-T at doses of up to and including 10 mg/kg/day did not cause any increase in preimplantation loss or the rate of dead implants, and did not have any effect on the fertilization quota. Cytogenetic analysis of the spermatogonia of male Chinese hamsters orally dosed five times at 24-hr intervals with 2,4,5-T at levels of up to and including 100 mg/kg did not provide any indication of 2,4,5-T having chromosome-damaging effects. Therefore, none of the three test systems provided any indication of 2,4,5-T having a mutagenic effect.

Electroretinographic assessment of early retinopathy in rats

Amoscanate, a substance which damages photoreceptors, was administered orally to Wistar rats in doses of 10, 40, and 125 mg/kg body weight once daily for 3 or 10 days. At both times electroretinographic, ophthalmological, and histopathological examinations of the retina were carried out to compare the sensitivity of conventional methods and to test electroretinography (ERG) for suitability for use in toxicity studies. Time-dependent and dose-dependent effects were found by electroretinography and light microscopy. However, signs of retinal changes appeared earlier and more distinctly in the electroretinogram. Ophthalmological fundus examination in albino rats yielded no characteristic correlate. In conclusion, electroretinography constitutes a valuable supplement to histopathology and is suitable for use in toxicity studies.

Inhalationstoxikologische Untersuchungen mit tertiärem Butylisonitril an Ratten und Mäusen

The inhalation toxicity of tertiary butylisonitril (TBIN) was evaluated in rats and mice. In addition, pregnant rats were exposed to TBIN aerosols to test its embryotoxic effects and male mice were exposed to TBIN aerosols to evaluate its mutagenic effects using the dominant lethal test.Inhalation of TBIN aerosols caused death in rats only at high concentrations. The following inhalation LC50 values were determined for rats and mice following inhalation of TBIN aerosols: 4-hr exposure: male rats 715, female rats 710, male mice 377; five 4-hr exposures: male and female rats between 356 and 583 mg/m3 air. The animals were impaired for a long period of time, and death occurred up to 10 days after the exposure.Female rats were exposed to TBIN aerosols from the 6th to the 15th day of gestation daily for 4 hrs. The used concentrations were not toxic to the pregnant rats. It became evident that only the lowest concentration (14 mg/m3) was not effective to the development of the fetus. A TBIN concentration of 36 mg/m3 definetely increased the resorption in embryos, and with 71 mg/m3 a complete loss of the fetus occurred due to resorption. A teratogenic effect could not be determined.A single 4-hr inhalation of 125 mg TBIN/m3 air caused changes of the sperms of male mice. There was a decreased fertilization capability during the first week of mating after the exposure and a decreased implantation rate with a simultaneous increase of pre-implantation losses in the females.With respect to industrial hygiene it is important that concentrations of TBIN in the air show embryotoxic and anti-spermatogenic effects in animals, which they tolerated without symptoms of poisoning.ZusammenfassungDie Toxicität von tertiärem Butylisonitril (TBIN) wurde nach dynamischer Inhalation an Ratten und Mäusen untersucht. Ferner erfolgten inhalationstoxikologische Untersuchungen an Ratten zur Prüfung auf embryotoxische Wirkungen und an Mäusen zur Prüfung auf mutagene Wirkungen im Dominant-Letal-Test nach Behandlung der männlichen Mäuse.Die Einatmung von tert. Butylisonitril-Aerosolen wirkte bei Ratten erst in hohen Luftkonzentrationen letal. So betrugen bei der einmaligen, 4stündigen Exposition die LC50-Werte für männliche und weibliche Ratten 715 bzw. 710 mg/m3 und bei der 5mal 4stündigen Exposition zwischen 356 und 583 mg/m3. Mäuse waren empfindlicher als Ratten, bei der einmaligen 4stündigen Exposition betrug die LC50 für männliche Mäuse 377 mg/m3. Die Tiere waren lange Zeit geschädigt, und Todesfälle traten bis zu 10 Tagen nach der Exposition auf.Im Embryotoxicitätsversuch wurden weibliche Ratten vom 6. bis 15. Tag der Trächtikgeit täglich für 4 Std verschiedenen, für die Muttertiere nicht toxischen Konzentrationen von TBIN ausgesetzt. Es zeigte sich, daß nur die niedrigste untersuchte Dosis, 14,8 mg/m3, schädigungslos für die Fruchtentwicklung war. Bei einer TBIN-Konzentration von 36 mg/m3 war die Resorption von Embryonen sehr deutlich gegenüber den Kontrollen erhöht, und bei 71 mg/m3 trat vollständiger Keimverlust durch Resorption ein. Eine teratogene Wirkung war nicht nachzuweisen.Im Dominant-Letal-Test führte eine einmalige, 4stündige Inhalation von 125 mg TBIN pro m3 Atemluft bei Mäuseböcken zu einer Schädigung der Spermien, die sich in der 1. Paarungswoche nach der Exposition in einer herabgesetzten Befruchtungsfähikgeit und einer herabgesetzten Implantationsrate bei gleichzeitigem Anstieg des präimplantativen Verlustes bei den befruchteten Weibchen äußerte.Gewerbehygienisch bedeutsam ist nach diesen Ergebnissen, daß tert. Butylisonitril in Konzentrationen, die symptomlos toleriert wurden, im Tierversuch die Keimlinge und die männlichen Samenzellen schädigen kann, nachdem es über die Atemwege in den Organismus gelangt ist.