Twenty-four-month oral carcinogenicity study of ebrotidine in rats.

Three groups of Sprague-Dawley CD rats (males and females) were initially administered p.o. with ebrotidine, a novel H2-receptor antagonist, mixed with the diet, at 50, 200, and 500 mg/kg/d, respectively. Two concurrent control groups of animals were used. After 13 months, initial 200 mg/kg was lowered to 150 mg/kg, and a new group was administered with 300 mg/kg, due to the body weight reduction observed in the top dose group. After 24 months, survivors were killed and necropsied, and a histopathological study was performed. The frequencies of the different tumour types that were found were not raised due to the treatment. Lower frequencies of some types of pituitary and mammary gland tumours, in the groups treated with the higher doses, were the only statistically significant changes. Among the non-neoplastic effects, a lower body weight increment and food consumption (500 and 300 mg/kg, both sexes), lower survival (500 mg/kg, males), presence of lipoid pneumonia (500 mg/kg, only in males, and 300 mg/kg, both sexes), and lithiasis in urinary system (500 mg/kg) were observed. No changes in gastric mucosa (the main target organ) were attributable to ebrotidine. Regarding the non-neoplastic effects, 150 mg/kg was the no observed adverse effect level. According to the previous results of the carcinogenicity study in mice, conjointly with those of the study in rats reported here, there is no evidence of carcinogenic risk either in males or in females in these species.

Acute toxicity studies of ebrotidine.

The acute toxicity of two formulations of ebrotidine (N-[N-[(E)-[[2-[[[2-[(diaminomethylene)amino]-4-thiazolyl] methyl]thio]ethyl]amino]methylene]-4-bromo-benzenesulfonamide, CAS 100981-43-9, FI-3542) was studied in rats and mice by different routes of dosing: oral and intraperitoneal routes in rats and mice (suspension), intravenous and oral routes in mice and intravenous route in rats (injectable solution). LD50 values for the oral route were indeterminable in all cases. For the intraperitoneal route. LD50 values were 316 mg/kg (rat) and 366 mg/kg (mouse), and for the intravenous route LD50 values were 100 mg/kg (rat) and 107 mg/kg (mouse).

Subacute toxicity of ebrotidine in rats and dogs.

Subacute toxicity studies of ebrotidine (N-[(E)-[[2-[[[2-[(diaminomethylene)amino]-4-thiazolyl]methyl] thio]ethyl]amino]methylene]-4-bromo-benzenesulfonamide, CAS 100981-43-9, FI-3542) were performed in Spragu-Dawley rats and Beagle dogs. Both animal species were administered with the same dose levels (50, 200 and 500 mg/kg) for 4 and 7 weeks, respectively. In a previous 4-week subacute toxicity study in the rat, ranitidine and cimetidine at 500 mg/kg were used as reference drugs. The results indicated that ebrotidine was well tolerated at 50 mg/kg, while there were dose-related effects at 200 and 500 mg/kg. Probably due to its pharmacokinetics, ebrotidine was more toxic in dogs than in rats, since the most severe effects were the death or sacrifice in extremis of two dogs from the high dose group which had undergone rectal prolapse, while no deaths occurred in the rats. The changes that were very likely related to treatment (500 mg/kg) were a lower weight in both species, a slight decrease of hematocrit and red blood cells in rats, single increments of transaminases, alkaline phosphatase and lactate dehydrogenase in dogs (some animals of the 200 mg/kg dose group were also affected) and a higher liver weight. These effects with a few exceptions were found to be common to cimetidine and ranitidine.

Chronic toxicity of ebrotidine in rats and dogs.

The results obtained in the chronic toxicity studies of ebrotidine (N-[(E)-[[2-[[[2-[(diaminomethylene)amino]-4-thiazolyl] methyl]thio]ethyl]amino]methylene]-4-bromo-benzenesulfonamide, CAS 100981-43-9, FI-3542) by oral route in rats and in Beagle dogs are reported. Rats were administered for 6 months and dogs for 12 months. The doses were 50, 200 and 500 mg/kg in rats and 50, 200 and 400 mg/kg in dogs. The dose of 400 mg/kg was reduced to 350 mg/kg after 3 months of treatment, due to its toxicity. The effects probably related to the administration of ebrotidine were as follows: three dogs from the high dose group died after 3, 4.5 and 8 months of treatment (in rat there was no mortality); occult blood in faeces; lower weight gain in the high dose group (in rats only females were affected); lower food consumption in rats from the high dose group (and also females from the middle dose group); reduction of erythrocyte count and packed cell volume, only in rats and at the end of the study; alkaline phosphatases increment in rats and dogs; proteinemia decrease in rats; and a tendency to decrease in the testicular weight, which was not statistically significant (p > 0.05). The only histopathological changes observed were moderate erosions or ulcerations in the intestinal mucosa of some dogs from the high dose group. These effects coincide with those published for other competitive H2-receptor inhibitors. The maximum toxic effect-free level was 50 mg/kg for both rats and dogs, which provides a wide safety margin with respect to the therapeutic dose.

Toxicity of ebrotidine on reproduction. Toxicity on fertility and general reproductive performance, embryo-fetal toxicity and peri- and postnatal toxicity.

Reproduction toxicity studies of ebrotidine (N-[(E)-[[2-[[[2-[(diaminomethylene)amino]-4-thiazolyl]methyl] thio]ethyl]amino]methylene]-4-bromo-benzenesulfonamide, CAS 100981-43-9, FI-3542) are presented in this paper. Rats dosed with 50, 200 and 500 mg/kg p.o. of ebrotidine were used for the fertility and peri- and postnatal toxicity studies, and rabbits dosed with 25, 100 and 250 mg/kg and rats dosed with 50, 200 and 500 mg/kg of ebrotidine were used for the embryotoxicity study. The fertility study was designed in accordance with a 2-generation study protocol. The results showed that ebrotidine did not interfere with male and female gametogenesis, fertility, organogenesis, postnatal development and lactation in F0 or F1 animals. Only general or non-specific effects were attributed to treatment, such as a lower weight gain in parents or fetuses in rats, or a somewhat slower bone calcification in rats, which was shown to be recoverable and had no peri- or postnatal repercussions. Neither did the fertility study reveal a possible longer duration of gestation nor did the peri- and postnatal study show a lower weight of the F1 offspring. There was only an increase in rabbit embryonic mortality, probably related to some cases of abortion at the high dose. No potential antiandrogenic effect on the reproductive function has been found. Among the different doses used in both animal species, the maximum toxic effect-free dose was that of 25 mg/kg.

Carcinogenicity studies on ebrotidine.

The results from two carcinogenicity studies on ebrotidine (N-[2-(E)-[[2-[[[2-[(diaminomethylene)amino]-4-thiazolyl] methyl]thio]ethyl]amino]methylene]-4-bromo-benzenesulfonamide, CAS 100981-43-9, FI-3542) conducted in mice and rats are reported. Oral doses of 50, 200 and 500 mg/kg were administered to mice for 18 months and 50, 200 (150), 300 and 500 mg/kg were administered to rats for 24 months. The study design was prepared according to EEC guidelines, and the recommendations by the International Agency for Research on Cancer were used for the statistical analysis of data. Weekly palpations were made along the course of studies and general parameters were monitored. The only effects attributed to ebrotidine administration were a slight decrease in the survival rate of female mice given the 500 mg/kg dose and a lower weight gain in rats of both sexes. The histopathological data revealed that lipoid pneumonia and kidney calculi are more frequent in rats treated with doses of 500 and 300 mg/kg. No increment in the spontaneous occurrence of tumours or significant presence of tumours in treated animals differing from that in control animals was observed, and a decrease in the time required for their onset that could be related to ebrotidine was not observed either. There were no differences in hyperplastic and/or dysplastic changes between treated and control animals. Therefore, it is deduced that ebrotidine does not induce neoplastic or preneoplastic effects in rats or mice even at doses of 500 mg/kg, at which some general toxicity effects are seen.

Ocular tolerance of sertaconazole gel.

The in vitro and in vivo tolerance of sertaconazole gel, a new topical azole antifungal, was studied. Ketoconazole gel (Panfungol) was used as a reference substance. The methods applied for tolerance assessment were the bovine corneal opacity and permeability test for the in vitro assay and a modified Draize test for the in vivo assay. The results obtained show that both substances can be classified as slightly irritant and with acceptable tolerance. However, unlike ketoconazole gel, sertaconazole gel did not cause a positive lesion index in vivo. Ketoconazole was 5.25 times more irritant in vitro than sertaconazole gel, whose effect was similar to that of saline solution. Consequently, the negligible irritant effect of sertaconazole gel on a type of epithelium that is extremely sensitive, i.e. the cornea, confirms the good tolerance of this new antifungal gel on other structures such as the skin and mucous membranes.

Acute toxicity studies of sertaconazole.

The acute toxicity of 7-chloro-3-[1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethoxy- methyl]benzo[b]thiophene (sertaconazole, FI-7045, CAS 99592-32-2) was evaluated in mice and rats after single administration by oral, subcutaneous and intraperitoneal routes. The latter intended to ensure maximum blood levels, since intravenous route was unfeasible due to drug insolubility in water. LD50 was indeterminable (greater than 8000 mg/kg) in all routes of dosing. According to these results, and having furthermore proved the absorption of the test substance after oral administration, sertaconazole was concluded to be very safe in the event of overdose or accidental ingestion.

Subacute toxicity and maximum tolerable dose of sertaconazole in repeated administration studies.

28-Day oral and dermal subacute toxicity studies of 7-chloro-3-[1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl) ethoxy-methyl] benzo [b] thiophene (sertaconazole, FI-7045, CAS 99592-32-2), were carried out. The oral studies included the evaluation of subacute toxicity in rat (dose levels of 50, 150 and 300 mg/kg) and maximum tolerable dose in repeated administration in ferrets (consecutive dose levels in accordance with a geometric progression of 50, 75, 112.5, 168 and 250 mg/kg), which were the animal species intended for chronic toxicity studies. The dermal studies included the evaluation of subacute toxicity in rats and rabbits (1 ml/kg of a 2% cream). The results, in general, have shown low toxic effects, which can be summarized as a slight non-significant hepatomegalia in the rat with increased gamma-GTP and alkaline phosphatase values and a high urinary pH value; no histopathological changes were observed. These effects are characteristic of azole derivatives and are therefore common to other antifungals with this chemical group.

Chronic toxicity studies of sertaconazole after oral administration to rats and ferrets.

Six-month chronic oral toxicity studies of 7-chloro-3-[1-(2, 4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethoxy-methyl] benzo[b]thiophene (sertaconazole, FI-7045, CAS 99592-32-2) were carried out in rats and ferrets. The dose levels used were 50, 150 and 300 mg/kg in rats and 50, 150 and 250 mg/kg in ferrets. There was no mortality associated with the drug in either of the two species. The results obtained show that the toxic effects may be summarized as a smaller body weight increase in rats at 150 and 300 mg/kg and in male ferrets at 250 mg/kg. Food consumption decreased significantly in rats at 300 mg/kg, and was not proportional to the doses of 150 and 50 mg/kg. In serum biochemistry, increases in alkaline phosphatase in rats, ALT in male ferrets at 150 and 250 mg/kg and AST only at 250 mg/kg were observed. BUN increased at 150 and 250 mg/kg in ferrets.

Reproduction toxicity of sertaconazole. Segment II (teratology) and Segment III (peri-postnatal toxicity).

The reproduction toxicity of 7-chloro-3-[1-(2,4-dichlorophenyl)-2- (1H-imidazol-1-yl)ethoxy-methyl]benzo[b]thiophene (sertaconazole, FI-7045, CAS 99592-32-2) (50, 100 and 150 mg/kg by oral route) has been investigated by performing two studies: the embryotoxicity or teratology study in rats and rabbits, and the peri-postnatal toxicity study in rats. According to the results obtained from the embryotoxicity studies, there was no maternal toxicity in either of the two species studied. The only embryofoetal abnormalities with statistical and toxicological significance were observed at the dose of 150 mg/kg in the teratology study on rabbits: hepatomegalia, pericardial oedema and peritoneal and hepatic haemorrhages. The results of the peri-postnatal study showed that the only maternal effect relating to the drug was an increase, proportional to the dose, in the weight of the ovaries, which was significant at the dose of 150 mg/kg. With reference to the offspring, only a reduction in the viability index at 150 mg/kg was observed. The non observed effects level (NOEL) for all three studies can be estimated at 100 mg/kg.

Dermal tolerance and phototoxicity studies of sertaconazole.

The local dermal tolerance of 7-chloro-3-[1-(2,4-dichlorophenyl)-2-(1H- imidazol-1-yl)ethoxy-methyl]benzo[b] thiophene (sertaconazole, FI 7045, CAS 99592-32-2) was evaluated in hairless rats and albino rabbits. Test substance was applied in 2% powder (0.5 g), gel (0.5 ml) and solution (0.5 ml) formulations to rats (this is the concentration intended for the pharmaceutical preparation) and in 6% cream (0.5 ml) to rabbits (this concentration was used to estimate an overdose maximal effect). After 3- and 24-h exposure of the test substance to animals, they were observed for erythema and oedema. Sertaconazole may be considered as non irritant, since its primary irritation index was less than 0.5 in all the tests. A phototoxicity study was also carried out on guinea pigs with 1 ml 2% cream per animal, compared with psoralene as a positive control. Sertaconazole did not present any risk, since the phototoxicity level reached was null, with 2 being the minimum value considered as having toxic significance.

Relationship between fetal weight and litter size in rats: application to reproductive toxicology studies.

The inverse relationship between mammalian fetal weight and litter size has been discussed by many authors, but their opinions reveal no agreement at all. As in toxicity studies of reproduction, both parameters must be correctly evaluated. We investigated the existence of such a relationship in 2466 fetuses from 203 litters of Sprague-Dawley CD control rats. The frequency distribution of fetal weights had a normal adjustment. From the mean weight of fetuses in each litter, the mean fetal weights in each litter size and correlation coefficient were calculated and the regression line was plotted; the correlation coefficient (r = 0.677) was highly significant (P = 0.002), which made evident that there was an inverse relationship between fetal weight and litter size. If fetal weight/litter size inverse relationship is not taken into account when toxicity on the fetal weight is analyzed, wrong conclusions may be reached if the test substance reduces the litter size, provoking embryofoetal mortality. The iatrogenic decrement in fetal weight can be masked by an increment due to the litter size reduction. We suggest that in all three segments of reproductive toxicity studies, litter size must be considered as a covariate to the effect of the test substance on the fetal weight, in order to perform a correct analysis of covariance (ANCOVA), in addition to the dose factor commonly used in common ANOVA.