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

Teratogenesis, Carcinogenesis, and Mutagenesis 18:263–277 (1998)

© 1998 Wiley-Liss, Inc.

Twenty-Four-Month Oral CarcinogenicityStudy of Ebrotidine in Rats

Alfonso Romero, 1,2* Frederic Villamayor, 1 Antonio Rives, 3

María-Teresa Grau, 1 Aurelio Sacristán, 1 and José-Alfonso Ortiz 1

1Centro de Investigación Farmacéutica Grupo Ferrer, Barcelona, Spain2Department of Toxicology and Health Legislation, Barcelona University,Barcelona, Spain

3Department of Cellular Biology and Pathological Anatomy, Barcelona University,Barcelona, Spain

Three groups of Sprague-Dawley CD rats (males and females) were initially admin-istered p.o. with ebrotidine, a novel H2-receptor antagonist, mixed with the diet, at50, 200, and 500 mg/kg/d, respectively. Two concurrent control groups of animalswere used. After 13 months, initial 200 mg/kg was lowered to 150 mg/kg, and a newgroup was administered with 300 mg/kg, due to the body weight reduction observedin the top dose group. After 24 months, survivors were killed and necropsied, and ahistopathological study was performed. The frequencies of the different tumour typesthat were found were not raised due to the treatment. Lower frequencies of sometypes of pituitary and mammary gland tumours, in the groups treated with the higherdoses, were the only statistically significant changes. Among the non-neoplastic ef-fects, 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 (500mg/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 wasthe no observed adverse effect level. According to the previous results of the carci-nogenicity 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 spe-cies. Teratogenesis Carcinog. Mutagen. 18:263–277, 1998.© 1998 Wiley-Liss, Inc.

Key words: Ebrocit™; histamine-2 receptor antagonists; cytoprotection

INTRODUCTION

Ebrotidine(N-[(E)-[[2-[[[2-[(diaminomethylene)amino]-4-thiazolyl]methyl]-thio]ethyl]amino]methylene]-4-bromo-benzenesulfonamide) is a novel competitive H2-

*Correspondence to: Dr. José-Alfonso Ortiz, Centro de Investigación Farmacéutica Grupo Ferrer, Juande Sada, 32, 08028-Barcelona, Spain.

264 Romero et al.

receptor antagonist with gastroprotective properties [1,2]. Carcinogenicity studies arespecially important in this therapeutic group due to the hyperplastic and neoplasticrisk in the gastric mucosa that has been demonstrated in rodents, for excessivelypowerful inhibitors of the gastric acid secretion, with an unsurmountable or long-acting mechanism of action [3–5]. In these cases, carcinoids are found, which arecompletely formed by enterochromaffin-like cells—the cells responsible for the se-cretion of histamine. This proliferative response is not attributed to a carcinogeniceffect of the gastric secretion inhibitors per se but to a trophic effect due to thehypergastrinemia that achlorhydria induces [6–8].

Neither preneoplastic nor neoplastic effects attributable to ebrotidine were found inthe previous carcinogenicity study in mice [9]. Thus, our objective in the study reportedhere is to complete the pre-clinical evaluation of the carcinogenetic risk of ebrotidine, ifany, by the long-term oral administration of ebrotidine to a second species (rat), with aspecial attention to the changes that could occur in the gastric mucosa.

MATERIALS AND METHODS

The study reported here was conducted according to current European guide-lines [10] for carcinogenicity testing. Test substance was ebrotidine (batch D-12),synthesized by Ferrer Internacional S.A. (Barcelona, Spain). Test system was Spe-cific Pathogen Free (SPF) OFA Sprague-Dawley rat from Iffa-Credo (Lyon, France).Rats from both sexes were used. After quarantine, animals were randomly allocatedto the experimental groups (50 male/50 female each), and individually housed in theSPF animal facilities. Care and handling of the animals were according to relevantEuropean guidelines [11], as well as environmental conditions, that were controlledand kept within the recommended ranges. Animals were fed ad libitum with A04feed for mice/rats from UAR (Paris, France), controlled and sterilized by g-radiation.Sterile water was supplied ad libitum. Cage positions were periodically rotated ineach rack and room during the course of the study.

Ebrotidine was administered for 104 weeks and was mixed with the diet. Theebrotidine concentration was periodically adjusted to variations in body weight andfood consumption in order to maintain the dosing regime. Initially, three groups ofanimals were treated with ebrotidine 50, 200, and 500 mg/kg/d, respectively. Therewere two concurrent control groups, which were exactly handled as treated groups,but received the food without ebrotidine. After 13 months, a body weight reductionbecame evident, threatening the survival of the top dose group. Consequently, theaddition of another experimental group to be treated with ebrotidine 300 mg/kg/dwas decided, as well as the reduction of the dose from the 200 mg/kg/d group downto 150 mg/kg/d.

The in-life observations in this study included periodical monitoring for theappearance of clinical signs and palpable masses (weekly), and recording of bodyweight evolution (weekly for the first 3 months, then monthly) and diet consumption(weekly for the first 3 months and then quarterly). Mortality was monitored dailyand recorded.

Necropsy was performed on all the animals that died or were sacrificed in ex-tremis before week 104, as well as on all the animals that survived to week 104.Liver, prostate, stomach, and testes (considered as the main target organs) wereweighed in all terminal animals. Specimens were taken from all the organs and tis-

Carcinogenicity Study of Ebrotidine 265

sues specified in Table I, and processed for histopathological analysis. This was per-formed on all the organs from all the animals in both control groups and those treatedwith ebrotidine 300 and 500 mg/kg. Animals treated with ebrotidine 50 and 200(150) mg/kg were completely analysed when they died before week 104. Otherwise,only those organs presenting macroscopic lesions suspected to be tumors, as well asall palpable masses that had been recorded in-life were examined for histopathologi-cal evaluation.

The statistical methods used to analyse general parameters included Kaplan-Meier’s statistical analysis of survival, Repeated Measures Analysis of Variance, andcontingency table analysis (Spearman’s c

2, and Mantel-Haenszel’s tests). In all thesetests, a = 0.05 was adopted as significance level, and results were corrected accord-ing to Bonferroni’s criterion for multiple comparisons. Tumour frequency was analysedaccording to the methods described by Peto et al. [12] and recommended by Gart etal. [13]. Statistical tests to compare tumor frequencies among the different experi-mental groups were performed only on those types with a total absolute frequency(in one sex) higher than 1. Otherwise, chance was considered the responsible for thepresence of the tumor on any particular group. In order to avoid the occurrence offalse positive errors, a dual signification level was adopted: a1 = 0.01, for frequenttumours (more than five in the study), and a2 = 0.05, for less frequent tumors. Thiscriterion has been recommended by Haseman [14].

RESULTS

Results reported here are those that have shown statistically significant differ-ences between treated and control groups, with a dose-related trend, as well as thosethat in our opinion, are biologically relevant, irrespective of its statistical signifi-cance. “Terminal animals” will refer to those animals that survived to week 104 ofthe study, and “non-terminal animals” to those that did not.

Figure 1 displays Kaplan-Meier estimates of the survival function, and meansurvival times are shown in Table 2. Survival in males exhibits a non-linear trendwith dose, with a maximum at the group treated with 200 (150) mg/kg, and mini-mum at 500 mg/kg. In contrast, the survival of females is linearly dose-related, witha maximum at 500 mg/kg.

Clinical signs showing a perceptible dose-related trend (either positive or nega-tive) in their frequency were i) alopecia, whose frequency was lower as the doseincreased; ii) haematuria, whose highest frequency was on the group treated with

TABLE I. Organs and Tissues That Were Examined*

Adrenals Kidneys Parathyroid SpleenBone Lymph nodes Peripheral nerve StomachBone marrow Liver Pituitary TestesBrain Lung Prostate ThymusCerebellum Mammary gland Salivary gland ThyroidColon Oesophagus Skin TracheaEyes Ovaries Small intestine Urinary bladderHeart Pancreas Spinal cord Uterus

*Additionally, isolated specimens were examined from gross lesions present at peritoneal cavity,meninges, pubic region, rectum, and soft/subcutaneous tissues.

266 Romero et al.

Fig. 1. Kaplan-Meier estimates of the survival function. Closed circle, control-I; closed square, con-trol-II; open inverted triangle, 50 mg/kg; open diamond, 200(150) mg/kg; open triangle, 300 mg/kg;open square, 500 mg/kg.


ebrotidine 500 mg/kg; and iii) excessive lacrimation, with a slightly increased fre-quency in the animals treated with ebrotidine. These three effects were detected inboth sexes.

Mean body weight changes with time in terminal animals is shown in Figure 2.There were progressively increasing differences between both males and femalestreated with ebrotidine 300 and 500 mg/kg and the corresponding control groups. Itshould be mentioned that both female control groups differed significantly from thetwelfth month until the end of the study. Differences in male control groups did notreach statistical significance.

The evolution of food consumption with time is shown in Figure 3. The con-sumption on the group treated with ebrotidine 500 mg/kg (both males and females)was lower than that on both control groups. When consumption relative to the meanbody weight was calculated and analysed, the highest values were on the groupstreated with ebrotidine 300 and 500 mg/kg (both males and females).

The concentration of the test substance was periodically adjusted to the foodconsumption and body weight changes as the study progressed. Once the study wascompleted, the calculation of the mean dose that each animal received was performed.Table III shows a summary of these results for each treated group.

The monitoring of palpable masses included any masses and nodules that couldbe detected grossly, with no concern about their nature: They could be real tumors,as well as abscesses or other non-neoplastic lesions. Figure 4 graphically shows thedecay of the percentage of animals that were free of palpable masses as their onsetrate increased with age, in the different experimental groups. As can be seen, thelowest onset rates were on the groups treated with ebrotidine 300 and 500 mg/kg(both sexes).

Macroscopic observations were recorded when necropsy was performed. In or-der to clarify their significance, the observations from terminal and non-terminal

TABLE II. Mean Survival Times in the Different Experimental Groups

MSTa SEMb 95% CIc

MalesControl-I 88 3 (82; 94)Control-II 92 2 (88; 96)50 mg/kg ebrotidine 93 3 (88; 99)200 (150) mg/kg ebrotidine 103* 2 (98; 107)300 mg/kg ebrotidine 95 3 (90; 100)500 mg/kg ebrotidine 76 4 (68; 84)

FemalesControl-I 93 3 (87; 99)Control-II 95 3 (89; 101)50 mg/kg ebrotidine 87 3 (81; 93)200 (150) mg/kg ebrotidine 94 2 (90; 98)300 mg/kg ebrotidine 99 2 (95; 103)500 mg/kg ebrotidine 102** 2 (98; 106)

aMean survival time.bStandard error of mean.c95% Confidence interval.*P £0.05 (referred to both control groups).** P £0.05 (referred to only one control group).

268 Romero et al.

Fig. 2. Body weight evolution. Legend as in Figure 1.

animals were analysed separately. The only changes that were attributed to a toxicorigin were i) lithiasis in the urinary system from males and females treated withebrotidine 500 mg/kg; ii) lipoid pneumonia, which was more frequent in the malestreated with ebrotidine 300 mg/kg and in the females treated with 500 and 300 mg/kg (this change, which is progressive, had a higher prevalence in the terminal ani-


mals); iii) a lower prevalence of mammary and subcutaneous palpable masses in theterminal females and non-terminal males from the group treated with ebrotidine 500mg/kg; and iv) a higher prevalence (but not statistically significant) of polycysticovaries in the females treated with ebrotidine 500 mg/kg.

According to the histopathological evaluation, the only statistically significant

Fig. 3. Food consumption evolution. Legend as in Figure 1.

270 Romero et al.

findings were a dose-related decrement in pituitary adenomas in males and a decre-ment in mammary fibroadenomas in females (Tables IV and V). Pituitary anaplastictumours were also less frequent in the treated males, but differences with controlgroups were not significant.

The most relevant non-neoplastic lesions revealed by the histopathological studythat were related to the treatment with ebrotidine have already been identified aboveas macroscopic lesions: lipoid pneumonia (groups of foam macrophages), which hada higher prevalence in females treated with ebrotidine 500 mg/kg and males andfemales treated with 300 mg/kg, and renal and/or vesical lithiasis, in males and fe-males treated with ebrotidine 500 mg/kg. No other histopathological alterations thatcould be related to the treatment with ebrotidine were observed.

DISCUSSION

European guidelines on long-term carcinogenicity assays recommend that finalsurvival should be higher than 50% at the end of the study in order to preservestatistical power (other sources consider the absolute number of terminal survivorsas more determinant for this purpose). Nevertheless, a progressive reduction in sur-vival in the main strains of rats that are being used as experimental systems in thiskind of studies has been detected in recent years and is widely documented. Amongthe causes of such reduction in longevity, two kinds of factors are mentioned: aprobable genetic drift [15], and environmental factors, mainly competition for food[15–17].

Sprague-Dawley CD rats (the strain we have chosen) is one of the most suitablemodels for two reasons: i) its longevity is lower than that of other strains (WistarHan), so the recommendation that the administration period (2 years) covers 80% oflife-span [17] is accomplished, and ii) it is one of the most widely used strains and(except for pituitary and mammary glands) the natural prevalence of tumours is notas high as in Fischer 344 rats [18–20]. Many papers show that, currently, survival islower than 50% in many control groups from 104 week carcinogenicity studies. Swal-low [15] has reviewed 124 104-week carcinogenicity studies on Sprague-DawleyCD rats, classifying them in 14 groups, according to the year they were performed.

TABLE III. Mean Doses (mg/kg) in the Groups Treated With Ebrotidine as Computed FromBody Weight, Food Consumption, and Product Concentration Data

Real dose

Sex Expected dose Mean SDa Sample size

Males 50 36 3,2 50177b 136 10,0 50300 296 18,9 50500 394 41,1 50

Females 50 39 3,1 50177b 152 12,7 50300 301 21,3 50500 487 35,1 50

aStandard deviation.bMean expected dose if receiving 200 mg/kg for the first 13 months and 150 mg/kg for the last 11months.


Fig. 4. Kaplan-Meier estimates of the onset rate of palpable masses. Legend as in Figure 1.

From these, in 8 groups of males and 6 groups of females, survival was lower than50%. In another database containing 58 studies [17], survival ranged from 7 to 73%for males, and from 29 to 68% for females. In our study, survival in both controlgroups has been 30% for males, and 44% and 52% for females. In treated groups,

272 Romero et al.

TABLE IV. Frequency of Neoplastic Lesions in Males†

Control Dose of ebrotidine (mg/kg)

200I II 50 (150) 300 500

Adrenals (44) (47) (29) (20) (49) (47)Cortical adenoma 2 4 1 2 3 4Pheochromocytoma 2 4 1 1 2 0

Spleen (45) (46) (26) (19) (47) (43)Lymphoma 0 0 0 0 1 0

Stomach (47) (48) (31) (23) (48) (45)Squamous papilloma 2 1 0 0 0 0

Lymph nodes (46) (46) (28) (18) (43) (43)Lymphoma 2 3 4 0 1 0

Liver (49) (50) (29) (18) (49) (48)Hepatocellular carcinoma 0 1 0 0 0 1Hepatocellular adenoma 0 1 0 2 0 0Cholangioma 1 0 0 0 1 0

Pituitary (45) (50) (31) (22) (47) (46)Anaplasitc tumour 10 15 9 6 14 4Adenoma 6 6 4 2 3 0*Haemorrhagic adenoma with

isolated anaplasia 0 0 3 2 0 0Unidentifiable tumour 0 2 1 0 0 0

Small intestine (38) (36) (17) (16) (32) (29)Adenocarcinoma 1 1 1 1 0 0

Pancreas (47) (45) (26) (34) (46) (40)Insular adenoma 6 4 0 2 1 1Exocrine adenoma 1 0 1 0 0 0Insular carcinoma 0 1 1 0 0 0

Parathyroid (35) (39) (19) (11) (33) (31)Adenoma 2 3 2 1 1 0

Skin (12) (11) (3) (9) (1) (11)Squamous carcinoma 1 2 0 3 0 0Trichoepitelioma 0 1 1 0 1 0

Prostate (44) (43) (45) (49) (49) (47)Carcinoma 1 3 1 2 6 3

Lung (50) (50) (30) (24) (49) (48)Adenocarcinoma 2 1 0 1 0 1

Soft/subcutaneous tissues (2) (4) (4) (4) (3) (2)Fibrosarcoma 1 3 1 2 1 0Fibroma 0 0 1 1 1 0Lipoma 1 0 1 0 0 1

Thymus (49) (44) (25) (20) (47) (44)Lymphoma 0 0 1 0 0 0

Thyroid (47) (47) (28) (18) (47) (45)Carcinoma 0 2 1 0 1 0Adenoma 9 5 1 1 11 2

Urinary bladder (41) (45) (26) (18) (37) (41)Transitional cell papilloma 0 2 1 0 5 3

†Number examined in brackets. Only lesions with a total frequency greater than 1 are shown.*P £ 0.05, Peto’s test for linear trend.


TABLE V. Frequency of Neoplastic Lesions in Females†

Control Dose of ebrotidine (mg/kg)

200I II 50 (150) 300 500

Adrenals (49) (48) (35) (29) (49) (50)Cortical adenoma 5 3 4 3 1 7Pheochromocytoma 0 1 0 0 3 1

Spleen (47) (46) (32) (30) (44) (50)Lymphoma 2 0 1 1 0 0

Cerebellum (49) (50) (35) (30) (50) (50)Glioma 1 0 0 0 0 1

Brain (49) (50) (35) (30) (50) (50)Malignant glioma 0 2 0 0 0 0

Stomach (49) (49) (33) (31) (50) (50)Squamous papilloma 1 0 1 0 0 0

Lymph node (49) (49) (33) (30) (44) (50)Lymphoma 2 2 0 1 0 1

Mammary gland (50) (50) (46) (44) (50) (49)Fibroadenoma 26 19 20 24 10 5*Adenocarcinoma 13 14 17 13 18 11Fibroma 1 0 1 1 2 1Adenoma 0 1 3 1 0 0

Liver (50) (50) (35) (29) (50) (50)Hepatocellular carcinoma 0 3 0 2 1 2Cholangioma 1 1 0 0 2 3Haemangioma 0 1 1 0 1 1

Pituitary (47) (50) (41) (37) (47) (49)Anaplasitc tumour 14 17 11 11 14 12Adenoma 6 3 10 6 6 5Haemorrhagic adenoma with

isolated anaplasia 0 1 4 3 0 0Small intestine (43) (39) (28) (26) (36) (47)

Leiomyoma 1 0 0 1 0 0Peripheral nerve (45) (45) (22) (28) (23) (42)

Glioma 0 1 0 0 0 0Ovaries (50) (47) (37) (34) (47) (49)

Papillar cystadenoma 4 2 0 0 3 6Papillar cystadenocarcinoma 0 1 0 1 2 1Carcinoma 1 0 1 0 0 0

Pancreas (47) (46) (32) (29) (46) (49)Insular adenoma 0 2 0 0 1 1

Parathyroid (33) (38) (25) (23) (35) (30)Adenoma 0 1 1 0 0 1

Skin (48) (46) (34) (30) (49) (48)Squamous cell carcinoma 1 0 0 2 0 0Soft/subcutaneous tissues (0) (2) (2) (1) (1) (0)Fibrosarcoma 0 2 1 1 1 0

Thyroid (48) (48) (32) (30) (50) (50)Carcinoma 2 1 0 1 0 0Adenoma 3 2 0 1 5 1

Uterus (48) (47) (35) (30) (48) (50)Adenosquamous carcinoma 0 0 0 0 2 0

Urinary bladder (45) (45) (32) (26) (39) (48)Transitional cell papilloma 1 1 3 1 5 3

†Number examined in brackets. Only lesions with a total frequency greater than 1 are shown.*P £ 0.05, Peto’s test for linear trend.

274 Romero et al.

survival has ranged from 22 to 72%. In most of the experimental groups, survivalhas been higher in females than in males, coincidently with published data.

The dose of ebrotidine had inverse effects on survival in both sexes. A probabletoxic effect has been detected in males treated with 500 mg/kg, but this dose, infemales, has shown a curious beneficial effect: that group of animals was the onewith the highest final survival (72%); furthermore, and suggestively, there is a sig-nificant dose-effect relationship in this sex. It is possible that it could be due to thesignificant action that ebrotidine has exerted against the development of mammarytumours. These tumours have been one of the main causes of death in female ani-mals, not by their malignancy (since they are benign), but indirectly, due to the greatsize of the masses that they formed. A possible mechanism of action of this apparentanti-neoplastic effect will be discussed below.

The effect of ebrotidine on body weight is clear in both sexes at doses of 200and 500 mg/kg. It is very probable that the lower food consumption in males treatedwith 500 mg/kg is related to body weight evolution and is difficult to resolve thecause from the effect. It has been confirmed that conversion of consumed food tobody weight has been less efficient in the animals treated with the top dose. Theseeffects could be related to the organoleptic properties of ebrotidine.

Haematuria, renal and vesical calculi, hydronephrosis, and renal microlithiasisare attributable to a single lesion affecting the renal function in the high dose groups.This lesion has evolved more rapidly in males, as we deduce from its prevalence,which is much higher in non-terminal males. Possibly, it has been one of the maincauses of death in this sex. Prevalence of this lesion in females is higher amongterminal animals, so presumably it was not a main cause of death in this sex. Itshould be noted that this kind of lesion has not been found in previous long-termstudies with ebrotidine: 6-month and 12-month chronic toxicity in rats and dogs,respectively, and 78-week carcinogenicity in mice [9,21].

Origin of lipoid pneumonia, which also seems a consequence of the long treat-ment period, can be attributed to the lipophilic nature of ebrotidine. This propertycould have produced fatty deposits in lungs, that were later phagocytised by mac-rophages. The accumulation of these cells had the macroscopic appearance of whiteareas, as observed in necropsies. This change is also induced after long-term treat-ments with ranitidine [22,23].

Prostatic hypertrophy is macroscopically described in necropsies of some malestreated with the top dose and it is not attributed to an effect of ebrotidine treatment.This statement is based on the fact that other products from the same therapeuticgroup tend to originate prostatic hypotrophy [24,25]. Furthermore, prostatic hyper-trophy has been detected exclusively on non-terminal animals with a low body weight,which could alter the appreciation of the size of the prostate, making it greater thanit was. The absence of any effect on this organ is supported on the lack of statisti-cally significant differences on its weight among the different experimental groups.Hereby, it is an advantage in a therapeutic group like H2-receptor antagonists, whereanti-androgenic effects are widely discussed [24,26–28].

The frequency of neoplastic lesions, the endpoint of concern in this study, didnot show any significant increment related to the administration of ebrotidine in anyof the different tumor types that were histologically diagnosed. The frequency ofexternally palpable masses did not either show any rise that could be attributable toebrotidine, nor was its mean latency period shortened by the treatment, but com-


ments should be made on the statistically significant decrements in the frequenciesof mammary fibroadenomas and pituitary adenomas, as well as the (not statisticallysignificant) decrement in the frequency of pituitary anaplastic adenomas, occurringin the animals treated with the higher doses of ebrotidine. Tumor incidence reduc-tions in these organs have been reported [29–34], and related to a lower body weight.Establishing simple relationships among both parameters in treated animals can behazardous, since effects of the test substance can be exerted only on one of them, oron both, simultaneously. In any case, interpretation is very difficult. This can be thecase for H2-receptor antagonists, ebrotidine among them, which, as we have seenbefore, induce a lower body weight increment when administered at high doses.

But other mechanisms, related to immunodepression, can also be acting on tu-mour incidence. Its reduction can be related to the role of histamine on the neoplas-tic development. It has been proved that high levels of this biogenic amine inducethe growth of tumoural cells [35,36]. This can be an explanation for the “anti-neo-plastic” effect described for cimetidine [36–39] and for ranitidine [40,41], whichseems to be related (in a way not yet satisfactorily clarified) with a rise in the levelsof T-lymphocytes acting as cytotoxicity suppressors. Therefore, the lowest frequencyin several tumour types that has been reported in studies on H2-receptor antagonistsdoes not seem to be a fortuitous finding.

Nevertheless, as it was the main objective of this study, the results prove the ab-sence of general preneoplastic and neoplastic changes and, very particularly, in the gas-tric mucosa. These results were expected, in view of those obtained in previous studies[42,43], demonstrating that ebrotidine does not possess any hyperplastic effect on differ-ent endocrine cell populations from the gastric mucosa of mice and rats. Carcinogeniceffects on the rodent gastric mucosa have focused the attention on the antiulcerous thera-pies in recent years, due to the risk inherent to unsurmountable or excessively powerfulH2-antagonists [6,7,8,44], as well as proton pump inhibitors [5,45].

The present work completes the studies on the carcinogenic risk of ebrotidine.Hereby, it is proper now to render an overall evaluation of the results obtained fromthe studies performed both in rats and in mice. U.S. Food and Drug Administrationuses a scale ranging from 0 to 4 [5] that is obtained by cross tabulating the overallresults of the carcinogenicity assays in the two species (rats and mice) by sex andcounting the number of positive cells, i.e., those indicating an increment in tumourfrequencies attributable to the test drug. According to this, ebrotidine is classified inthe group with 0 positive results for carcinogenicity in rodents.

CONCLUSIONS

The present study demonstrates that orally administered ebrotidine to rats for24 months has not induced preneoplastic or neoplastic effects in any of the organsand tissues examined, even at doses that are approximately 100 times higher than thetherapeutic dose. Non neoplastic changes have been found at very high doses, and150 mg/kg can be stated as lacking any adverse effect.

ACKNOWLEDGMENTS

We thank Miss Mercè Recio for her collaboration.

276 Romero et al.

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