Centre International de Toxicologie, BP 563, Miserey, 27005 Evreux and
1 Besins Iscovesco, 5 rue du Bourg l'Abbé, 75003 Paris, France
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Abstract |
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Introduction |
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4-hydroxytamoxifen (Figure 1) is an active metabolite of tamoxifen (7) and is currently undergoing clinical evaluation for the treatment of various non-malignant breast diseases by topical application. The objective of the work reported here was to evaluate the potential carcinogenicity of 4-hydroxytamoxifen after daily cutaneous application for 2 years to female SpragueDawley rats. In the light of the findings described above for tamoxifen, it was of particular concern to establish that 4-hydroxytamoxifen does not induce proliferative or neoplastic lesions of the rat liver. This study was undertaken using the cutaneous route of application since it is the proposed therapeutic route of administration. As 4-hydroxytamoxifen is intended for clinical use in women, only female animals were included in this study. The systemic exposure of the animals was also evaluated.
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Materials and methods |
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Chemicals
4-hydroxytamoxifen was supplied by Laboratoires Besins Iscovesco (Paris, France). Treatment solutions were prepared in ethanol/water (65/35 v/v) on a weekly basis and stored protected from light and under argon pending use.
Test animals and husbandry
A total of 250 female SpragueDawley Crl CD (SD) BR rats were selected for the study on the basis of body weight and clinical examinations. The animals were acclimatized for 2 weeks to the study conditions and habituated to wearing an Elizabethan collar. They were randomly allocated into three treatment groups and two control groups of 50 females each, using a computerized stratification procedure to ensure similar mean body weights for each group. At the beginning of the treatment period, the animals were aged ~6 weeks with a mean body weight of 182 g.
The animals were individually housed in suspended wire mesh cages (43.0x21.5x18.0 cm) in a barriered rodent unit and had free access to pelleted diet (A04C diet; UAR, Villemoisson-sur-Orge, France) and to filtered tap water. Cages were placed in numerical order vertically on the racks and the racks were rotated clockwise within the animal room every fortnight. Environmental conditions were set at: temperature 21 ± 2°C; relative humidity 50 ± 20%; light/dark cycle 12 h/12 h; ventilation ~13 cycles/h of filtered, non-recycled air.
Cutaneous administration
Hair was clipped from the retroscapular dorsum 24 h before the first application and as necessary during the duration of the study (at least 4 h before application of the test substance). Care was taken to avoid abrading the skin, since this could alter its permeability. The solution of 4-hydroxytamoxifen was applied uniformly over the retroscapular area, using a glass syringe fitted with a metal probe. A constant dosage volume of 0.5 ml/kg/day was used. The area of administration was adjusted on the basis of body weight to ~25 cm2/kg. No dressing was applied to the area of administration but the animals were fitted with a protective Elizabethan collar for a period of 6 h after each application to avoid oral ingestion. After removal of the collar, any test substance remaining was wiped off using isotonic NaCl solution and absorbent paper. Treatments were performed at approximately the same time each day.
Study conduct
The animals were checked daily for clinical signs or mortality. After 2 months of treatment, the animals were palpated every 2 weeks in order to record any palpable masses.
Food consumption and body weight were recorded once a week during the first 13 weeks of treatment, then once every 4 weeks. Ophthalmologic examinations were performed once before the treatment period and during weeks 26 and 100 in all groups.
Differential white cell counts were investigated during weeks 52, 78 and 101 in all surviving animals. Plasma samples were taken during weeks 26, 52 and 101, from 10 animals/group, ~24 h after application, for the determination of plasma levels of 4-hydroxytamoxifen and oestradiol. Determination of 4-hydroxytamoxifen levels was performed at Cemaf (Poitiers, France) by mass spectrometry (8). Oestradiol levels were determined by radioimmunoassay at the National Veterinary School of Lyon (Department of Endocrinology, Marcy l'Etoile, France).
The study was terminated in week 101, when mortality in one of the control groups reached 80%. All animals (both deceased animals and animals killed at termination of the experiment) were submitted to a full macroscopic necropsy examination. Histopathological examination was performed on adrenal glands, brain, caecum, colon, duodenum, eyes, femoral bone with articulation, heart, ileum, jejunum, kidneys, liver, lungs with bronchi, lymph nodes, mammary glands, oesophagus, ovaries, pancreas, pituitary gland, rectum, salivary glands, sciatic nerve, skeletal muscle, skin (treated and non-treated areas), spinal cord, spleen, sternum with bone marrow, stomach, thymus, thyroid gland with parathyroids, trachea, urinary bladder, uterus and vagina and on all macroscopic lesions.
Statistical analysis
For the statistical analysis of body weight, food consumption and haematology data, a check was made for a normal distribution of the data (Kolmogorov's test) and data that was not normally distributed was logarithmically transformed. The data were tested for homogeneity of variance between groups using Bartlett's test. Comparisons between treated and control groups were made using Dunnett's test (for data with homogeneous variance) or Dunn's test (for data with heterogeneous variance). If logarithmic transformation did not normalize the data, comparisons were made using the original data and Dunn's test. Comparison of the number of neoplasms/group/organ were made using Peto's test (9).
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Results |
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Mortality
Throughout the study the survival rate was higher in 4-hydroxytamoxifen-treated groups than in the control groups. At the end of the study, the mean mortality was 75% in the control groups, whereas the percentage mortality was lower (46%) in the animals given 20 µg/kg/day and dramatically lower (6% in both groups) in the animals given 140 or 1000 µg/kg/day (Figure 2 and Table I
). These differences were statistically significant starting from weeks 85, 69 and 65 for the treatment groups at 20, 140 and 1000 µg/kg/day, respectively. The major factors contributing to death (or killing) of treated and control animals were pituitary adenomas and mammary gland tumours (adenomas or carcinomas).
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During the study, moderately lower mean body weight gain than in the controls was observed at all dose-levels, from week 11 onwards at the low dose-level and from week 2 onwards at the intermediate and high dose-levels (Figure 3). At termination of the study, mean body weight values were lower than the control values in all 4-hydroxytamoxifen-treated groups (Table I
). The differences in body weight were statistically significant on almost every occasion throughout the study in groups given 140 or 1000 µg/kg/day and from week 30 were frequently statistically significant in the treatment group given 20 µg/kg/day.
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Plasma levels of 4-hydroxytamoxifen and oestradiol
After cutaneous administration, 4-hydroxytamoxifen was absorbed at all dose-levels, as shown by the resultant plasma levels (Table II). Over the course of the study the plasma levels obtained 24 h after application did not alter. There was good dose proportionality between plasma concentrations at 24 h (C24 h) and administered dose at 20 and 140 µg/kg/day, whereas the C24 h was slightly less than proportional at 1000 µg/kg/day. No 4-hydroxytamoxifen was detected in the plasma samples of control animals. At the high dose-level of 1000 µg/kg/day, the plasma levels corresponded to ~60-fold the clinical plasma levels (2967 compared with 50 pg/ml in clinical use).
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Necropsy examination
In females given 140 or 1000 µg/kg/day, absence of prominent mammary glands or reduced size of the mammary glands was noted, whereas these findings were not seen in control animals. There was a greater incidence of animals with small ovaries in the 1000 µg/kg/day treatment group. At 140 and 1000 µg/kg/day there was also a lower incidence of animals with an enlarged pituitary gland. Subcutaneous masses which correlated at microscopic examination with mammary tumours were noted in both control groups and in the 20 µg/kg/day group, but not in the 140 or 1000 µg/kg/day groups. Greyish or whitish foci or areas were seen in the lungs of females given 140 or 1000 µg/kg/day, with a higher frequency than in control females.
Histopathologic examination
Non-neoplastic lesions.
Histopathological examination revealed non-neoplastic findings in the mammary glands, ovaries, thymus, bile duct and liver, lungs and kidneys (Table IV).
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Persistent epithelial cells (including clear epithelial cells) were noted in the thymus, with a higher incidence and/or severity in animals of all treated groups than in the controls.
In the liver, the incidence and severity of pre-neoplastic lesions (acidophilic foci of hepatocellular alteration and nodular hyperplasia) were similar in the control and the low dose-level females, but were decreased in animals of the intermediate and high doses. At these dose-levels, biliary proliferation and peribiliary fibrosis were increased in incidence and severity when compared with controls. There were no other non-neoplastic changes in the livers of 4-hydroxytamoxifen-treated animals which were attributable to treatment.
In the lungs, there was an increased incidence and severity of foamy alveolar macrophages, microgranulomas and/or cholesterol clefts in 4-hydroxytamoxifen-treated animals.
In the kidneys, the incidence of chronic tubulo-interstitial nephropathies, a common cause of mortality, was reduced by treatment with 4-hydroxytamoxifen (controls 28%, treated 14, 6 and 2%). In the adrenals, 4-hydroxytamoxifen-treated animals showed a reduced incidence and severity of vacuolated cortical cells, altered cell foci in the cortex (at 140 and 1000 µg/kg/day) and cortical nodular hyperplasia (at 20, 140 and 1000 µg/kg/day) when compared with the control animals.
Some other minor histopathological findings, including differences in the incidence of myeloid cell hyperplasia, bone marrow lipomatosis, extramedullary haemopoiesis in the spleen and plasmocytosis in the mandibular lymph nodes, were considered to be related to the lower incidence of eschars and resultant inflammatory changes found at the higher dose-levels.
Neoplastic lesions.
In 4-hydroxytamoxifen-treated animals there was a lower incidence of benign and malignant tumours. As shown in Table V, the number of animals with neoplasms, the number of animals with more than one primary neoplasm and the number of benign and malignant tumours were all lower in 4-hydroxytamoxifen-treated groups than controls. This difference was due to lower incidences of mammary gland tumours and pituitary adenomas (Table VI
). No tumours were observed in the mammary glands at the intermediate and high dose-levels of 4-hydroxytamoxifen. At the low dose-level, the incidence of benign (fibroadenoma, adenofibroma and adenoma) or malignant (adenocarcinoma and ductular carcinoma) mammary tumours was lower than that of control females. At the intermediate and high dose-levels of 4-hydroxytamoxifen the incidence of acidophilic, small or mixed cell adenomas of the pituitary gland was markedly lower than that in controls or low dose females.
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In the liver, hepatocellular adenoma was observed for 3/50 females given 20 µg/kg/day (6%) and for 1/50 females given 1000 µg/kg/day (2%), compared with none in the control animals. Since there was no dose relationship for the incidence of this tumour and the observed incidences fall within the range of historical data for our laboratory and that reported in the literature, this neoplasm was considered to bear no relation to treatment with 4-hydroxytamoxifen. No other liver tumours were noted in any 4-hydroxytamoxifen treatment group.
Some other neoplasms which are observed spontaneously in senile laboratory rats were noted with a sporadic incidence in this study and are detailed in Table VII.
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Discussion |
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4-hydroxytamoxifen is an active metabolite of tamoxifen found in the rat and man (7). In the present study, daily cutaneous application of 4-hydroxytamoxifen to female SpragueDawley rats over 101 weeks at a dose-level of 20, 140 or 1000 µg/kg/day did not result in evidence of a carcinogenic action at any site. These dose-levels resulted in marked pharmacological actions and a relatively severe loss of body weight gain over the course of the study. The terminal mean body weight values were 25, 32 and 38% lower than the combined control group value at the low, intermediate and high dose-levels, respectively.
Treatment with 4-hydroxytamoxifen resulted in a marked reduction in the number of palpable masses and at the mid and high dose-levels very few such masses were observed. On histopathological examination, there was a corresponding low incidence of benign and malignant tumours in 4-hydroxytamoxifen-treated animals, when compared with controls. This difference was due to lower incidences of mammary gland tumours and pituitary adenomas. No tumours were observed in the mammary glands at the intermediate and high dose-levels of 4-hydroxytamoxifen and the incidence of acidophilic, small or mixed cell adenomas of the pituitary gland was markedly lower than the controls. In addition, the incidence of preneoplastic lesions of the adrenals (foci of altered cells and nodular hyperplasia) was lower in treated animals than in controls.
This study provided no evidence of tumour induction in the liver, the target organ for tumour induction by tamoxifen in the rat, and the incidence of preneoplastic hepatic lesions (foci of altered cells and nodular hyperplasia) was lower in treated animals than in controls. Similarly, there was no evidence for the induction of tumours in the uterus or endometrium, the putative site of tumour induction by tamoxifen in women, and there was no evidence of a carcinogenic action of the skin, the site of application for the proposed clinical use.
It appears that the increased longevity of the intermediate and high dose-level animals may be attributed largely to the absence of mammary and pituitary tumours, since these tumours were among the major causes of death in control animals. In addition, the incidence of chronic tubulo-interstitial nephropathy, another cause of death in control animals, was reduced in 4-hydroxytamoxifen-treated animals. It cannot be excluded that the reduced body weight gain of 4-hydroxytamoxifen-treated animals may have contributed to the increased longevity through other mechanisms, since it is known that reduced body weight gain is associated with increased lifespan in rats.
Oestrogens are known to play a significant role in the development of pre-neoplastic and neoplastic lesions of the pituitary, mammary glands, adrenals and liver (16). It may therefore be proposed that the reduced incidence in tumours of these sites in 4-hydroxytamoxifen-treated animals can be attributed to its anti-oestrogenic properties. Plasma levels of oestradiol were not influenced by treatment with 4-hydroxytamoxifen and the tumour reducing action may therefore have been mediated directly by occupation of the receptor or by an antagonistic action at the receptor. The same mechanisms may be involved in explaining the lower incidence of nephropathies and the lower food consumption (17).
Although 4-hydroxytamoxifen produced pharmacological changes consistent with the action of the parent compound, tamoxifen, the non-pharmacological histopathological findings were not entirely consistent with those caused by tamoxifen. Expected changes resulting from the pharmacological action included reduced development of the mammary glands and atrophy of the ovaries and uterus. Changes which could not be attributed to the pharmacological action of 4-hydroxytamoxifen included pulmonary lipidosis, which has been reported previously for tamoxifen (18). Biliary proliferation and peribiliary fibrosis were observed and have not been described previously in rats after chronic treatment with tamoxifen; the bile is the principal route of elimination of 4-hydroxytamoxifen (unpublished data). The reduced incidence of renal tubulo-interstitial nephropathy was reported previously for rats treated with tamoxifen (5). Some changes which have been associated with treatment of rats with tamoxifen were not observed in this study (or in the previous general toxicology studies we have performed with 4-hydroxytamoxifen; unpublished observations): these include alopecia, lenticular degeneration and the induction of cataracts.
The hepatocarcinogenic action of tamoxifen is believed to be mediated by genotoxic mechanisms (14). It has also been shown that 4-hydroxytamoxifen can be activated to reactive intermediates capable of forming adducts with DNA both in vitro and in vivo in the rat (19). Accordingly, in the present study it was of interest to determine whether chronic treatment with 4-hydroxytamoxifen resulted in damage to DNA. Consistent with absence of a hepatocarcinogenic action of 4-hydroxytamoxifen, there was no evidence of increased DNA adduct formation (when compared with control animals) in the livers of 4-hydroxytamoxifen-treated animals (E.A.Martin and J.A.Styles, unpublished observations).
In this study, the high dose-level of 4-hydroxytamoxifen resulted in a reduction in body weight gain and in plasma drug levels which were 60-fold greater than those found in clinical use. Hence, the study fulfils regulatory criteria for the design, dose selection and performance of carcinogenicity bioassays and demonstrates the lack of carcinogenicity of 4-hydroxytamoxifen when administered by the cutaneous route.
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Acknowledgments |
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Notes |
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3 Biologie Servier, BP 255, Gidy, 45403 Fleury les Aubrais, France
4 To whom correspondence should be addressed Email: roy_forster{at}compuserve.com
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References |
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