Animal toxicity studies performed for risk assessment of the once-a-month injectable contraceptive Mesigyna®

Animal toxicity studies performed for risk assessment of the once-a-month injectable contraceptive Mesigyna@ B. Seibert and I? Giinzel Institute

for Experimental

Toxicology,

Schering AG, Berlin, Germany

Results from toxicity studies performed for risk assessment of the combined injectable hormonal preparation Mesigyna@ are reviewed. Both components of Mesigynam, i.e., estradiol valerate (E,Val) and norethisterone enanthate (NET-EN), have been thoroughly investigated as individual compounds and some limited toxicity data have been obtained for the combination. Most findings which were gathered in these studies from different animal species occurred in the gonads, accessory genital and endocrine organs and can be related to the known species-specific pharmacological activity of a typical estrogen or progestin, respectively. No additional or unexpected information which might indicate a possible estrogenlprogestin interaction was gained from the administration of the combined preparation to animals. Based on the results from toxicity testing, there were no objections to the long-term therapeutic use of Mesigyna@ for hormonal contraception. The predictive value of the effects (including the tumorigenicity) observed in the common laboratory animals with regard to human safety is critically discussed, taking the vast amount of previous experience with hormonal contraceptives into consideration. The conclusion is drawn that there is no animal model for safety assessment of sex steroids that adequately represents the human situation. Quantitative extrapolations from animal toxicity findings to humans, therefore, are not possible. Especially, the value of long-term studies and of toxicity studies on estrogenlprogestin combinations is put into question. Like endocrine pharmacology studies, the toxicity studies with these steroid hormones are useful for the characterization of the possible endocrine pharmacological profile only. Keywords: Monthly combined injectable contraception, Mesigyna, valerate, Norethisterone enanthate, Toxicology, Animal pharmacokinetics, Tumorigenicity, Mutagenicity.

0 1994 Butterworth-Heinemann

Contraception

1994:49,

Estradiol

April

303

Toxicology of Mesigyna:

Seibert and Giinzel

Introduction Mesigyna@ is a monthly injectable hormonal contraceptive for women. The two pharmacologically active components of Mesigyna@, E,Val (estradiol valerate) and NET-EN (norethisterone enanthate), are esters of steroid hormones and fatty acids (see Figure 1). The esterification of a steroid with fatty acids of varying length changes the lipophilic properties of the former and results in an effectively controlled release from a depot after intramuscular injection as an oily solution (in castor oil:benzyl benzoate, 6:4). In vivo, these steroidal esters are split by unspecific esterases into the steroid hormone alcohol and the respective fatty acid. Therefore, E,Val and NET-EN display the pharmacological activity profile of 17P-estradiol and norethisterone, respectively. The free fatty acids, valeric acid and heptanoic acid undergo p-oxidation and are eliminated as water and carbon dioxide. Up to the present, extensive human experience has been gathered for both components of Mesigyna@ used either individually or in combination products. E,Val has been used for several years either as monopreparation or as a component with a progestin, for the oral or parenteral treatment of climacteric disorders. Norethisterone (NET), in the form of the acetate ester (NET-AC) and in the free form, has now been utilized for 25-30 years as the progestin component in a series of oral contraceptives. Its enanthate ester, which is the form contained in Mesigyna @‘, has already been in widespread use as a long-acting progestin-only injectable preparation for hormonal contraception (Noristeratem). The development of Mesigyna@ as a combined once-a-month injectable hormonal contraceptive was initiated because the addition of estrogens to long-acting progestins has proven to be a successful strategy for over-

EZVal

FIGURE 1. Structural formula of estradiol valerate EN). E,Val NET-EN

304

NET-EN

(E,Val) and norethisterone

Contraception

enanthate

(NET-

1994:49, April

Toxicology

of Mesigyna:

Seibert and Giinzel

coming the endometrial bleeding problems associated with the use of long-acting progestin-only contraceptives. Human studies using either different total doses of E,Val and NET-EN and altered estrogen/progestin ratios have confirmed that the finally chosen composition and content of Mesigyna@ affords effective ovulation inhibition with optimum cycle control and is well tolerated.’

Prerequisites for the Evaluation of Toxicity Studies and Human Risk Assessment Pharmacodynamics During the early developmental phase of Mesigyna@, the pharmacodynamic profile of each of its components was assessed in relevant animal models. The estrogenic actions of E,Val were experimentally determined in mice and rats by its ability to stimulate endometrial proliferation and uterine growth, to provoke premature vaginal opening and to decrease the sialic acid production in the vagina. Further, E,Val had antigonadotropic effects as indicated by its ability to inhibit ovulation or testis growth. NET-EN proved in similar experiments also to have antigonadotropic effects. Furthermore, it has progestogenic effects as measured by its ability to transform the endometrium from the proliferative into the secretory phase and to maintain pregnancy in ovariectomized rats. However, in rats and mice, NET-EN also had inherent estrogenic effects (i.e., stimulation of vaginal epithelium proliferation], which could even outweigh the progestogenic effects if very high doses were applied. In humans, up to now, no partial estrogenic effects of NET-EN were noted. Since the pharmacological properties were investigated separately for E,Val and NET-EN, respectively, the possibility of interactions had to be assessed for the planned simultaneous use of the two compounds. This question was investigated by in vitro receptor binding studies. Steroid hormones are thought to act on their target tissues by binding to cellular receptor molecules. As a consequence, cellular events may be stimulated, leading to alterations of cellular function. The competitive binding of E,Val and NET-EN as well as of the respective unesterified steroids, estradiol and norethisterone, to the estrogen- (uterus rat] or progestin- (uterus rabbit, hamster) receptor was examined. The results showed first, that no receptor binding of the esterified parent compounds occurred and second, that no interaction between the respective free steroids is expected at the receptor level. Pharmacokinetics A comparison of characterisic pharmacokinetic parameters of the free steroid 17l3-estradiol in different species after injection of E,Val is com-

Contraception

1994:49, April

305

Toxicology

of Mesigyna:

Seibert and Giinzel

piled in Table 1. A number of differences between the species were noted. In dogs, E,Val is released from the depot faster and its main portion is eliminated faster than in humans. Due to the higher rate of metabolism in the dog, far smaller areas under the plasma concentration curves are reached in this species when compared to the human. Moreover, particularly in rats, but also in dogs, estradiol metabolites are eliminated predominantly via the liver, whereas in humans most metabolites are eliminated via the kidneys. It has been shown in laboratory animals and humans that, following intramuscular injection, E,Val is completely released from the oily solution at the site of administration. The steroid ester is then split by enzymatic hydrolysis into 17p-estradiol and the fatty acid. This can take place not only in the liver but also in the blood and tissues. The 17p-estradiol arising in vivo from E,Val behaves in the organism like endogenous 17pestradiol and is subject to intermediate metabolism. It was apparent from comparisons of the areas under the curves representing the estradiol levels in plasma following i.v. or i.m. administration of E2Val, that 17p-estradiol becomes completely available in humans and to more than 90% available in dogs after intramuscular administration. The characteristic pharmacokinetic parameters of the progestagenic component of Mesigyna @, NET-EN, after i.m. injection in rats, dogs, monkeys and humans are compiled in Table 2. As described for estradiol, it can be seen that the areas under the plasma concentration curves of the free steroid norethisterone are also smaller in all animals tested than in humans due to a higher metabolic clearance rate in the experimental

TABLE 1. Comparative and humans*

pharmacokinetics

of estradiol after i.m. injection of E,Val in rats, dogs

Species Pharmacokinetic

parameter

C max (ng/ml) t max (d) AUC (ng x d / ml) t ‘/i a (h) t ‘h p (h) bioavailability (%) Ratio of elimination

(feces: urine)

Rats’

Dogs

1.5 1 .o 3.52 nd. 11 n.d.

2.7 1 .o 11.5 2 8 >90

5:l

2:l

Humans 5-7 2 49.6 4-5 n.d. 100 1:4

lAUC (area under the plasma concentration over time curve) and C max corrected for a dose of 1 mg/kg E,Val i.m. n.d. = Values could not be determined. Values for rats determined after combined administration of 0.02 mg E,Val + 2 mg NET-EN/kg i.m. ZCorrected for naturally occurring E, areas

306

Contraception

1994:49, April

Toxicology of Mesigyna:

Seibert and Giinzel

TABLE

2. Comparative pharmacokinetics of norethisterone after i.m. injection of NET-EN in rats, dogs, monkeys and humans, corrected for a dose of 1 mg/kg NET-EN i.m.

Soecies Pharmacokinetic

parameter

C max (nglml) t max (d) AUC (ng x d / ml) t % a (d) t % 6 (d) bioavailability (%) Ratio of elimination

(feces:urine)

Rats’

Dogs

Monkeys

Humans

0.95 1 10 nd. 13 nd.

3.7 4 47 6 18 45

0.81 3-7 21 n.d. 12-20 20

2-5 3-10 100 4-5 15-20 100

4:l

1:2

1.5:1

1.5:1

n.d. = Values could not be determined. ‘Values for rats determined after combined administration of 0.02 mg E,Val + 2 mg NET-EN/kg i.m.

animals. Furthermore, it was shown that norethisterone is eliminated mainly via the liver in rats, mainly via the kidneys in dogs, whereas in monkeys and humans, both elimination routes are of the same order. In all species tested, including humans, the ester NET-EN is completely released from the oily solution, whereby the release rate differs markedly from species to species and is slowest in rats. From the comparisons of the areas under the curves representing the norethisterone levels in plasma following subcutaneous administration of free norethisterone or i.m. administration of the ester NET-EN, it could be shown that the bioavailability of norethisterone in dogs after i.m. injection is 45% and about 20% in monkeys. The rest was probably metabolized and eliminated via urine and feces without ester cleavage. In humans, however, the bioavailability of norethisterone was about 100% and the hydrolysis of the ester NET-EN by non-specific esterases was complete. In rats, the ester cleavage is even far quicker than in humans, indicated by the fact that no measurable amount of the unsplit ester NETEN could be detected in this species in contrast to humans. In all species tested, the elimination rate of NET is more dependent on the release rate of NET-EN from the depot than on the administered doses. With regard to the injection intervals used in toxicity studies, it is important to consider that in rats, due to the slow release of NET-EN from the depot, cumulation factors of 3.5, 1.5 or 1.3 were determined by analog computer simulation for intervals of 2, 8 or 12 weeks, respectively. These cumulation rates in the rat were not dependent on the doses tested. No cumulation was found in the monkey or was anticipated in the dog during the dosing intervals used in toxicity studies. In humans, no cumula-

Contraception 1994149,

April

307

Toxicology

of Mesigyna:

Seibert and Giinzel

tion of norethisterone was noted after repeated dosages of 6 mg/kg at injection intervals of 8 or 12 weeks. From the species differences in the pharmacokinetics of NET-EN, differences in the response of the target organs towards the compound have also to be expected. In rats, e.g. the relatively high cumulation rate of norethisterone and, in addition, the fact that norethisterone in this species is mainly eliminated via the liver indicates that an overburden of this organ is more likely in this species than in humans. For the characterisation of Mesigyna @, it is also essential to analyse the possibility of a pharmacokinetic interaction between the two components E,Val and NET-EN when administered simultaneously as an oily solution. Table 3 compiles comparative pharmacokinetic data of estradiol and norethisterone after individual versus combined i. m. administration. No interaction between both compounds occurs in humans. For both estradiol and norethisterone, the respective pharmacokinetic data are in good agreement and it can be concluded that neither the release rate of compounds from the oily depot nor their systemic availability are influenced by the presence of the respective other compound. As far as the situation in rats is concerned, studies similar to those in humans are not available. However, the figures calculated from fitted curves derived from experiments with different doses do not suggest an influence of E,Val on the release and availability of NET-EN and vice versa. Theoretically, a mutual interaction of both compounds cannot be expected because the compounds are slowly released from the depot and only low amounts per unit of time become systemically available. Therefore, saturation

parameters of E,Val and NET-EN given as TABLE 3. Comparison of some pharmacokinetic single compounds or in combination to humans or rats; the dose-dependent parameters (Cmax, AUC) are corrected for the dose of 1 mg/kg; all values are calculated for the respective free steroids, estradiol (E2) and norethisterone (NET)

Combination

E,

NET

E2

NET

Humans

Cmax (ng/ml) tmax (day) AUC (ng x h/ml)

5-7 2 1190

2-5 3-l 0 2400

4.8 16 860

3.6 3-12 1670

Rats

Cmax (nglml) tmax (day) AUC (ng x h/ml)

1-2’ -

0.2 (7th day) 3-10 90

1.5 1 85

0.45’

- = Data not available. Calculated concentration on day 7.

308

Single Compounds

Pharmacokinetic Parameter

Species

239

from excretion data. 2Taken from fitted curves, calculated

Contraception

1994:49, April

Toxicology

of Mesigyna:

Seibert

and Giinzel

processes in metabolism will not occur. On the other hand, the rate of release is determined predominantly by the physicochemical properties of the compounds (solubility, lipophilicity) and, therefore, is independent of the presence of the other compound.

Species Differences and the Predictive Value of Animal Toxicity Data for Human Risk Assessment The comparative summary of characteristic pharmacokinetic parameters given above for E,Val and NET-EN indicates profound differences between the commonly used laboratory animals and the human. Generally, as can be seen from comparison of the areas under the plasma concentration curves, the systemic exposure to either of the steroids is always lower in animal species than in humans if equal doses on the basis of body weight are injected. Therefore, one might conclude that, accordingly, higher doses would have to be administered to laboratory animals in order to achieve an at least similar biological effect-provided the sensitivity of the animal species was also similar. A special peculiarity in the testing of exogenously administered steroida1 estrogens and progestins is the fact that in every mammalian organism, they encounter target organs, feedback mechanisms and endocrine control systems which are already controlled by endogenously produced hormones. A comprehensive comparative analysis of these factors in laboratory animals and in humans with regard to human risk assessment in the field of steroidal sex hormones has been published previously.2 Therefore, only a few basic points relevant for the interpretation of the toxicity data obtained for Mesigyna@ will be summarized here. With regard to the interpretation of effects after administration of exogenous sex steroids, the knowledge of the endogenously occurring hormone levels in each species during non-pregnant reproductive cycles and during pregnancy can give a good estimation as to what extent variations in plasma levels occur under physiological conditions, i.e., it would suggest the limits of the hormone levels normally tolerated in each species. From the compilation in Table 4, it can be seen that estradiol levels during the normal reproductive cycle and especially during pregnancy show species differences with lower values and smaller periodic rises in all animal species as compared to women. Thus, from normal endocrine physiology, it may be concluded that the human organism is able to handle much higher levels of estradiol properly than the common laboratory animal species. It also indicates that the rat and dog which were used for toxicity studies with E,Val, the estrogenic component of Mesigyna@, might be expected to be very sensitive towards exogenous administration of estradiol. Also, the comparison of endogenous progesterone levels during the

Contraception

1994~49, April

309

Toxicology

of Mesigyna:

Seibert and Giinzel

TABLE 4. Comparison of estradiol plasma levels between animal species and humans throughout normal reproductive cycles and pregnancy

Plasma Levels of 176-Estradiol Cycle Phase Early Middle Late Pregnancy

(pg/ml)

Mouse

Rat

Dog

Monkey

23 n.d. 4-39

30-80 1.2-25 2.3-30

n.d. 12-18 23-57

42-80 69-500 18-l 00

16 20 30

600 500 n.d.

20 24 8

170 550 550

Human 2-l 20 80-650 20-250

phase

Early Middle Late

1000-7000 6000-l 2000 12000-29000

n.d. = No data.

normal reproductive cycle and during pregnancy indicates species differences of normal physiology, although not as extreme as observed with estradiol (see Table 5). It is particularly interesting to note that the monkeys regulate pregnancy at a significantly lower progesterone level than all the other species, while in humans there is a striking increase in progesterone levels during the last third of pregnancy. This might indicate that the administration of human doses of progestins (or even multiples thereof) during toxicity tests on the basis of mg/kg body weight, especially in the monkey, would exceed by far the amount which this species is accustomed to handle. For contraceptives containing estrogen/progestin combinations, it is still recommended by health authorities to perform animal toxicity tests

TABLE

5. Comparison of progesterone plasma levels between throughout normal reproductive cycles and pregnancy

animal species and humans

Plasma levels of oroaesterone Cycle Phase Early Middle Late Pregnancy Early Middle Late

Mouse

Rat

Dog

2-7 15 5-27

35-40 3-11 5-30

20-40 15-60 80

60-87 loo-118 5-20

&ra/ml)

Monkey

Human

n.d. l-5 23-35

51.2 2-4 0.5-l 1

51.1 ‘<8-l 0
20 50 10

l-3 2-5 2-5

25 30 120-250

phase

n.d. = No data

310

Contraception

1994:49, April

Toxicology

Seibert and Giinzel

of Mesigyna:

on the combination. And it is advised that the same estrogemprogestin ratio should be used as in the preparation which is developed to be marketed.3 Therefore, it is of interest to compare the physiological estrogen/ progestin ratio usually encountered in test species with the human situation. The compilation in Table 6 shows that the estrogen/progestin ratio is far more extended in mice, rats and dogs when compared to monkeys and humans, especially during pregnancy. From these data, it is quite obvious that the exogenous administration of steroid combinations to animals which were selected on the basis of an optimum pharmacological response in humans does not yield predictive data for human risk assessment. This also holds true for results obtained in the monkey, as could be conceived from differences in the range of absolute endogenous hormone levels which this species is accustomed to handle. The problem of interspecies extrapolation for combined preparations is additionally complicated by the fact that either of the components has independent speciesspecific pharmacokinetic features and is subject to plasma protein-binding which usually does not parallel the human situation. Further profound species differences were reported with regard to the sensitivity of control systems and target organ reactions. For example, the absolute doses and ratios of progesterone and estradiol required for optimum transformation of the endometrium shows a trend to reflect similar relationships in each species, as noted in the pattern of endogenous estradiol and progesterone levels described above. However, if doses are compared between the rat and the human which are necessary for inhibition of ovulation, it becomes apparent that there is a substance-specific as well as a species-specific difference in the sensitivity of the ovulation inhibiting mechanism. Thus, in rats, the absolute doses necessary for this mechanism can be more than two orders of magni-

TABLE 6. Estradiollprogesterone pregnancy (Estradiol 9 1)

ratio in plasma throughout

normal reproductive

cycles and

Species Cycle Phase

Mouse

Early Middle Late

1:85 nd. 1:1200

Pregnancy

Rat n.d. 1:180 1: 1200-5000

Dog

Monkey

Human

n.d. 1:55-833 1:384-l 333

1:3-12 1:4-57 1:50-600

1:2-53 1:15-125 16-600

1:lOOO 1:3600 1:1300

1:24 1:7 1:7

phase

Early Middle Late

1:1900 1:2000 1:2700

1:122 1:218 n.d.

1:6 1:3 117

n.d. = No data.

Contraception

1994:49, April

311

Toxicology

of Mesigyna:

Seibert

and Giinzel

tude higher than in humans. If the areas under the plasma concentration curves are compared, they may be in approximately the same range in rats and humans (e.g., levonorgestrel, gestodene), or it may differ by one (norethisterone) or two (cyproterone acetate) orders of magnitude. The results of chronic toxicity studies performed with E,Val and NETEN provide further examples for the species-specific sensitivity of some target organs and feedback mechanisms; these are discussed below.

3. Toxicity Studies Performed Mesigyna@

for Risk Assessment

of

Introduction The toxicological studies were carried out on rats (Wistar, Charles River CD, Sprague-Dawley), mice (NMRI, Charles River CD), rabbits (White New Zealand), dogs (Beagle) and monkeys (Rhesus) from various breeders. The animals were kept in individual cages or up to 5 animals (mice and rats) per cage under conventional conditions in air-conditioned rooms with controlled lighting (12-hour day/l2-hour night rhythm). The animals were given tap water to drink and were fed with the appropriate standard diet. For the sake of clarity, the experimental results are presented separately for the studies performed on the individual compounds (E,Val, NET-EN) and the combination, respectively. In each section, the specific study details are presented before the corresponding results. Furthermore, the salient findings are briefly discussed.

Toxicity

Studies

Performed

with E,Val

Single Dose Toxicity. Acute toxicity tests with E,Val revealed a low toxicity after administration of single high doses as is generally kown for steroid hormones.4 The approximate values for the acute toxicity after a single dose (LD5,,) of E,Val in rats and mice were r 4 g/kg after oral or subcutaneous application. In acute toxicity studies in dogs, the LDSo values were >l.O g/kg after oral and >0.25 g/kg after i.m. application. These results confirm the wide safety margin between acute adverse effects of E,Val in animal species and the therapeutic dose which is administered to humans. Therefore, no severe risk due to accidental overdosage needs to be assumed. Repeated Dose Toxicity and Tumorigenicity. Rodent studies-The findings observed in rats after repeated oral application of E,Val are mainly based on a 90-week study (interim sacrifice at 26 weeks). Some additional data on food intake and body weight changes result from a 40-day rat study in which the effects of three different routes of oral application

312

Contraception

1994:49,

April

Toxicology

of Mesigyna:

Seibert and Giinzel

(intragastric, sublingual and by month) were compared. The results of intramuscular application of E,Val were obtained from a 28-week study in rats during which E,Val was administered either alone (4 mg/kg every 3 weeks) or in combination with dehydroepiandrosterone enanthate (DHE) in a ratio of 5O:l (DHE:E,Val). Details on the design of the studies are summarized in Table 7. Even if the application of Mesigyna@ in humans is an intramuscular injection, the results on the systemic tolerance after oral application of E,Val to rats are equally relevant since it was shown that the areas under the plasma concentration curves (AUC) were of about a similar order after the highest doses used in these studies for either route of administration. Generally, all findings noted after repeated dosages of E,Val in rats (see Table 8 for a compilation) including those which were related to tumorigenicity were to be expected, as they represent well known effects of systemically effective estrogens in these species and have therefore been described before.4-6 Moreover, most of them are exaggerated pharmacological rather than toxic effects. Selected findings have been discussed in detail, particularly in light of their relevance for human risk evaluation. The decrease of body weight gain and food intake in rats is a well known effect of estrogens in rodents, the mechanism of which is not yet fully understood. However, it has no analogue in the human since generally, e.g. after the use of oral contraceptives, body weight increase is more

TABLE 7.

Experimental

design of repeated dose studies in rats treated with E,Val

Dose (mg/kg) and Application Interval

Animal Number Per Group and Sex

Route of Application

30 Ml50 F’

p.o. (diet)

10 10 10 30

intragastric sublingual p.0. i.m.

M M3 M M/30 F

E,Val 0 1.2lday 3.6Iday 12.0Iday 1.2lday 1.2Iday 1 Zday 0.24 0.72 4.0 4.0

DHE2

Duration of Treatment 90 weeks

40 days

12.0 36.0 200.0 -

25-28 weeks

/every 3 weeks ‘10 M/20 F interim sacrifice after 26 weeks. *DHE = dehydroepiandrosterone enanthate. %omparison different routes of oral application forms-only food intake and body weights were determined.

Contraception

1994:49,

April

of three

313

Toxicology of Mesigyna: TABLE

8.

Seibert and Giinzel

Salient findings observed

in rats after repeated oral and/or intramuscular

treatment

with E,Val

Clinical observations:

1 food intake and body weight gain T mortality’ t alopecia’ ?’ incidence of dullness of the lens’ 1‘ diuresis (high dose females only)’

Hematology:

1 hematocrit, hemoglobin and erythrocyte count 1‘ neutrophilic granulocytes and macrophages, lymphopenia’ 1 thrombocyte and leucocyte counts2

Organ weights:

1 ovaries 1 testes, prostrate, T pituitary

Histopathology:

preputial gland and musculus levator ani

-atrophy of the ovaries (inhibition of the maturation of follicles and corpora lutea, concurrent stromal hyperplasia) -atrophy of the glandular tissue of the uterus with concurrent signs of hypertrophy of the fibromuscular tissue and partial squamous cell metaplasia -atrophy of the testes (inhibition of spermatogenesis) -atrophy of the epididymis, glandular epithelium of the seminal vesicle and prostrate (concurrent hyperplasia of the fibromuscular tissue elements) -stimulation of mammary ductular growth (specifically at the high dose level of 12 mg/kg/day p.0.) with duct dilation, cyst formation and epithelial hyperplasia -earlier appearance of mammary tumors (adenomas, fibroadenomas, carcinomas) in both sexes and an increased incidence in mid dose males’ -pituitary hyperplasia and/or hypertrophy of the chromophobe cells of the anterior lobe -increased incidence of pituitary adenomas -focal atrophy of the fascicular zone of the adrenals and hypertrophy of the reticular zone (from 3.6 mg/kg/day onwards) -hemosiderosis of the spleen and the kidneys* -focal atrophy of erythropoetic’foci of the bone marrow2

‘only observed after oral administration 2only observed after intramuscular administration 1‘ = increase. 1 = decrease

often reported than decrease in relation to the intake of estrogens or estrogen-progestin combinations. The same observation holds true for the hematological disturbances which lead to a distinct anemia in rats (and also in the dog, see below). For more than 25 years, it has been an established fact that estrogens affect the hemopoietic organs of rodents. 7-9 As in our studies performed

314

Contraception

1994:49, April

Toxicology

of Mesigyna:

Seibert

and Giinzel

with E,Val, anemia was observed after estradiol treatment in rats and mice and could be experimentally related to depressed erythropoietin production. In these experiments, erythropoietin synthesis was stimulated in the animals by hypoxia. From such experiments, either a direct inhibition of erythropoietin action on stem cells? or a mechanism via suppression of the production of an extrarenal precursor of the erythropoiesis stimulating factor9 was suggested. In humans, neither severe anemia nor effects on the myelopoiesis have been described after estrogen treatment. In contrast, a slight increase in hematocrit was observed in one study with postmenopausal women treated with estradiol valeratelO and hormonal contraceptives are generally considered to protect against iron deficiency anaemia due to reduced menstrual blood 10ss.l~ Hair loss is a characteristic sign after treatment with estrogens in the rat6 and is often associated with atrophy of all integumental structures, epidermis, hair follicles and glands.5 Similar changes in humans are not known after estradiol valerate use. Rather, androgens are considered to play a part in the occurrence of human female baldness, usually in combination with genetic factors and exogenous damage to the hair follicles.12 Pharmacologically, the main effect to be expected from E,Val administration, the active hormone of which is the naturally occurring 17@-estradiol, is of course the estrogenic effect. The consequences of estrogenicity are growth stimulation via estrogen-dependent and/or -sensitive organs and influence on regulatory mechanisms. One of these consequent effects is the antigonadotropic effect with the consequence of involution of gonadotropin-dependent tissue. Therefore, the observed changes in organ weights and the histopathology of testes, prostate, preputial gland in male rats or of the ovaries, uterus and vagina in females are considered as direct pharmacological effects of E,Val and have been described earlier for this species after administration of estrogens.5s6 An increased incidence of hyperplastic changes including adenomas of the anterior pituitary gland was noted both in male and female rats as compared with controls, even if these controls had a high spontaneous rate of such alterations (20% in males and about 50% in females after a 90-week observation period). These findings are consistent with many other reports on the stimulating effect of systemically effective estrogens in rodents on the prolactin-secreting pituitary cells.2,5J3 In mice and rats, a positive feedback mechanism between estrogens and prolactin is known: Estrogens inhibit at least one of the prolactin-inhibiting factors (dopamine) and stimulate directly the prolactin synthesis of the anterior pituitary gland cells. Thus, they enhance prolactin secretion in two different ways and, therefore, can cause adaptive hyperplasia and eventually adenomas of the pituitary gland.14 Prolactin, in turn, is the important luteotropic and mammotropic hormone in rodents and is thus able to induce mammary activity and mammary growth stimulation and eventually tumors.2J5 The

Contraception

1994:49,

April

315

Toxicology

of Mesigyna:

Seibert and Giinzel

incidence of mammary gland adenomas, however, was only increased in male rats at mid dose level (3.6 mg/kg/d p.o.), whereas an earlier appearance of these tumors was noted in both sexes and all treatment groups after oral administration of E,Val. Moreover, female rats treated with E,Val had some mammary gland carcinomas (4 at the mid, 3 at the high dose level) which were not observed in the control or male rats. Even if, in individual cases, it was suggested that estrogens either contained in contraceptives or used for other therapeutic reasons might provoke the clinical expression of otherwise clinically silent tumors’7 or even induce adenoma growth of the pituitary, if administered at extremely high dosages, I8 in healthy women, an association between the incidence of pituitary adenomas and the use of sex steroids, as in oral contraceptives, could not hitherto be supported by epidemiological studies.16 For the preparation Mesigyna@, it was shown in humans that after chronic administration for more than one year, only a very marginal and transient increase in serum prolactin levels was noted after the injection and it was concluded that prolonged use of this preparation does not affect the baseline prolactin secretion in women.19 Therefore, in contrast to the rat studies, no comparable permanent stimulation on the pituitary prolactin cells is found in women after application of therapeutic doses of E,Val. Also with regard to the mammary tumors frequently observed in rodents after treatment with estrogens, there is no conclusive evidence from epidemiological studies that this finding bears a relation to the development of breast tumors in women. While there is a general consensus in the literature that the use of sex steroids as combined oral contraceptives does not impose a risk to mature women, there is still some concern expressed with regard to the effects of early use before a first-term pregnancy.llmzOTo the best of our knowledge, these doubts are not well founded. Because of discrepancies noted in available data up to the present, further epidemiological investigations are needed before definitive conclusions can be drawn. Non-rodent studies--Systemic tolerance of E,Val after repeated administration was assessed in dogs on the basis of a 52-week study (with an interim sacrifice at 20 weeks) at oral dosages of 1.2, 3.6 and 12.0 mg/kg/ day and two studies with repeated intramuscular injections. In one of the latter two studies, the compound was tested either alone (2.4 mg/kg once a week for 4 weeks and every 3 weeks thereafter) or in combination with DHE in a ratio of 150 over a period of 54 weeks. In the second study, the effects of E,Val were compared with those of 17B-estradiol after a treatment period of 65 weeks. Details of the design of these studies are summarized in Table 9. Among the changes seen after repeated treatment of dogs with E,Val (see Table lo), the severe alterations in peripheral blood cells and in the

316

Contraception

1994:49, April

Toxicology TABLE 9.

Experimental

Animal Number Per Group and Sex

Seibert and Giinzel

of Mesigyna:

design of repeated dose studies in dogs treated with E,Val

Dose (mg/kg) and Application Interval Route of Application

DHE2

E,Val

5 Ml5 F’

p.0. (gelatine capsules)

0 1.2lday 3.6lday 12.0lday

3 M/3 F

i.m.

0.24 0.72 2.4 2.4

Duration of Treatment 52 weeks

-

54 weeks

12.0 36.0 120.0 -

/once a week for 1 month, every 3 weeks thereafter 0 2.41 every 3 weeks3

65 weeks

‘2 M/2 F interim sacrifice after 12 weeks. 2DHE = Dehydroepiandrosterone in comparison with the effects of 176estradiol.

enanthate. 3This study was performed

i.m.

6F

bone marrow are a common finding in this species after administration of estrogens.5 Regardless of whether they are treated with naturally occurring estrogens or synthetic estrogenic compounds such as diethylstilbestrol, dogs develop a decrease in erythrocytes, reticulocytes and particularly thrombocytes as well as granulocytopenia, often after initial leucocytosis, in the peripheral blood accompanied by severe alterations to the bone marrow which can ultimately result in complete anaplasia as was observed in one dog of the above studies. Prolonged bleeding time, general hemorrhagic diathesis and increased rate of infections can therefore be considered as secondary effects due to the bone marrow damage caused by estrogens in dogs. Although anemia is also a common finding in rats, the pronounced sensitivity of the dog and the underlying mechanism for this finding is considered to be unique for this species2* and has not been described for humans. The increase in serum levels of cholesterol and glucose has not been described earlier as an estrogenic effect5 but rather is a common finding in this species after treatment with progestins.22 In general, the results of toxicity studies with sex steroids and their effect on lipidand carbohydrate metabolism have been very inconsistent and cannot be considered as predictive for human risk evaluation.4 All changes of gonads and accessory sex organs observed in the present study have been described before in this species after treatment with

Contraception

1994:49, April

317

Toxicology

of Mesigyna: TABLE 10.

Seibert and Giinzel

Salient findings observed in dogs after repeated oral and/or intramuscular

treatment

with E,Val

Clinical observations:

7 mortality (mostly due to secondary infections of the urogenital system but also two cases of anaplastic anemia after 12 mg/kg/d p.o.) L food intake and body weight gain (slight and inconsistent) -vomiting, diarrhea* -loss of hair -hemorrhages of different tissues, blood in the urine and feces2 -drop in blood pressure’ -swelling of the anal glands and vulva or preputial glands -suppression of estrus’

Hematology/Biochemistry:

? serum glucose and serum cholesterol2 -anemia t coagulation time and maximum amplitude thromboelastogramz 1 thrombocyte and leucocyte counts

in the

Organ weights:

1 ovaries or testes 7 uterus or prostrate

Histopathology:

-atrophy of ovarian follicles with concurrent interstitial fibrosis -hyperplasia of rete ovariP -increased incidence of ovarian carcinomas* -hypertrophy and fibrosis of the uterine myometrium -thickening and fibrosis of the uterine endometrium, atrophy of endometrial glands, endometritis, pyometra -stimulation’ or papillary hyperplasia* of the mesothelial layer of the ovaries, uterus and pelvis -atrophy of testicular germ cells with suppression of gametogenesis and concurrent interstitial fibrosis -enlargement of the prostrate with cysts -squamous cell metaplasia, occasionally with cornification of the prostate, chronic inflammation’ -stimulation of mammary growth with cystic dilatation2 and secretory activity -increase in number and size of pituitary prolactin cells and atrophy of gonadotropic cells2 -depletion of lipid content of the adrenals and nodular hyperplasia of the cortex2 -aplasia or hypoplasia of the bone marrow (3 high dose animals)’

‘only observed after oral administration *only observed after intramuscular administration f = increase, 1 = decrease

318

Contraception

1994:49, April

Toxicology

of Mesigyna:

Seibert and Giinzel

estrogens, including the tumorigenic response observed in the ovaries.5 This tumorigenic response clearly bears no relevance to humans since numerous epidemiologic studies have uniformly demonstrated a protective effect of hormonal contraceptives against ovarian epithelial cancer in women.llJo As in the present studies with E2Val, other investigators also did not find an increased incidence of pituitary adenomas in dogs due to estrogens.5 However, a stimulating effect on prolactin cells combined with a marked inhibitory effect on gonadotropic cells was observed23 which is far more distinct after prolonged progestin treatment than after estrogens in dogs. The stimulatory effect of E,Val on mammary growth in dogs has also been described earlier for this species after administration of various estrogens including 17P-estradiol .5,24 The underlying mechanism of this finding might either be a direct effect on the mammary tissue and/or an indirect effect on endocrine regulation (i.e., through the stimulation of prolactin cells). Reproduction toxicity-Embryotoxicity studies with E,Val were performed on rats and rabbits using daily subcutaneous (rats) or intramuscular (rabbit) administration during the period of organogenesis. The results did not indicate teratogenic effects on non-genital organs even at partly embryolethal doses. Embryolethality, on the other hand, is a known effect of many sex steroids in rodents and rabbits. Even if the precise mechanism of this is not yet understood, it might be related to the severe disturbance of the hormonal balance in these species which regulate their pregnancy at far lower basal estrogen levels and at a different estrogen:progesterone ratio than humans.2 With E2Val, a mild feminisation of male fetuses was reported in mice at extremely high dosages (1 mg/animal S.C. or 10 mg/animal p.o.) which are not relevant for humans, particularly if the basal 17p-estradiol levels in the different species are considered. In the test animals used, these levels are 2 to 3 orders of magnitude lower during pregnancy than in humans. In humans, these levels range during pregnancy from about 5 ng/ml (early stage) to 20 ng/ml E, (late period).2 After i.m. application of Mesigyna@ to human volunteers, maximum concentrations of up to 1.0 ng/ml estradiol were determined. In addition to the aforementioned studies, the embryotoxicity of E,Val was also investigated in monkeys. The compound was administered intramuscularly at weekly intervals in combination with hydroxyprogesterone caproate (in a ratio E,Val:hydroxyprogesterone caproate of 150) because of the therapeutic use of this combination during embryonic and fetal development. No effect on prenatal development was observed from 0.002 to 0.06 mg/kg/d, whereas embryolethality was complete at 0.25 and 2.5 mg/kg/d. As already discussed above, this finding was considered to be due to the severe hormonal imbalance caused by the administered compounds

Contraception

1994:49, April

319

Toxicology of Mesigyna:

Seibert and Giinzel

because the monkey also displays much lower endogenous estrogen level during pregnancy when compared to humans.2 Mutagenicity testing-It was not considered necessary to test E,Val itself for mutagenicity since the ester in humans is completely split into 17p-estradiol and valeric acid. The fatty acid undergoes p-oxidation and, thus, is completely eliminated as carbon dioxide and water. The steroid 17p-estradiol was assessed in the Ames test. Five different strains of Salmonella typhimurium with and without metabolic activation were used in this test and all had negative results on mutagenicity. These results are consistent with those of many other test systems such as V 79 HGPRT and V 79 ATPase assay,25 sister chromatid exchange in human or mouse fibroblasts2’ and chromosomal aberrations28 relymphocyte+ ported in the literature to be negative on mutagenicity. Local tolerance-Studies on the local tolerance of the solvent itself (castor oil: benzylbenzoate, 6:4) has been performed in rabbits and caused mild inflammatory reactions at the injection sites. While no separate local tolerance studies have been performed for E2Val when dissolved in this vehicle, the results of the chronic toxicity testing using intramuscular injections of thZ compound did not indicate an increase in irritation due to the compound. Furthermore, during extensive clinical experience with the compound dissolved in this vehicle for intramuscular injection, no unacceptable irritating effects in humans at the application site were noted. Toxicity

Studies

Performed

with NET-EN

As expected, NET-EN showed a very low acute Single Dose Toxicity. toxicity in mice and rats using the p.o., s.c., i.p. and i.m. routes of application. The LDSo values were above 1.0 g/kg (i.p., i.m., p.o.) or >12.5 g/kg (s.c.), respectively. Th e p 1anned human dose of 50 mg per woman i.m. (= ca. 1 mg/kg), therefore, lies far below the dose range where severe acute adverse side effects would be expected. Repeated Dose Toxicity and Tumorigenicity. Rodent studies-For the assessment of toxicity after long-term treatment, combined systemic tolerance and tumorigenicity studies were performed in mice and rats. The details of the study design are given in Table 11. A compound-related effect observed in mice was an increase in body weight gain over the first 30 weeks of treatment. As this was not associated with a similar increase in food consumption, some metabolic alterations in this species are indicated. Further signs of systemic effects were the expected atrophic changes of the ovaries with an increased incidence of absent corpora lutea at the high dose level. At the highest dose level, an increased incidence of transitional cell

320

Contraception

1994:49, April

Toxicology of Mesigyna: TABLE 11. NET-EN

Experimental

Animal Species, Number per Group and Sex

design

of repeated

Duration of Treatment

Mice 40 M, 40 F

78 weeks

Rats 40 M, 40 F

2 years

Rats 60 F

2 years

Rats 60 F

2 years

Rats

2 years’

dose studies

Seibert and Giinzel

in mice and rats treated

intramuscularly

Dose (mgkg)

Injection Intervals

0 10 30 100 0 10 30 100 0 1 10 50 0 (0) 6 (4) 60 (40) 300 (200) 0 200 200

weekly

with

weekly

every 2 weeks

every 12 (8) weeks for 5 (6) intervals’

every 8 weeks every 12 weeks

Comparative study in which the effects on rat liver after i.m. treatment were compared to those after p.0. treatment with the combination of NET-AC plus mestranol. *Initially, the rats received 5 injections at intervals of 12 weeks. Later, they received 6 injections at intervals of 8 weeks at the reduced dose levels which are given in brackets.

carcinomas of the urinary bladder was noted. Other investigators observed an increased incidence of urinary bladder carcinomas in mice and rats under estrogen influence .29 Therefore, it was suspected that the inherent estrogenic effects of NET-EN in rodents30 had a similar effect in this study, particularly as a high body burden due to accumulation of NET-EN had to be suspected when NET-EN was injected at weekly intervals. In humans, neither an estrogenic effect of NET-EN nor a stimulation of urinary bladder tumors by steroid hormones has been described to our knowledge. A significant decrease of tumors of the lympho-reticular system was also noted in the high dose group. No further statistically significant differences in tumor incidences were noted. However, there was a slight, but dose-dependent increase in liver cell adenomas and mammary gland carcinomas in mice treated with NET-EN if compared to controls. These findings might indicate some tumor-promoting activity on these organs in mice, as is also known for other sex steroids.4 The relevance of these

Contraception

1994149, April

321

Toxicology

of Mesigyna:

Seibert

and Giinzel

findings to humans is discussed below together with the similar changes observed in rats. The results obtained after long-term treatment of rats are summarized in Table 12. The decrease in body weight gain, the hematological and blood-biochemical alterations, the increased incidence of alopecia, and the stimulatory effect on the endometrium and mammary glands are indicative of the inherent estrogenicity of NET-EN in rats because these alterations are typical estrogenic effects reported for this species.4 The histological changes at the ovaries and the endothelial changes at the cervix and the vagina, on the other hand correspond well to the gestagenic activity of the compound.31 In the combined systemic tolerance and tumorigenicity studies carried out on rats and mice, the incidence of proliferative liver lesions including benign adenomas was more or less distinctly in-reased. In the rat studies, it was shown that this incidence was directly dependent on the time intervals between the NET-EN i.m. injections. The incidence of benign adenomas was increased only at injection intervals of one or two weeks, that is when extremely high accumulation of the compound occurs in this species due to the pharmacokinetics of NET-

TAELE 12. EN

Salient findings observed

Clinical observations:

t 1 T t

in rats after repeated intramuscular

treatment

with NET-

mortality body weight gain alopecia swellings at the injection site

Ophthalmoscopy:

t cataracts

Hematology/Biochemistry:

1 erythrocyte counts 1 serum cholesterol ‘T’serum alkaline phosphatase

Organ weights:

L ovaries t liver

Histopathology:

-atrophy of ovarian follicles -endometrial hyperplasia -mutinous cervical transformation -decreased incidence of mammary fibroadenomas and increased incidence of cystic mammary adenomas and mammary carcinomas -proliferative and/or mutinous changes in the vagina decreased incidence of pituitary adenomas -liver ceil hypertrophy and hyperplasia, increased incidence of liver adenoma -inflammatory reaction at the application site

‘T = increase, J = decrease

322

Contraception

1994:49,

April

Toxicology

of Mesigyna:

Seibert and Giinzel

EN in rats. It was not observed in one tumorigenicity study using 8- or 12-week intervals for injection. However, in a further rat study using a different rat strain, an increase in benign liver cell adenomas was also noted at 8- and 12-week injection intervals. Thus, not only the body burden of the compound, but also other factors, presumably genetically different sensitivity of target organs, may play a role. The apparent sensitivity of the rodent liver towards NET-EN caused a series of experiments to study possible underlying mechanisms. NET-EN as well as many other non-mutagenic xenobiotic compounds such as phenobarbital and other steroidal hormones have presumably a promoting effect on spontaneously occurring preneoplastic liver lesions in mice and rats and can, thus, increase the incidence of benign liver tumors in this species.s2 It could be shown that some of the pre-stages of such neoplasms were reversible after NET-EN withdrawal. In humans, an association between benign liver cell adenomas and the prolonged use of oral contraceptives has to be considered in very rare individual cases,33 whereas a causal relationship between malignant liver cell tumors and prolonged treatment with oral contraceptives, even if postulated repeatedly, could not be shown. In the United States, for example, by 1980 more than 5 million women had used oral contraceptives for more than 8 years. The number of deaths from primary liver cancer among US women, however, remained unchanged from 1968 to 1980.“4 Non-rodent studies-Studies on long-term toxicity and tumorigenicity were also performed in dogs and monkeys using the intramuscular route of administration. Details of the study designs such as duration of treatment, doses, and injection intervals are compiled in Table 13. All findings which were clearly or suspectedly related to treatment over periods up to 7 or 10 years, respectively, are listed in Table 14. In the dogs, the findings related to the systemic tolerance of NETEN as well as the tumorigenic response in the mammary glands were to be expected as they are well-known effects of prolonged treatment with progestins in this species including the naturally occurring progesterone.21,35 NET-EN like other progestagens led to hematological changes, to increases in serum fibrinogen and to severe cystic changes of the endometrium with the consequence of increased occurrence of infections, and thus the necessity to hysterectomize the animals. Neither of these findings were considered to indicate a possible intolerability in humans. The increased incidence of mammary tumors in dogs after chronic exposure to NET-EN confirmed the results of many other investigators who showed that prolonged treatment with progestins, including the naturally occurring progesterone, enhances tumor formation in the dog.35 The mechanism has been previously elucidated by a number of experiments including some on hypophysectomized beagle dogs. It could be

Contraception

1994~49,

April

323

Toxicology TABLE 13. NET-EN

of Mesigyna:

Experimental

Animal Species, Number per Group and Sex Dogs 4 M, 4 F

Dogs 24 F

Seibert

and Giinzel

design of repeated dose studies in dogs and monkeys treated intramuscularly

Duration of Treatment

Dose (mg/kg)

1 year

0 10 30 100

7 years

0 6

(0) (4)

60 150

(40) (100)

Injection Intervals weekly for 8 weeks; every three weeks thereafter

Monkeys 4F

90 days

0 6 60 300

Monkeys 24 F

10 years

0 6

(0) (4)

60 300

(40) (200)

every 12 weeks for 7 injections (every 8 weeks thereafter)

every 12 weeks for 7 injections (every 8 weeks thereafter)‘)

Dogs

Clinical observations: mortality body weight gain alopecia alopecia swellings at the injection site mammary nodules and masses mammary discharge red vaginal discharge gingival hypertrophy, gingivitis

324

by a reduction

of the

in dogs and monkeys after repeated intramuscular

Findings

Hematology/Coagulation erythrocyte count erythrocyte sedimentation fibrinogen

intervals

2 injections at 45day

‘The shortening of the injection intervals (as indicated in brackets) was accompanied administered doses (also indicated in brackets in previous column).

TABLE 14. Salient findings observed ment with NET-EN

with

t

r :

z

treat-

Monkeys

r t t .LT* ‘T‘

t t

rate

Contraception

1994:49, April

Toxicology TABLE 14.

of Mesigyna:

Seibert and Giinzel

Continued

Findings

Monkeys

Dogs

Biochemistry serum cholesterol,

-

(Q

:. -

i

T

T

T -

r

-

T

Cervix: mucus production

-

T

Vagina: atrophy

-

T

Clitoris: prolapse

-

T

;

T -

alanin aminotransferase

Organ weights: ovaries liver adrenals, thyroid/parathyroid, kidneys, pituitary

and protein

uterus

Histopathology: Ovary: atrophic changes Endometrium: cystic hyperplasia glandular atrophy, decidual adenocarcinomas Myometrium:

reaction

atrophy, adenomyosis

Mammary gland: nodules, adenomatous hyperplasia benign and malignant tumors Pituitary: enlargement,

increase in prolactin ceHs and chromophobe

adenoma

Liver: hypertrophy, vacuolation of hepatocytes red foci, reactive round cell infiltration and teleangiectasis Gall bladder: hyperplasia Thymus:

involution

Kidneys: glomerulosclerosis,

glomerulonephritis

and chronic nephritis

Cardiovascular system: thrombosis in blood vessels, endocarial lesions reactive round cell infiltrates and teleangiectasis Serum growth hormone

levels

in the heart

i

Ta

T T

r -

-

T

T

-

T -

r

T

n.d.

‘2 high-dose animals with endometrial carcinoma, one of which had lung metastases and died during the study, the other was terminally sacrificed. OOne high dose animal with chromophobe adenoma. T = increase. J = decrease. - = no effect. ( ) = suspected marginal change. = inconsistent finding. n.d. = Not determined. l

Contraception

1994:49, April

325

Toxicology

of Mesigyna:

Seibert

and Giinzel

shown that in the dogs progestogenic compounds lead to an increased secretion of pituitary growth hormone, which in turn is able to stimulate mammary growth in this species .2f21Serum growth hormone in the present study was also increased, specifically at the mid and high dose levels and, particularly, in those dogs with mammary tumors. No changes in growth hormone levels were observed in women receiving intramuscular injections of 200 mg NET-EN, i.e., four-fold the dose contained in [email protected] The finding of mammary tumors in dogs after treatment with progestins or progestin/estrogen combinations has caused a long-lasting controversial discussion in the scientific community with regard to the relevance for human risk assessment. Nowadays it is generally agreed that the results obtained in long-term studies with contraceptive steroids in the dog are of no predictive value for the human9’ The findings in monkeys related to the systemic tolerance did not indicate salient toxic organ effects of NET-EN, even at the high dose (200 times the human dose on the basis of body weight). Rather, most of the findings could be related to the endocrine pharmacological action of the compound and have been described before as typical organ alterations in monkeys treat-d repeatedly and over a prolonged time with progestagenic compounds.4,3” This holds true not only for findings in the endocrinedependent organs, such as ovaries, accessory sex organs, mammary gland and pituitary, but also for most of the very few and non-relevant changes of laboratory parameters.36 Further, in the high dose animals, some additional findings could be related to the intramuscular route of administration over a period of 10 years. In this group, the addition of the test compound to the vehicle aggravated the mild reactive inflammatory lesions which were seen after the vehicle alone, as noted to about the same degree in controls, low and mid dose animals. This had the consequence of more pronounced chronic inflammatory reactions not only at the injection sites, but in addition in the liver and heart in the high dose group animals. There were virtually no signs of intolerability as demonstrated by the survival rate which, if it was affected at all, increased with increasing dosage (10, 12, 12 and 14 animals out of 16 survived the lo-year period in the control, low mid and high dose groups). The only findings which might be related to a possible tumorigenic effect of NET-EN in the monkeys were endometrial carcinomas in two animals and probably the one pituitary adenoma, all of which occurred in the high dose group. Endometrial carcinomas in monkeys as a consequence of repeatedly intramuscularly administered high doses of progestogens have been reported before and this is considered to be a unique response of the monkey’s uterine endometrium.“’ The majority of endometrial cancers in women are adenocarcinomas associated with endometrial hyperplasia in patients whose ovaries do not produce progesterone.38

326

Contraception

1994:49,

April

Toxicology

of Mesigyna:

Seibert and Giinzel

Progestins, therefore, are considered rather to protect women from endometrial carcinomas and are even used for tumor therapy. The cause of the chromophobe adenoma in one high dose monkey in the lo-year monkey study remains unclear. On the one hand, spontaneous pituitary adenomas have been described in Rhesus monkeys,39 on the other hand, treatment-related proliferative alterations (increase in prolactin cell numbers) were noted in the pituitary anterior lobe in most of the high dose animals. Therefore, the adenoma might also be suspected to be related to the treatment with NET-EN, even if it had no signs of hormonal activity in the immunohistochemical examination. In humans, progestogens are neither suspected nor known to increase the incidence of pituitary adenomas. Furthermore, as mentioned in the discussion on pituitary findings in rodents after treatment with E,Val, the preparation Mesigyna@ did not elicit a stimulatory effect on prolactin baseline levels in women, even after treatment periods longer than a year. Reproduction toxicity-Embryotoxicity of NET-EN was examined in rats and rabbits. In addition, studies in mice, rats, rabbits and monkeys have been performed with the closely related compound NET-AC; they are also considered to be relevant as this compound contains the same pharmacologically active principle (norethisterone) as NET-EN. No teratogenic effects on non-genital organs were noted in any of these studies. Like other progestogenic hormones of the nortestosterone type, NET-EN at high doses caused a mild virilisation of female fetuses in the rat and the monkey, a problem which has been investigated extensively. However, the doses applied to induce these effects gave far higher plasma concentrations and far greater values of the areas under the plasma concentration curves than those determined in women after the injection of either 200 mg NET-EN or especially 50 mg NET-EN, as contained in Mesigyna@. Therefore, even in the unlikely event that Mesigyna@ will be accidentally injected at the period when sexual differentiation takes place in human fetuses (from day 45 p.c. onwards during human pregnancy), from the data gathered so far, feminisation or virilisation of the fetuses seems very unlikely. This is consistent with the clinical experience on the outcome of pregnancies when NET-EN was either administered before or after conception and no treatment-related malformations were observed. Mutagenicity testing-It was not considered necessary to test NETEN itself for mutagenicity since the ester in humans is completely split into norethisterone and heptanoic acid. The fatty acid undergoes p-oxidation and thus is completely eliminated as carbon dioxide and water. The steroid norethisterone was (as its acetate ester) assessed in the Ames test. Five different strains of Salmonella typhimurium with and without metabolic activation were used in this test and all had negative results on mutagenicity. In addition, in vitro unscheduled DNA synthesis (UDS) tests were performed on hepatocytes isolated from male and female rats

Contraception

1994:49, April

327

Toxicology

of Mesigyna:

Seibert

and Giinzel

with the free steroid norethisterone and gave no indication of a genotoxic potential of the compound. Local tolerance--Separate studies to test the local tolerance of intramuscularly administered NET-EN were not performed, as the local tolerance could be evaluated within the various chronic studies carried out on mice, rats, dogs and monkeys. Based on these results, NET-EN increased the mild inflammatory reactions observed with the vehicle alone to a tolerable degree. These results are, therefore, not considered prohibitive for usage in humans. Furthermore, broad clinical experience with this vehicle, which is used as oily solutions in many marketed injectable preparations including Noristerata containing NET-EN, confirms the above toxicological results of a tolerable and only slight local irritation.

Toxicity Studies Performed with the Combination E,Val: Experience from Limited Studies

of NET-EN and

A 2-year combined systemic tolerance and tumorigenicity study in rats with weekly intramuscular administration of the combination of NETEN and E,Val at the intended ratio for human use of 1O:l was initiated by the Toxicology Group of the WHO Special Programme of Research, Development and Research Training in Human Reproduction (for details of the study design see Table 15). The high dose group had to be prematurely terminated in week 62 because survival in the main group fell below 20%, which was previously designated as borderline for continuation of treatment. All high dose animals that died before week 62 and all low dose animals that died up to week 78 (latest interim report) showed pituitary enlargement, which can, therefore, be considered as the major cause of death. When it was decided to terminate the study, the survival rate in the remaining treatment group had already decreased to 30%. Additional treatment-related findings are compiled in Table 16. Body weight gains in the high dose group tended to decrease from about TABLE 15. Experimental design of repeated dose studies in female Sprague-Dawley rats treated intramuscularly with the combination NET-EN and E,Val at a ratio of lO:l, respectively

Animal Number’ and Sex 70 F 70 F 70 F l50 main group animals

328

Dose Levels NET-EN + E,Val (mgikg)

injection Interval

Time Point of Sacrifice

0 (controls) 0.91 + 0.09 4.55 + 0.45

once very 4 weeks

week 96 week 96 week 62

+ 20 satellite animals

Contraception

1994:49,

April

Toxicology TABLE 16. Salient findings observed combination NET-EN + E,Val

of Mesigyna:

in rats after repeated

Seibert and Giinzel intramuscular

treatment

with the

Clinical observations:

t 1 t t

mortality body weight gain water consumption alopecia

Hematology/Biochemistry*:

1 erythrocyte counts, blood hemoglobin, packed cell volume 1 total white blood cell counts, lymphocyte counts ‘? neutrophil counts 1‘ total serum protein T serum urea

Organ weights*:

1 adrenals f pituitary

? = Increase. 1 = Decrease. *Determined in satellite animals.

week 30 onwards, and after about week 60, a decline in weight gain was also observed in low dose animals when compared to controls. No clearcut concomitant alteration in group mean food consumption was observed. It even tended to be transiently increased rather than decreased versus control values. Also, water consumption was increased in treated animals. Other major alterations were the changes in hematological parameters (determined in weeks 13, 26 and 52) and alterations in organ weights determined in satellite animals at week 52 at scheduled interim sacrifice. As would be expected, these findings demonstrate the predominance of estrogenicity of the combination in rats and revealed no additional knowledge to results obtained for E,Val alone. This study was prematurely terminated because, during a meeting that was held in Geneva in October 1986, the WHO Toxicology Review Panel came to the conclusion that the results of this study would be irrelevant for human risk assessment. This view was already previously held by the German Federal Health Authorities (BGA). Rather than adhering to formal regulatory rules, the conviction of the irrelevance of animal toxicity studies using the combination was based on scientific considerations. The activity profiles for either component of Mesigynam obtained from extensive toxicological, pharmacokinetic and pharmacological investigations including receptor binding studies gave no indication of possible interactions between the two substances which could be meaningfully further assessed in animal species. As outlined earlier, the requirement of using the same estrogen/progestin ratio in animal studies as intended for human use poses quite an unphysiological condition on most animal species, with the probable exception of the monkey. However, as previously out-

Contraception

1994:49, April

329

Toxicology

of Mesigyna:

Seibert and Giinzel

lined, even the monkey by its normal physiology is not accustomed to handle such large variations in hormone levels as the human. It must be considered that during clinical use, it is rather the subtle changes in lipid and carbohydrate metabolism and in coagulation parameters which are of prime interest for evaluation of long-term tolerability of various estrogen/ progestin combinations in humans. Since this requires proper dosing and balancing of both components according to the human physiology, it is impossible to reliably derive predictive data from animal toxicity studies.

4. General Discussion and Conclusion The extensive experimental studies performed on the components of the monthly injectable hormonal contraceptive Mesigyna@ provide a broad basis for risk assessment. Thereby, the emphasis should be on the characterisation of the various pharmacodynamic and toxicodynamic properties of both compounds rather than on the attempt of a quantitative risk assessment for humans. Both compounds displayed a toxicological activity profile in each of the species examined which showed them to act as a typical steroidal estrogen or progestin, respectively, and that they do not differ from other comparable compounds used, e.g., in oral hormonal contraceptives. This was to be expected from the basic pharmacological characterisation performed on either substance. Most of the findings gathered in the different species for sex steroids, including the tumorigenic effects that were observed in long-term studies, were retrospectively not predictive for human risk evaluation. This is in accordance with the wealth of knowledge which is already available from the evaluation of toxicity data for various steroida1 estrogens and progestins, on the one hand, and human clinical data and the results from human epidemiological investigations, on the other hand.4 The reason for the lack of predictivity of animal toxicity data is the fundamental species differences, specifically of the endocrinology, but also of the pharmacokinetics and metabolism of the compounds.2 Thus, the accumulated knowledge on E,Val and NET-EN as isolated compounds as well as on their possible interaction gained in various in vitro and in vivo studies does not provide a safety profile of Mesigyna@ in humans which differs principally from that of oral contraceptives. It is to be hoped that the public knowledge about the low predictive value of animal toxicity testing in the field of contraceptive sex steroids will increase through retrospective analysis of the already available data. This should lead to a revision of regulatory guidelines in that standardised long-term studies, including tumorigenicity studies in rodents, will no longer be required for this class of compounds. Rather the emphasis should be on a basic toxicodynamic and pharmacokinetic characterisation of newly developed steroidal compounds, which can best be achieved by

330

Contraception

1994:49, April

Toxicology

of Mesigyna:

Seibert and Giinzel

performing carefully and individually designed animal studies (not necessarily exceeding 6 months duration) prior to a thorough investigation in humans which should commence as early as possible.

Acknowledgment The studies reviewed under “Pharmacodynamics” and “Pharmacokinetits” were conducted and evaluated in the respective departments of Schering AG, Berlin. The toxicological evaluation is partly based on previous summaries by Dr. B. Putz, Schering AG, Berlin.

References 1. 2.

3. 4.

9. 10.

11.

12. 13. 14.

Contraception

Garza-Flores J, Hall PE, Perez-Palacios G. Long-acting hormonal contraceptives for women. J Steroid Biochem Molec Biol 1991;40:697-704. Gi.inzel P, Putz B, Lehmann M, Hasan SH, Hiimpel M, El Etreby MF. Steroid toxicology and the ‘pill’: Comparative aspects of experimental test systems and the human. In: Dayan AD, Pain AJ, eds. Advances in Applied Toxicology. London, New York, Philadelphia: Taylor and Francis, 1989: 19-49. Jordan A. FDA requirements for nonclinical testing of contraceptive steroids. Contraception 1992; 46:499-509. Lehmann M, Putz B, Poggel HA, Gtinzel P. Experimental toxicity studies with contraceptive steroids and their relevance for human risk estimation. In: Dayan AD, Pain AJ, eds. Advances in Applied Toxicology, London, New York, Philadelphia: Taylor and Francis, 1989:51-79. Heywood R, Wadsworth PF. The experimental toxicology of estrogens. Pharmacol Ther 1980;8:125-42. Gibson JP, Newberne JW, Kuhn WL, Elsea JR. Comparative chronic toxicity of three oral estrogens in rats. Toxic01 Appl Pharmacol 1967;11:489-510. Piliero SJ. The interrelationships of the endocrine and hemopoietic systems in the Long-Evans rat. Lab Anim Care 1969;19:702-9. Jepson JH, Lowenstein L. Inhibition of the stem cell action of erythropoietin by estradiol valerate and the protective effect of 17a-hydroxyprogesterone caproate and testosterone propionate. Endocrinology 1967;80:430-4. Miraud EA, Gordon AS. Mechanism of estrogen action in erythropoiesis. Endocrinology 1966;78:325-32. Lageder H. Carbohydrate metabolism and hormonal replacement therapyProblems and clinical results. Acta Obstet Gynecol Stand Suppl. 1977;65: 57-63. Vessey MI? The Jephcott Lecture, 1989: An overview of the benefits and risks of combined oral contraceptives. In: Mann RD, ed. Oral Contraceptives and Breast Cancer. The proceedings of an international Symposium held at the Royal Society of Medicine, London, 1989:121-35 Lubowe II. The treatment of female pattern baldness. Clin Med 1972;79:25-7. McConnell RF. Comparative aspects of contraceptive steroids: Effects observed in rats. Toxic01 Path01 1989;17:385-8. El Etreby MF, GrHf KJ, Giinzel P, Neumann F. Evaluation of effects of sexual

1994:49, April

331

Toxicology of Mesigyna:

15.

16. 17. 18. 19.

20. 21. 22. 23. 24.

25. 26.

27.

28.

29.

30. 31.

332

Seibert and Giinzel

steroids on the hypothalamic-pituitary system of animals: Mechanisms of toxic actions on some target organs. Arch Toxic01 1979;2:1 l-39. Welsch CW, Adams C, Lambrecht LK, Hassett CC, Brooks CL. 17B-estradiol and Enovid mammary tumorigenesis in C3H/HeJ female mice: Counteraction by concurrent 2-bromo-alpha-ergocryptine. Br J Cancer 1977;35:322-8. Wingrave SJ, Kay CR, Vessey MP. Oral contraceptives and pituitary adenomas. Br Med J 1980;280:68.5-6. Shermann BM, Schlechte J, Halmi NS et al. Pathogenesis of prolactinsecreting pituitary adenomas. Lancet 1978;2:1019-21. Gooren, LIG et al. Estrogen-induced prolactinoma in man. J Clin Endocrinol Metab 1988;66:444-6. Garza-Flores J, Alba VM, Cravioto MC, Hernandez L, Perez-Palacios G. Estrogen-progestogen once-a-month injectable contraceptives and serum prolactin. Contraception 1989;39:519-29. Schlesselmann JJ. Cancer of the breast and reproductive tract in relation to use of oral contraceptives. Contraception 1989;40:1-38. Johnson AN. Comparative aspects of contraceptive steroids: Effects observed in the Beagle dog. Toxic01 Path01 1989;17;389-95. Weikel JH, Nelson LW. Problems in evaluating chronic toxicity of contraceptive steroids in dogs. J Toxic01 Environment Health 1977;3:167-77. El Etreby MF, Fath El Bab MR. Effect of 17B-estradiol on cells stained for FSHB and/or LHJ3 in the dog pituitary gland. Cell Tiss Res 1978;193:211-8. Bergink EW, Attia M, de Jager E. Effects of estradiol-17B and progesterone on the mammary gland of the Beagle dog: Morphology and receptor levels in the cytosol fraction. In: Wittcliff JL, Dapunt 0, eds. Steroid Receptors and Hormone-Dependent Neoplasia. New York: Masson, 1980:219-22. Devron C, Piccoli C, Montesano R. Mutagenicity assays of estrogenic hormones in mammalian cells. Mutat Res 1981;89:83-90. Hill A, Wolff, S. Sister chromatid exchange and cell division delays induced by diethylstilbestrol, estradiol, and estriol in human lymphocytes. Cancer Res 1983, 43:4114-8. Hillbertz-Nilsson K, Forsberg J.G. Estrogen effects on sister chromatid exchanges in mouse uterine, cervical and kidney cells. J Nat1 Cancer Inst 1985;75:575-80. Banduhn N, Obe G. Mutagenicity of methyl-2-benzimidazolecarbamate, diethylstilbestrol and estradiol: Structural chromosomal aberrations, sisterchromatid exchanges, C-mitoses, polyploidies and micronuclei. Mutat Res 1985;156:199-218. MC Connell RF. General observations on the effects of sex steroids in rodents with emphasis on long-term oral contraceptive studies. In: Michal F, ed. Safety Requirements for Contraceptive Steroids. Cambridge: Cambridge University Press, 1987:21 l-229. Markiewicz L, Hochberg RB, Gurpide E. Intrinsic estrogenicity of some progestagenic drugs. J Steroid Biochem Molec Biol 1992;41:53-8. Nishino Y, Neumann F. The sialic acid content in mouse female reproductive organs as a quantitative parameter for testing the estrogenic and antiestrogenie effect, antiestrogenic depot effect, and dissociated effect of estrogens on the uterus and vagina. Acta Endocrinol Suppl 1974;187:1-62.

Contraception

1994:49, April

Toxicology 32. 33. 34. 35.

36.

37. 38. 39.

Contraception

of Mesigyna:

Seibert and Giinzel

Schulte-Hermann R. Tumor promotion in the liver. Arch Toxic01 1985; 57:147-58. Rooks JB, Ory HW, Ishak KG et al. Epidemiology of hepatocellular adenoma. J Am Med Ass 1979;242:644-8. International Planned Parenthood Federation. The pill and possible liver cancer. IPPF Medical Bulletin 1986; 2O:l. Capel-Edwards K, Hall DE, Fellowes KP et al. Long-term administration of progesterone to the female Beagle dog. Toxic01 Appl Pharmacol 1973; 24:47&88. Dhall K, Kumar M, Rastogi GK, Devi PK. Short-term effects of norethisterone oenanthate and medroxyprogesterone acetate on glucose, insulin, growth hormone, and lipids. Fertil Steril 1977;28:156-8. Valerio MG. Comparative aspects of contraceptive steroids: Effects observed in the monkey. Toxic01 Path01 1989; 17:401-10. Gurpide E. Endometrial cancer: Biochemical and clinical correlates. J Nat Cant Inst 1991;83:405-16. MC Clure HM. Neoplasia in rhesus monkeys. In: Bourne GH, ed. The Rhesus Monkey, Vol. II, New York: Academic Press, 1975:369408.

1994:49, April

333