13
Section of Oncology
tendency in present circumstances for cases to be reported where the patient has been on the pill, and not otherwise unless there are clinical or pathological features of exceptional interest. Furthermore, some cases have almost certainly been reported more than once (Baum et al. 1973, Ameriks et al. 1975) and this distorts the figures. Both hepatic adenoma and focal nodular hyperplasia are well known to be commoner in women than in men, and to be commonest in women during reproductive life. Since it is estimated that, in the United Kingdom, about 30 % of women of child-bearing age have taken oral contraceptives, there is quite a high chance that a woman with any condition whatsoever is on, or has been on, the pill. In addition, many of the patients in whom the association has been suggested have been taking oral contraceptives for only a short time. For instance, one of Baum's original patients had been on the pill for only six months, and another of her patients had only a two-year history of oral contraceptive ingestion. Most recognized human carcinogens have latent periods of 10-15 years and in some the time between exposure and tumour development is considerably longer. There are two published reports of women on the pill developing hepatocellular carcinoma (Christopherson et al. 1975, Hermann & David 1973). One woman (Thalassinos et al. 1974) developed hepatocellular carcinoma after being on an 'cestrogen-like' drug for infertility, and there has been a case of hepatoblastoma (Meyer et al. 1974) in a patient on oral contraceptives. Until many more cases are reported, it would be wrong to posit any association between oral contraceptive ingestion and malignant hepatic tumours. In summary, the case for an association between the pill and either hepatic adenomas or focal nodular hyperplasia of the liver must be regarded as 'not proven'. The question is clearly important, and there is a strong case for the setting up of a panel to which all British cases of these lesions, without selection of patients either by sex or by age, be referred for pathological assessment. Only in this way will it be seen whether there is, a true predilection of either lesion for women taking oral contraceptives. Acknowledgment: I am grateful to Professor Sir Theo Crawford for his encouragement and advice.
Bisson A, Duron J-J, Fagniez P-L, Pinaudeau Y & Germain A (1974) Nouvelle Presse Mddicale iii, 2079 Christopherson W M, Mays E T & Barrows G H (1975) Obstetrics and Gynecology 46, 221 Committee on Safety of Medicines (1972) Carcinogenicity Tests of Oral Contraceptives. HMSO, London Contostavlos D L (1973) Lancet ii, 1200 Edmondson H A (1958) Atlas of Tumor Pathology. Armed Forces Institute of Pathology, Washington, DC; Section 7, fascicle 25, pp 12, 193 Farrell G C, Joshua D E, Uren R F, Baird P J, Perkins K W & Kronenberg H (1975) Lancet i, 430 Fisher A W F, Curry B & Jacques J (1975) Canadian Medical Association Journal 112, 1196 Henson S W jr, Gray H K & Dockerty M B (1956) Surgery, Gynecology and Obstetrics 103, 23 Hermann R E & David T E (1973) Surgery 74, 715 Horvath E, Kovacs K & Ross R C (1974) Lancet i, 357 Ishak K G & Rabin L (1975) Medical Clinics of North America 59, 995 Kelso D R (1974) Lancet i, 315 Knapp W A & Ruebner B H (1974) Lancet i, 270 Mays E T, Christopherson W M & Barrows G H (1974) American Journal of Clinical Pathology 61, 735 Meyer P, Li Volsi V A & Cornog J L (1974) Lancet ii, 1387 Model D G, Fox J A & Jones R W (1974) Lancet i, 865 O'Reilly K (1974) Australian and New ZealandJournal ofSurgery 44, 142 (1975) A ustralian and New Zealand Journal of Surgery 45, 76 O'Sullivan J P & Wilding R P (1974) British MedicalJournal iii, 7 Perez V, Gorodisch S, De Martire J, Nicholson R & Di Paola G (1969) Science 165, 805 Phillips M J, Langer B, Stone R, Fisher M M & Ritchie S (1973) Cancer 32, 463 Shojania N G & Hogg G R (1975) Gastroenterology 69, 28 Sorensen T I A & Baden H (1975) Scandinavian Journal of Gastroenterology 10, 1 13 Thalassinos N C, Lymberatos C, Hadjionnou J & Gardikas C (1974) Lancet i, 270 Tountas C, Paraskevas G & Deligeorgi H (1974) Lancet i, 1351 Zurbriggen S & Tylen U (1975) Fortschritte aufdem Gebiete der Rontgenstrahlen und der Nuklearmedizin 122, 404
REFERENCES Adlercreutz H & Tenhunen R (1970) American Journal of Medicine 49. 630 Ameriks J A, Thompson N W, Frey C F, Appelman H D & Walter J F (1975) Archives of Surgery 110, 548 Anthony P P (I1975) Lancet i, 685 Antoniades K & Brooks C E jr (1975) Surgery 77, 137 Baum J K (1975) Journal of the American Medlical Association 232, 1329 Baum J K, Holtz F, Bookstein J J & Klein E W (1973) Lancet ii, 926 Berg J W, Ketelaar R J, Rose E F & Vernon R G (1974) Lancet ii, 349
353
Dr N P Bishun (Research Department, Tissue Culture and Cytogenetics Unit, Marie Curie Memorial Foundation, The Chart, Oxted, Surrey)
Chromosomes and Oral Contraceptives The difficulty of anticipating the biological effects of chemicals in the environment applies to longterm effects like hereditary changes, cancer induction and degenerative cellular effects leading to poor health as well as to teratogenic effects. Also, mutagenic effects, in the broad sense, are of central importance. The ability of certain chemicals to alter hereditary material has been known for three decades; in fact, this knowledge has constituted a corner-stone in the development of modern experimental genetics. In spite of this, many geneticists were for a long period slow to appreciate the practical consequences of chemical mutagenesis and possible risks to human populations. There is now an increasing demand for chromosomal evaluation of chemicals released in the environment and for the elimination of those products which may cause mutations in human beings.
Proc. roy. Soc. Med. Volume 69 May 1976
14
It is now known that oral contraceptives produce profound and fundamental changes in the endocrine system of the patients to whom they are given, through their action on the hypothalamus. Dodds (1961), in a review written soon after the introduction of these contraceptive agents, suggested 'that it would be unlikely that one could submit the delicately balanced hormonal system of a woman to such a violent alteration for 40 years without something serious happening'. On account of this state of affairs, and bearing in mind the possible relationship between mutagenicity and carcinogenicity, one would expect the molecular nature of the two processes to exhibit some parallelism, especially if the chemicals concerned are initiators of malignant cell transformation. We therefore investigated the possibility of any chromosomal abnormalities occurring in women who had taken oral contraceptives over long periods, and also in their progeny, as a means to contributing further to the accumulating evidence on the safety of these synthetic steroids.
The subjects, who were chosen from the maternity ward of a general hospital, were (a) women who had taken one or more types of oral contraceptive at some time during their reproductive life (test mothers), or (b) women who had never practised any form of oral contraception (control mothers) (for details see Table 1). Both groups had entered hospital for the imminent birth of their babies which were included in the survey as test and control babies respectively. Triplicate blood cultures from control and test mothers and their respective babies were set up.
354
Results and Discussion The frequency of chromosome abnormalities in control subjects in the present study is comparable with that reported in other series. On the whole, the total results from the four groups, namely control mothers and babies, and test mothers and babies, show no significant differences in the percentage of cells with more or less than 46 chromosomes (P> 0.05). In our study (Table 2), the percentage of cells with >46 chromosomes (excluding tI iploid and
Table 1 Various factors associated with the length of time the oral contraceptive was taken
Oral contraceptive Ovulen Gynovlar Minovlar Anovlar LyndiQl
Mean length of time Length of tinme oral contraceptive betweenz ceasing oral was taken (mlonths) contraceptiv e and pregnancy (miionths) Mean Range 11.3 2-84 22.8 9.9 3-63 22.4 9.1 2-28 9.0
No. of mothers
34 19 8 9 12 15
20.4 17.6 10.1 14.9 29.8 14.7 26.0 6.3 2.0 16.3 17.9 21.3
Norinyl 10 Conovid 7 Ortho-Novin 3 Volidan 3 Sequens 3 Nuvacon l Normenon Combination 29 14 VariouLs Not known 18
14.8 10.0 5.7 9.9 3.2 4.3 8.5 6.3 0.0 7.6 2.9 18.6
1-72 2-48 1-24
3-30 2-72 2-36 12-36 3-12 2 3-48 5-60 1-36
Percentage of' niothers taking oral contraceptives
continuously 69 79 100 89 75
'0O 100 100 100 66 100 100 79 71 85
Table 2 Total chromosome results in control and test mothers and babies Cells countel in
Control
Test
mizothers
mothers
No. 84
No.
Control babies
Test
babies
Chromosomal groups < 45 45 46 > 46
Triploid Tetraploid Endoreduplicated Polyploid Gaps Breaks Total cells counted
19 2009 8 2 28 14 0
11 11 2164
% 3.98 0.97 92.9 0.4 0.1 1.3 0.6 0 0.5 0.5
89 22
1963 9 2 32 7 2 8 12 2126
% 4.2 1.0 92.3 0.4 0.1 1.5 0.3 0.1 0.4 0.6
No. 159 4.3 37 1.0 3417 92.7 6 0.2 0.03 1 47 1.3 8 0.2 0.03 1 3 0.1 8 0.2
No. 169 4.86 54 1.5 3270 92.1 10 0.3 6 0.2 34 1.0 7 0.2 0 0 14 0.4 4 0.1
3686
3550
355
15
Section of Oncology
tetraploid cells) was similar in the control and test mothers group (0.4%), but possibly slightly larger in the test babies (0.28%) compared to controls (0.16%). Only a small number of these hyperdiploid cells had sex chromosome alterations and the majority had two or three extra abnormal chromosomes which could not be allocated to any of the groups of the Denver system of classification. Goh (1967) also found a higher percentage of cells with 47 chromosomes present in women who had been using oral contraceptives (1.05%) which was considerably higher than we found in the mothers in the present study (0.4 %). The frequency of triploid cells in the test and control cultures of the mothers in the present study (0.1 %) was identical, but the test babies showed over five times as many triploids as were present in the control group (P47 chromosomes, which probably includes triploidy, as 0.9% in 5 women on the pill compared with 0.3 % in control subjects. Mechanisms which can account for the increase in triploidy include the defective segregation of one haploid set of
chromosomes from one of the two metaphase plates before division of the zygote. Triploid cells have been found in human short-term leukocyte cultures (Bishun, personal observation), and these findings were thought to represent malsegregation of one haploid set from a 2n cell line but tripolar division of a tetraploid cell is another possible explanation. The occurrence of tetraploid cells found in both mothers and babies in our study showed no significant differences from controls (P> 0.05), and no further data concerning the frequency of tetraploidy following the taking of oral contraceptives have been found in the literature. The number of endoreduplicated cells was identical in the two groups of babies in our study, and although the number of these cells in the mothefs receiving the pill (0.6%) was twice that of the control mothers group, this difference is not thought to be significant (P> 0.05). In so far as structural abnormalities of the chromosomes were concerned, no significant differences were seen in the numbers of gaps and breaks occurring in the mother groups, and no differences in breaks as far as the babies were concerned, but nearly five times as many gaps were found in the chromosomes of the babies whose mothers had been taking oral contraceptives (0.39% and 0.08 %), which is highly significant (P< 0.001). The overall findings of this chromosome study suggest that there is no increase in chromosomal damage in human subjects who had taken oral contraceptive preparations before conceiving, at the normal prescribed dosage, for modetately prolonged periods of time. However, it may be that the lapse of time, 9 months to 1 year, between cessation of taking oral contraceptives and obtaining blood samples for the present chromosomal studies might invalidate these generally negative findings. Nevertheless, it is our view that, for a true evaluation of the mutagenic potential, a period of recovery should be allowed to enable normal repair and rejection mechanisms to operate effectively (cf. Goh). Although the potential of a chemical substance for mutagenicity is not the same as its potential for carcinogenicity, this is largely a matter of species differences in tissue distribution of the chemical and its metabolism. However, our studies of leukocyte chromosomal changes in humans at normal dosage are possibly more relevant to the evaluation of the risk of cancer in human subjects than is the determination of the carcinogenic potential of these chemicals at excessively high dosage in rodents. In any case there is considerable scepticism with regard to accepted methods of carcinogenicity testing in rodents as this species has a high level of endogenous oncogenic viruses present in their tissues.
Table 3 Percentages of abnormal cells in the mothers' and babies' groups from each type of oral contraceptive
Oral contraceptive Normenon (chlormadinone acetate) Ovulen (ethynodiol diacetate + mestranol) Gynovlar (norethisterone acetate + ethynylcestradiol) Lyndiol (lynestrenol + mestranol) Anovlar (norethisterone acetate + ethynylcestradiol) Conovid
(norethynodrel + mestranol) Minovlar (norethisterone acetate + ethynykcestradiol) Nuvacon (megestrol acetate + mestranol) Volidan (megestrol acetate +ethynyloestradiol) Ortho-Novin (norethisterone +mestran'ol) Sequens (chlormadinone + mestranol) Norinyl (norethisterone + mestranol) Mean for all test subjects Mean for all control subjects
Abnormal cells in mothers (%) 4.0
Abnormal cells in babies () 0.0
4.3
1.5
4.7
1.8
3.5 2.4
2.1 0.0
2.6
4.6
1.4
0.7
0.0
0.0
0.0
4.1
4.4
3.6
2.0
1.9
1.2
2.1
3.3 3.4
1.8 2.0
The abnormal cells included are those with > 46 chromosomes, triploid, tetraploid, polyploid and endoreduplicated cells, and those exhibiting gaps and breaks, and the figures are the total expressed as a percentage of the total cells counted
356
Proc. roy. Soc. Med. Volume 69 May 1976
16
Table 4 Percentages of mothers and babies with chromosomal abnormalities and percentages of abnormal cells found for each oral contraceptive constituent Mothers
Oral contraceptive
Ethinylcestradiol Mestranol Norethisterone acetate Lynestrenol Ethynodiol acetate Norethynodrel Megestrol acetate Chloramadinone
No. of blood cultures 22 51 34
Babies
Percentage with
No. of chronmosome cells abnornmalities counted 41 472 31 777 38 791
Percentage of
abnormnal cells 3.6 3.2 2.6
No. of blood cultures 27 71 43
Percentage with
Percentage
No. of chromosonme cells abnormalities counted 33 624 32 1691 30 1120
abnormal cells 1.9 2.3 2.1
of
7 22
43 27
116 314
3.4 4.5
11 31
27 23
241 347
2.0 2.0
8 3 I
25 0 100
78 27 25
2.6 0.0 4.0
7 6 1
71 50 0
174 181 25
4.6 2.3 0.0
114
33
2126
3.3
154
31
3550
1.8
124
27
2164
3.4
157
27
3686
2.0
acetate
Total for test subjects Total for control subjects
It is interesting to compare our findings on traceptives are seen largely to substantiate the mutagenicity of the oral contraceptive prepara- findings of a previous large-scale carcinogenicity tions with the evaluation of their carcinogenic study of these compounds in rodents, namely potential in rodents. In the Report by the that no overt risk to women taking the pill is Committee on Safety of Medicines (1972) on the proved (see Tables 2 and 3), although certain carcinogenicity testing of oral contraceptives, preparations merit further investigations. three preparations came under suspicion: (1) Megestrol acetate/ethinyl oestradiol and norethy- REFERENCES Medical Journal (1972) iv, 190 nodrel, which caused an increase of malignant British Committee on Safety of Drugs mammary tumours in mice. (2) Norethynodrel, (1970) British Medical Journal ii, 231 Committee on Safety of Medicines (1972) Carcinogenicity which caused an increase in mammary carcinoma. Tests of Oral Contraceptives. HMSO, London (3) Norethisterone/ethinyleestradiol,'which caused Dodds E C (1961) Journal of Endocrinology 23, 3 K G (1967) Chromosomal Breaks in Women Taking Birth an increase in benign and malignant mammary Goh Control 'Pills'. USAEC (Report), Oak Ridge Associated tumours in rats. It is perhaps significant that in Universities, 106, pp 97-104 our mutagenicity studies Conovid (mestranol/ norethynodrel), Volidan (megestrol acetate/ethinyloestradiol) and Ortho-Novin (mestranol/norethisterone) are the preparations which had a slight increase in abnormalities in babies. The following papers were also read: Several preparations show a slight, but insignificant increase of abnormalities in the mothers Sex Hormones and Breast Cancer: Epidemiological Aspects (Tables 3 and 4). In conclusion, it is becoming increasingly Professor Martin Vessey obvious that drug safety and toxicity is best (Department of Social Medicine, evaluated by methods which can be applied Oxford University, Oxford, OX] 3QN) directly to man rather than to experimental animals, and which are quickly and relatively The Effects of the Pill upon the Breasts: inexpensive. The method of examining the effect Physiological Considerations of drugs on chromosomal changes in circulating Dr R V Short leukocytes in humans has been subiected to (MRC Unit of Reproductive Biology, much criticism concerning the reproducibility Department of Obstetrics & Gyna?cology, and meaning of results and their implications as Edinburgh, EH3 9ER) regards carcinogenicity. The advantages of using relevant species (man), the relevant dosage The Effects of the Pill upon the Breasts: and the relevant circumstances of dosage (dura- Clinical Considerations tion, sex, age, &c.) outweigh this criticism. The Mr Ian Burn total results of the present study on oral con- (Charing Cross Hospital, London W6 8RF)
Section of Oncology
tendency in present circumstances for cases to be reported where the patient has been on the pill, and not otherwise unless there are clinical or pathological features of exceptional interest. Furthermore, some cases have almost certainly been reported more than once (Baum et al. 1973, Ameriks et al. 1975) and this distorts the figures. Both hepatic adenoma and focal nodular hyperplasia are well known to be commoner in women than in men, and to be commonest in women during reproductive life. Since it is estimated that, in the United Kingdom, about 30 % of women of child-bearing age have taken oral contraceptives, there is quite a high chance that a woman with any condition whatsoever is on, or has been on, the pill. In addition, many of the patients in whom the association has been suggested have been taking oral contraceptives for only a short time. For instance, one of Baum's original patients had been on the pill for only six months, and another of her patients had only a two-year history of oral contraceptive ingestion. Most recognized human carcinogens have latent periods of 10-15 years and in some the time between exposure and tumour development is considerably longer. There are two published reports of women on the pill developing hepatocellular carcinoma (Christopherson et al. 1975, Hermann & David 1973). One woman (Thalassinos et al. 1974) developed hepatocellular carcinoma after being on an 'cestrogen-like' drug for infertility, and there has been a case of hepatoblastoma (Meyer et al. 1974) in a patient on oral contraceptives. Until many more cases are reported, it would be wrong to posit any association between oral contraceptive ingestion and malignant hepatic tumours. In summary, the case for an association between the pill and either hepatic adenomas or focal nodular hyperplasia of the liver must be regarded as 'not proven'. The question is clearly important, and there is a strong case for the setting up of a panel to which all British cases of these lesions, without selection of patients either by sex or by age, be referred for pathological assessment. Only in this way will it be seen whether there is, a true predilection of either lesion for women taking oral contraceptives. Acknowledgment: I am grateful to Professor Sir Theo Crawford for his encouragement and advice.
Bisson A, Duron J-J, Fagniez P-L, Pinaudeau Y & Germain A (1974) Nouvelle Presse Mddicale iii, 2079 Christopherson W M, Mays E T & Barrows G H (1975) Obstetrics and Gynecology 46, 221 Committee on Safety of Medicines (1972) Carcinogenicity Tests of Oral Contraceptives. HMSO, London Contostavlos D L (1973) Lancet ii, 1200 Edmondson H A (1958) Atlas of Tumor Pathology. Armed Forces Institute of Pathology, Washington, DC; Section 7, fascicle 25, pp 12, 193 Farrell G C, Joshua D E, Uren R F, Baird P J, Perkins K W & Kronenberg H (1975) Lancet i, 430 Fisher A W F, Curry B & Jacques J (1975) Canadian Medical Association Journal 112, 1196 Henson S W jr, Gray H K & Dockerty M B (1956) Surgery, Gynecology and Obstetrics 103, 23 Hermann R E & David T E (1973) Surgery 74, 715 Horvath E, Kovacs K & Ross R C (1974) Lancet i, 357 Ishak K G & Rabin L (1975) Medical Clinics of North America 59, 995 Kelso D R (1974) Lancet i, 315 Knapp W A & Ruebner B H (1974) Lancet i, 270 Mays E T, Christopherson W M & Barrows G H (1974) American Journal of Clinical Pathology 61, 735 Meyer P, Li Volsi V A & Cornog J L (1974) Lancet ii, 1387 Model D G, Fox J A & Jones R W (1974) Lancet i, 865 O'Reilly K (1974) Australian and New ZealandJournal ofSurgery 44, 142 (1975) A ustralian and New Zealand Journal of Surgery 45, 76 O'Sullivan J P & Wilding R P (1974) British MedicalJournal iii, 7 Perez V, Gorodisch S, De Martire J, Nicholson R & Di Paola G (1969) Science 165, 805 Phillips M J, Langer B, Stone R, Fisher M M & Ritchie S (1973) Cancer 32, 463 Shojania N G & Hogg G R (1975) Gastroenterology 69, 28 Sorensen T I A & Baden H (1975) Scandinavian Journal of Gastroenterology 10, 1 13 Thalassinos N C, Lymberatos C, Hadjionnou J & Gardikas C (1974) Lancet i, 270 Tountas C, Paraskevas G & Deligeorgi H (1974) Lancet i, 1351 Zurbriggen S & Tylen U (1975) Fortschritte aufdem Gebiete der Rontgenstrahlen und der Nuklearmedizin 122, 404
REFERENCES Adlercreutz H & Tenhunen R (1970) American Journal of Medicine 49. 630 Ameriks J A, Thompson N W, Frey C F, Appelman H D & Walter J F (1975) Archives of Surgery 110, 548 Anthony P P (I1975) Lancet i, 685 Antoniades K & Brooks C E jr (1975) Surgery 77, 137 Baum J K (1975) Journal of the American Medlical Association 232, 1329 Baum J K, Holtz F, Bookstein J J & Klein E W (1973) Lancet ii, 926 Berg J W, Ketelaar R J, Rose E F & Vernon R G (1974) Lancet ii, 349
353
Dr N P Bishun (Research Department, Tissue Culture and Cytogenetics Unit, Marie Curie Memorial Foundation, The Chart, Oxted, Surrey)
Chromosomes and Oral Contraceptives The difficulty of anticipating the biological effects of chemicals in the environment applies to longterm effects like hereditary changes, cancer induction and degenerative cellular effects leading to poor health as well as to teratogenic effects. Also, mutagenic effects, in the broad sense, are of central importance. The ability of certain chemicals to alter hereditary material has been known for three decades; in fact, this knowledge has constituted a corner-stone in the development of modern experimental genetics. In spite of this, many geneticists were for a long period slow to appreciate the practical consequences of chemical mutagenesis and possible risks to human populations. There is now an increasing demand for chromosomal evaluation of chemicals released in the environment and for the elimination of those products which may cause mutations in human beings.
Proc. roy. Soc. Med. Volume 69 May 1976
14
It is now known that oral contraceptives produce profound and fundamental changes in the endocrine system of the patients to whom they are given, through their action on the hypothalamus. Dodds (1961), in a review written soon after the introduction of these contraceptive agents, suggested 'that it would be unlikely that one could submit the delicately balanced hormonal system of a woman to such a violent alteration for 40 years without something serious happening'. On account of this state of affairs, and bearing in mind the possible relationship between mutagenicity and carcinogenicity, one would expect the molecular nature of the two processes to exhibit some parallelism, especially if the chemicals concerned are initiators of malignant cell transformation. We therefore investigated the possibility of any chromosomal abnormalities occurring in women who had taken oral contraceptives over long periods, and also in their progeny, as a means to contributing further to the accumulating evidence on the safety of these synthetic steroids.
The subjects, who were chosen from the maternity ward of a general hospital, were (a) women who had taken one or more types of oral contraceptive at some time during their reproductive life (test mothers), or (b) women who had never practised any form of oral contraception (control mothers) (for details see Table 1). Both groups had entered hospital for the imminent birth of their babies which were included in the survey as test and control babies respectively. Triplicate blood cultures from control and test mothers and their respective babies were set up.
354
Results and Discussion The frequency of chromosome abnormalities in control subjects in the present study is comparable with that reported in other series. On the whole, the total results from the four groups, namely control mothers and babies, and test mothers and babies, show no significant differences in the percentage of cells with more or less than 46 chromosomes (P> 0.05). In our study (Table 2), the percentage of cells with >46 chromosomes (excluding tI iploid and
Table 1 Various factors associated with the length of time the oral contraceptive was taken
Oral contraceptive Ovulen Gynovlar Minovlar Anovlar LyndiQl
Mean length of time Length of tinme oral contraceptive betweenz ceasing oral was taken (mlonths) contraceptiv e and pregnancy (miionths) Mean Range 11.3 2-84 22.8 9.9 3-63 22.4 9.1 2-28 9.0
No. of mothers
34 19 8 9 12 15
20.4 17.6 10.1 14.9 29.8 14.7 26.0 6.3 2.0 16.3 17.9 21.3
Norinyl 10 Conovid 7 Ortho-Novin 3 Volidan 3 Sequens 3 Nuvacon l Normenon Combination 29 14 VariouLs Not known 18
14.8 10.0 5.7 9.9 3.2 4.3 8.5 6.3 0.0 7.6 2.9 18.6
1-72 2-48 1-24
3-30 2-72 2-36 12-36 3-12 2 3-48 5-60 1-36
Percentage of' niothers taking oral contraceptives
continuously 69 79 100 89 75
'0O 100 100 100 66 100 100 79 71 85
Table 2 Total chromosome results in control and test mothers and babies Cells countel in
Control
Test
mizothers
mothers
No. 84
No.
Control babies
Test
babies
Chromosomal groups < 45 45 46 > 46
Triploid Tetraploid Endoreduplicated Polyploid Gaps Breaks Total cells counted
19 2009 8 2 28 14 0
11 11 2164
% 3.98 0.97 92.9 0.4 0.1 1.3 0.6 0 0.5 0.5
89 22
1963 9 2 32 7 2 8 12 2126
% 4.2 1.0 92.3 0.4 0.1 1.5 0.3 0.1 0.4 0.6
No. 159 4.3 37 1.0 3417 92.7 6 0.2 0.03 1 47 1.3 8 0.2 0.03 1 3 0.1 8 0.2
No. 169 4.86 54 1.5 3270 92.1 10 0.3 6 0.2 34 1.0 7 0.2 0 0 14 0.4 4 0.1
3686
3550
355
15
Section of Oncology
tetraploid cells) was similar in the control and test mothers group (0.4%), but possibly slightly larger in the test babies (0.28%) compared to controls (0.16%). Only a small number of these hyperdiploid cells had sex chromosome alterations and the majority had two or three extra abnormal chromosomes which could not be allocated to any of the groups of the Denver system of classification. Goh (1967) also found a higher percentage of cells with 47 chromosomes present in women who had been using oral contraceptives (1.05%) which was considerably higher than we found in the mothers in the present study (0.4 %). The frequency of triploid cells in the test and control cultures of the mothers in the present study (0.1 %) was identical, but the test babies showed over five times as many triploids as were present in the control group (P47 chromosomes, which probably includes triploidy, as 0.9% in 5 women on the pill compared with 0.3 % in control subjects. Mechanisms which can account for the increase in triploidy include the defective segregation of one haploid set of
chromosomes from one of the two metaphase plates before division of the zygote. Triploid cells have been found in human short-term leukocyte cultures (Bishun, personal observation), and these findings were thought to represent malsegregation of one haploid set from a 2n cell line but tripolar division of a tetraploid cell is another possible explanation. The occurrence of tetraploid cells found in both mothers and babies in our study showed no significant differences from controls (P> 0.05), and no further data concerning the frequency of tetraploidy following the taking of oral contraceptives have been found in the literature. The number of endoreduplicated cells was identical in the two groups of babies in our study, and although the number of these cells in the mothefs receiving the pill (0.6%) was twice that of the control mothers group, this difference is not thought to be significant (P> 0.05). In so far as structural abnormalities of the chromosomes were concerned, no significant differences were seen in the numbers of gaps and breaks occurring in the mother groups, and no differences in breaks as far as the babies were concerned, but nearly five times as many gaps were found in the chromosomes of the babies whose mothers had been taking oral contraceptives (0.39% and 0.08 %), which is highly significant (P< 0.001). The overall findings of this chromosome study suggest that there is no increase in chromosomal damage in human subjects who had taken oral contraceptive preparations before conceiving, at the normal prescribed dosage, for modetately prolonged periods of time. However, it may be that the lapse of time, 9 months to 1 year, between cessation of taking oral contraceptives and obtaining blood samples for the present chromosomal studies might invalidate these generally negative findings. Nevertheless, it is our view that, for a true evaluation of the mutagenic potential, a period of recovery should be allowed to enable normal repair and rejection mechanisms to operate effectively (cf. Goh). Although the potential of a chemical substance for mutagenicity is not the same as its potential for carcinogenicity, this is largely a matter of species differences in tissue distribution of the chemical and its metabolism. However, our studies of leukocyte chromosomal changes in humans at normal dosage are possibly more relevant to the evaluation of the risk of cancer in human subjects than is the determination of the carcinogenic potential of these chemicals at excessively high dosage in rodents. In any case there is considerable scepticism with regard to accepted methods of carcinogenicity testing in rodents as this species has a high level of endogenous oncogenic viruses present in their tissues.
Table 3 Percentages of abnormal cells in the mothers' and babies' groups from each type of oral contraceptive
Oral contraceptive Normenon (chlormadinone acetate) Ovulen (ethynodiol diacetate + mestranol) Gynovlar (norethisterone acetate + ethynylcestradiol) Lyndiol (lynestrenol + mestranol) Anovlar (norethisterone acetate + ethynylcestradiol) Conovid
(norethynodrel + mestranol) Minovlar (norethisterone acetate + ethynykcestradiol) Nuvacon (megestrol acetate + mestranol) Volidan (megestrol acetate +ethynyloestradiol) Ortho-Novin (norethisterone +mestran'ol) Sequens (chlormadinone + mestranol) Norinyl (norethisterone + mestranol) Mean for all test subjects Mean for all control subjects
Abnormal cells in mothers (%) 4.0
Abnormal cells in babies () 0.0
4.3
1.5
4.7
1.8
3.5 2.4
2.1 0.0
2.6
4.6
1.4
0.7
0.0
0.0
0.0
4.1
4.4
3.6
2.0
1.9
1.2
2.1
3.3 3.4
1.8 2.0
The abnormal cells included are those with > 46 chromosomes, triploid, tetraploid, polyploid and endoreduplicated cells, and those exhibiting gaps and breaks, and the figures are the total expressed as a percentage of the total cells counted
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Proc. roy. Soc. Med. Volume 69 May 1976
16
Table 4 Percentages of mothers and babies with chromosomal abnormalities and percentages of abnormal cells found for each oral contraceptive constituent Mothers
Oral contraceptive
Ethinylcestradiol Mestranol Norethisterone acetate Lynestrenol Ethynodiol acetate Norethynodrel Megestrol acetate Chloramadinone
No. of blood cultures 22 51 34
Babies
Percentage with
No. of chronmosome cells abnornmalities counted 41 472 31 777 38 791
Percentage of
abnormnal cells 3.6 3.2 2.6
No. of blood cultures 27 71 43
Percentage with
Percentage
No. of chromosonme cells abnormalities counted 33 624 32 1691 30 1120
abnormal cells 1.9 2.3 2.1
of
7 22
43 27
116 314
3.4 4.5
11 31
27 23
241 347
2.0 2.0
8 3 I
25 0 100
78 27 25
2.6 0.0 4.0
7 6 1
71 50 0
174 181 25
4.6 2.3 0.0
114
33
2126
3.3
154
31
3550
1.8
124
27
2164
3.4
157
27
3686
2.0
acetate
Total for test subjects Total for control subjects
It is interesting to compare our findings on traceptives are seen largely to substantiate the mutagenicity of the oral contraceptive prepara- findings of a previous large-scale carcinogenicity tions with the evaluation of their carcinogenic study of these compounds in rodents, namely potential in rodents. In the Report by the that no overt risk to women taking the pill is Committee on Safety of Medicines (1972) on the proved (see Tables 2 and 3), although certain carcinogenicity testing of oral contraceptives, preparations merit further investigations. three preparations came under suspicion: (1) Megestrol acetate/ethinyl oestradiol and norethy- REFERENCES Medical Journal (1972) iv, 190 nodrel, which caused an increase of malignant British Committee on Safety of Drugs mammary tumours in mice. (2) Norethynodrel, (1970) British Medical Journal ii, 231 Committee on Safety of Medicines (1972) Carcinogenicity which caused an increase in mammary carcinoma. Tests of Oral Contraceptives. HMSO, London (3) Norethisterone/ethinyleestradiol,'which caused Dodds E C (1961) Journal of Endocrinology 23, 3 K G (1967) Chromosomal Breaks in Women Taking Birth an increase in benign and malignant mammary Goh Control 'Pills'. USAEC (Report), Oak Ridge Associated tumours in rats. It is perhaps significant that in Universities, 106, pp 97-104 our mutagenicity studies Conovid (mestranol/ norethynodrel), Volidan (megestrol acetate/ethinyloestradiol) and Ortho-Novin (mestranol/norethisterone) are the preparations which had a slight increase in abnormalities in babies. The following papers were also read: Several preparations show a slight, but insignificant increase of abnormalities in the mothers Sex Hormones and Breast Cancer: Epidemiological Aspects (Tables 3 and 4). In conclusion, it is becoming increasingly Professor Martin Vessey obvious that drug safety and toxicity is best (Department of Social Medicine, evaluated by methods which can be applied Oxford University, Oxford, OX] 3QN) directly to man rather than to experimental animals, and which are quickly and relatively The Effects of the Pill upon the Breasts: inexpensive. The method of examining the effect Physiological Considerations of drugs on chromosomal changes in circulating Dr R V Short leukocytes in humans has been subiected to (MRC Unit of Reproductive Biology, much criticism concerning the reproducibility Department of Obstetrics & Gyna?cology, and meaning of results and their implications as Edinburgh, EH3 9ER) regards carcinogenicity. The advantages of using relevant species (man), the relevant dosage The Effects of the Pill upon the Breasts: and the relevant circumstances of dosage (dura- Clinical Considerations tion, sex, age, &c.) outweigh this criticism. The Mr Ian Burn total results of the present study on oral con- (Charing Cross Hospital, London W6 8RF)
