American Journal of Pathology, Vol. 13 1, No. 2, May 1 988 The Morphologic Effects ofSynthetic Reproductive Steroids on the Mammary Gland ofRhesus Monkeys Mestranol, Ethynerone, Mestranol-Ethynerone, Chloroethynyl Norgestrel-Mestranol, and Anagestone Acetate-Mestranol Combinations F. A. TAVASSOLI, MD, From the Department of Gynecologic and Breast Pathology and the H. W. CASEY, COL, USAF, VC, Department of Veterinary Pathology, Armed Forces Institute of and H.J. NORRIS, MD Pathology, Washington, DC A total of213 treated and 16 control monkeys compris- 22 of 52 animals (42%), including five identical to in- ing 12 experimental groups was evaluated for determi- traductal carcinoma in the human and one identical nation of the long-term (10 years) effects of various to lobular neoplasia. Of the 40 monkeys administered dosages of a variety ofsynthetic oral contraceptive ste- anagestone acetate and mestranol, 20 (50%) developed roids on the mammary glands ofrhesus monkeys. The proliferative atypias; the atypias ranged from mild to steroid hormones included mestranol, ethynerone, a severe and included five resembling intraductal carci- combination ofmestranol and ethynerone, chlorethy- noma in human breast. The chloroethynyl norgestrel nyl norgestrel plus mestranol, and anagestone acetate and mestranol combination induced proliferative plus mestranol. Various degrees ofphysiologic lobular atypia in 25 of 52 monkeys (49%); six of these atypias hyperplasia and lactational changes were observed in were severe and indistinguishable from intraductal association with all of these steroid hormones; these carcinoma of the human breast; and one, if in the hu- changes appeared dose-dependent. Mestranol caused a man breast, would reflect a solid variant of an invasive proliferative atypia ranging from a minimal to a mod- carcinoma. Only 2 of the 16 control monkeys (12%) erate degree in 8 of 34 (23%) animals, but it was not developed proliferative atypias, and these were ofmin- dose-related. Eleven of 15 monkeys (73%) adminis- imal to mild degree. The occurrence of severe degrees tered ethynerone developed proliferative atypia, rang- ofatypia identical to intraductal carcinoma in the hu- ing in degree from minimal to severe, including one man breast and invasive carcinoma associated with invasive carcinoma and 2 lesions resembling intraduc- hormone administration suggests a carcinogenic tal carcinoma in the human. The mestranol and ethyn- effect. (AmJ Pathol 1988, 131:213-234) erone combination produced a proliferative atypia in THE LONG-TERM effect of estrogen and estrogen- Supported by National Institute ofChild Health and Hu- progestin combinations on the human and monkey man Development Contract NO l-HD-2-2729. breast have not been studied systematically. A num- The investigators adhered to the principles described in ber of divergent opinions have appeared in the litera- "Guide for the Care and Use of Laboratory Animals" pre- ture regarding the association of oral contraceptive pared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, use with fibrocystic changes and benign and malig- National Research Council. Accepted for publication December 16, 1987. The opinions and assertions contained herein are the pri- Address reprint requests to F. A. Tavassoli, MD, Vice vate views of the authors and are not to be construed as Chairman, Gynecologic and Breast Pathology Department, official or as reflecting the views of the Department of the Armed Forces Institute of Pathology, Washington, DC Army or the Department ofDefense. 20306. 213 214 TAVASSOLI ET AL AJP * May 1988 nant breast lesions in humans.1-6 Various results have The sources of chloroethynyl norgestrel and mestra- been reported in a small number ofanimal studies re- nol were the Department of Health, Education, and garding the effect ofdifferent reproductive steroid hor- Welfare and the FDA, Washington, DC; Merck and mones.71-3 This study describes the effects of mestra- Company, Inc., Rahway, New Jersey; and Merck, nol, ethynerone alone, and combinations of mestra- Sharpe and Dohme, West Point, Pennsylvania. The nol with ethynerone, chlorethynyl norgestrel, or anagestone acetate plus mestranol (10:1) was received anagestone acetate on the mammary glands ofrhesus from the Ortho Research Foundation, Raritan, New monkey over a 10-year period. With the exception of Jersey. those dying from various infections, all monkeys re- The steroids and steroid combination compounds ceived hormones for 10 years for comparison with were administered orally in 28-day cycles consisting control monkeys. Morphologic effects observed in- of 21 days of administration followed by 7 days with- cluded physiologic lobular hyperplasia, lactational out the drug. The steroids were mixed by means of a change, and an atypical proliferation that was graded mortar and pestle with an applesaucelike food vehicle from minimal to severe. The nature and significance at varying concentrations so that a constant volume ofthese findings are discussed. of 1.0 ml/kg could be administered at all dosage levels. The monkeys usually consumed the substances. Materials and Methods When the food vehicle was refused, the monkeys re- ceived the dosage by gastric intubation. Individual All specimens from the 229 rhesus monkeys and daily doses were calculated on the basis of body the clinical data for this study were provided by the weights obtained once every 4 weeks. Food and Drug Administration, which initiated a se- The first group consisted of the control monkeys. ries of long-term toxicity studies with oral contracep- The animals were 16 immature rhesus females that tive steroids in 1968. The experimental design and weighed 1.8-3.6 kg at the start of the 10-year study. methods used were according to a common protocol The animals underwent necropsy when they reached used in long-term studies ofthe toxicity ofsteroid con- 10 years on the study. Three monkeys died during this traceptives with guidelines established by the Food period, at 5.0, 5.4, and 5.5 years; and 2 animals were and Drug Administration. The purpose of the study sacrificed at 8 years for special eye studies. The mam- was to determine the effect, if any, of mestranol and mary glands from all 5 of these monkeys were sam- various other synthetic steroids on the monkey breast. pled, and the findings included in the final results. The The steroids were synthesized and tested because of next group consisted ofmonkeys receiving mestranol; intentions to utilize them in oral contraceptive com- these were divided into subgroups A and B. Subgroup pounds for human use. The protocol recommended A consisted of 15 monkeys receiving 0.02 mg/kg/day 12 monkeys per group for each dosage level of each of mestranol (10 times the proposed human dose); steroid. and Subgroup B, of 19 monkeys receiving 0.1 mg/kg/ The light- and electron-microscopic appearance of day of mestranol (50 times the human dose). Eight 213 female rhesus monkey (Macaca mulatta) mam- monkeys in Subgroup A died ofinfections, at 3.1, 3.4, mary glands comprising 11 experimental groups was 4.4, 4.8, 6.0, 7.0, 7.7, and 9.3 years after initiation of studied after 10 years ofexposure to various synthetic the study. Thirteen monkeys in Subgroup B died in a steroid hormones. The hormones used were mestra- similar manner, at 0.25, 0.25, 0.5, 0.9, 1.6, 2.3, 2.7, nol, ethynerone (MK-665), ethynerone plus mestra- 3.8, 5.3, 6.1, 7.9, 8.4, and 9.0 years. This left only 7 nol, chloroethynyl norgestrel (WY-4355) plus mes- and 6 monkeys, respectively, that completed 10 years tranol, and anagestone acetate (ORF- 1658) plus of the study in each subgroup. The third group con- mestranol. The progestational compounds included sisted of 15 monkeys on 2.0 mg/kg/day ofethynerone halogenated 19-nortestosterone derivatives (chloro- (50 times the proposed human dose). Five of these ethynyl norgestrel and ethynerone) and a 1 7-acetoxy monkeys died of infections, at 0.27, 4.0, 4.6, 4.8, and derivative of progesterone (anagestone acetate). Mes- 7.6 years. A fourth group received a combination of tranol, 3-methyl ester ofethynyl estradiol, is the estro- ethynerone and mestranol and was divided into three gen component of several marketed oral contracep- subgroups. Subgroup A consisted of 16 monkeys on tives, while the other compounds are investigational 0.084 mg/kg/day ofa combination ofethynerone and progestins that have never been marketed. The mestranol; B, of20 monkeys receiving the same com- sources of mestranol and ethynerone were the Food pound at a dose of0.42 mg/kg/day; and C, of 16 mon- and Drug Administration (FDA), Washington, DC; keys receiving 2.1 mg/kg/day of the same combina- Merck and Company, Inc., Rahway, New Jersey; and tion. The dosages represent 2, 10, and 50 times the Merck, Sharpe and Dome, West Point, Pennsylvania. proposed human dose, and the ratio of progestin to Vol. 1 31 * No. 2 STEROID-INDUCED MAMMARY LESIONS 215 mestranol was 20:1. Three of the monkeys in Sub- Table 1 -Number of Monkeys That Died in Each Group and Status of Mammary Glands at Autopsy group A died, at 4.2, 4.5, and 10 years. Six monkeys in Subgroup B died, at 0.2, 0.3, 0.4, 1.1, 6.5, and 7.7 Contraceptive and Number Autopsy findings in dosage (mg/kg/day) dead mammary tissue years. Four monkeys in Subgroup C died, at 2.6, 8.9, 9.0, and 9.6 years. The monkeys in the fifth group re- Control, no hormones 5 Normal ceiving chloroethynyl norgestrel plus mestranol (20: 1) Mestranol (0.02) 8 1, moderate PA; 7 normal Mestranol (0.1) 13 2, lactational change were divided into three subgroups, namely, Subgroup Ethynerone (2.0) 5 4, lactational change; A, consisting of 17 monkeys; B, of 17 monkeys; and 1, autolyzed C, of 18 monkeys.