Fd Cosmet. Toxicol. Vol. 10, pp. 29-34. Pergamon Press 1972. Printed in Great Britain

Effects of Cyclohexylamine on the Fertility of Male Rats

S. GREEN, K. A. PALMER and M. S. LEGATOR

Division of Toxicology, Food and Drug Administration, Department of Health, Education and Welfare, Washington, D.C. 20204, USA

(Received 4 August 1971)

Abstraet--Cyclohexylamine (CHA) was tested for possible dominant lethal mutations in male rats. Female rats were mated with CHA-treated males and were then examined for the number of corpora lutea, total number of implantations and the number of implantations regarded as early deaths. The results showed significant pre-implantation loss in females mated during wk 1 and 2 after intraperitoneal treatment of the males with 100 or 300 mg CHA/kg. The number of early deaths was not significant.

An average of 35 ~ of the ova flushed 48 hr after insemination from the oviducts of females mated with CHA-treated males showed no cleavage and did not exhibit two pronuclei. This indicated that fertilization had not occurred. It is concluded that the pre-implantation loss in females mated with CHA-treated males results from some mechanism other than that of dominant lethal mutation.

INTRODUCTION

Cyclohexylamine (CHA), a metabolite of cyclamate (Leahy, Wakefield & Taylor, 1967), has been reported to cause chromosomal abnormalities in t'itro in a mammalian cell culture (Green, Palmer & Legator, 1970) and in human leucocytes (Stoltz, Khera, Bendall & Gunner, 1970). Legator, Palmer, Green & Petersen (1969) have also reported that, in vivo, CHA induces chromosome breaks and gaps in bone marrow and spermatogonial cells of rats. Since these breaks seen in rat germinal cells are evidence of gross changes in chromo- somal constitution, subvisible or visible damage that is not amenable to cytogenetic analysis may also occur at other stages of spermatogenesis.

Cytogenetic abnormalities can only be seen in the spermatocytic and spermatogonial stages of spermatogenesis, but successive phases of spermatogenesis may be examined by the dominant lethal analysis. This procedure involves the mating of treated males at weekly intervals to virgin females and the examination of the females at mid-pregnancy for pre- and post-implantation loss. This study is more concerned with the pre-implantation loss, since this was the predominant effect seen with CHA.

Pre-implantation loss is defined as the difference between the number of corpora lutea formed and the number of implantations. This loss may be the result of two conditions: (1) the sperm fail to fertilize a large proportion of the eggs released, or (2) the eggs are fertilized but fail to become implanted because of some factor which renders them incapable of surviving to implantation. The former condition is one of altered fertilizing capacity of the sperm and the resultant egg death may not be ascribed to a genetic cause. The latter condition, on the other hand, does not result from altered fertilizing capacity but from some

29

30 S. GREEN, K. A. PALMER and M. S. LEGATOR

impairment of the genetic mechanism transmitted to the egg by the sperm and leading to egg death. In addition to the dominant lethal study, a separate investigation was undertaken to determine the cause of the pre-implantation loss associated with CHA.

EXPERIMENTAL

Random-bred Holtzman strain albino male and female rats, weighing 225-250 g and 190--200 g, respectively, were used. CHA was obtained from City Chemical Corp., New York, and was tested for purity by gas chromatography. No peaks were observed other than the peak from CHA. The solutions for injection were prepared by adjusting the CHA to pH 7.3 with 4 N-hydrochloric acid and diluting with water. All concentrations were prepared immediately before injection and adjusted so that each male received 1 ml/kg body weight. No animal received more than 0-3 ml at any one injection. All males were given two injec- tions, the second being administered 4 hr after the first.

The males of one group were injected intraperitoneally with doses of 200 and 100 mg/kg and males of the other groups were given equal injections of 150 or 50 mg CHA/kg. This resulted in two groups receiving total doses of 300 mg/kg and one group receiving a total of 100 mg/kg. The different initial doses of 200 and 150 mg/kg were given to obtain infor- mation as to whether the primary dose could influence the response, although the total dosage was the same. The males of the negative control group were given the same volumes of distilled water (0.3 ml) as males of treated groups. Triethylenemelamine (TEM), the positive control compound, was administered in a single intraperitoneal dose of 0.5 mg/kg. Each CHA-treated group consisted of a minimum of five males, and the control groups consisted of a minimum of ten males. Each male was mated to at least two females each mating day. All animals were given Purina laboratory chow and water ad lib.

Animals were mated on days 2, 4, 10 and 12 (Experiment I) or on days 1, 3, 5, 8, 10 and 12 (Experiment II), day 1 being the day after injection. In Experiment I, the matings in wk 2 were not begun until day 10 to allow the males a certain amount of recovery. In Experiment II, the waiting period was not observed. Vaginal smears were taken from virgin females before mating to ensure a proper stage of oestrus and immediately after mating to ensure the presence of sperm. Detection of sperm indicated day 1 of pregnancy. Females not showing positive evidence of mating were removed from the experiments.

Females were killed 14 days after sperm was observed in vaginal smears and were scored for the number of corpora lutea, living embryos and early deaths. Data from wk I matings of the same dosage were combined, as were those of wk 2. This procedure was appropriate since results in the dominant lethal assay are usually reported in terms of weekly effects; in addition, the distribution of the percentages of eggs implanting per female for a given control within a given week did not differ significantly.

The distribution of the percentages of eggs implanting per female for treated and control groups was compared by the non-parametric Mann-Whitney U test (Goldstein, 1969). Therefore the sampling unit in all cases was the individual female. The average pre-implanta. tion and post-implantation loss is included in the tables for ease of presentation, but the statistical evaluation was done by comparing the responses of individual females within dosage level within a certain week with those of the appropriate controls.

In a separate experiment, the effects of CHA on pre-implantation development wer studied in females mated during wk 1. All females from these matings were examined 48 h after insemination. Oviducts were flushed according to procedures outlined by F. J. Ca houn (personal communication, 1970). The ovary, oviduct and a small portion of t~

ANTIFERTILITY EFFECTS OF CYCLOHEXYLAMINE 31

uterine horn were removed and placed on an absorbent surface previously wetted down with physiological saline. The fatty tissue and ovarian bursa were trimmed from the ovary with small curved scissors and fine-pointed forceps, the ampulla end of the oviduct was found and the connecting ligament was cut. The mesentery was then carefully cut to uncoil the oviduct. Physiological saline was forced through the ampulla end by a 1-ml tuberculin syringe fitted with a 26-gauge needle. Small haemostats around the oviduct and needle served to prevent retrograde flow. Drops of saline containing ova were then collected in depression slides and examined under phase-contrast microscopy. The distribution of the percentage &eggs that had divided per female was compared by the Mann-Whitney U test.

RESULTS

Several deaths occurred within 4 hr in the group of males given the primary CHA dose of 200 mg/kg. No deaths were recorded for the group administered 150 mg/kg. Except for the initial toxicity, responses (weight and fertility) of the group given 200 mg/kg as the primary dose were similar to those of the group given 150 mg/kg. Rats surviving the initial injection usually recovered and sustained the second injection. At the time of the second injection, rats that had been given 200 or 150 mg/kg as the initial dose had lost approxi- mately 15 g body weight, and those injected at the 50 mg/kg level had lost approximately 12 g. At 24 hr, animals treated with total doses of 300 mg/kg had failed to recover from the weight loss caused by CHA, whereas those treated with a total dose of 100 mg/kg had regained the weight. This loss of weight may be attributed to the diuretic action of the CHA salt.

In Experiment I, females mated 2 or 4 days after injection of the males with CHA had a significant number of eggs that failed to implant (Table 1). Those mated 10 or 12 days afterwards showed a similar but less-marked response. In all cases the number of early deaths was not significant. Results of only two mating days in wk 1 and two in wk 2 were investigated.

In Experiment II, results of three mating days in wk 1 and three in wk 2 were recorded. Pre-implantation loss was significant in females mated to animals treated with total doses of 300 (200 + 100) or 100 mg/kg in wk 1 (Table 2). Post-implantation loss was significant only in the group given TEM. The number of females throughout this experiment was reduced because of the low number of sperm-positive animals.

Table 1. Fertility data from females mated to CHA-treated males: Experiment I

Total intra- Females with Average Average peritoneal Females pre-implantation pre- post-

dose of Wk pregnant] loss implanta- implanta- CHA of females Fertility tion loss1" tion loss

(mg/kg) mating mated (%) No. ~0 (~o) (~)

0 1 31]39 79.5 21 67.7 16"5 9.0 300 1 11]20 55.0 9 81.8 65-5** 12.7

(20O + 100) 0 2 19/40 47.5 lO 52.6 8.3 10.3 300 2 10]20 50.0 6 60.0 28.0* 20.4

(200 + 100)

tCalculated on the number of females with pre-implantation loss. Values marked with asterisks are significantly greater than the control value: *P<0.05; **P<0.01.

F.C.T. 10/I---C

32 S. GREEN, K. A. PALMER and M. S. LEGATOR

Table 2. Fertility data from females mated to CHA-treated males: Experiment II, wk 1 of mating

Total Females with Average Average intra- Females pre-implantation pre- post-

peritoneal pregnant/ loss implanta- implanta- Corn- dose females Fertility tion losst tion loss pound (mg/kg) mated (~o) No. ~o ( ~ ) (~o)

- - 0 18/60 30"0 4 22"2 46"3 12"2 CHA 300 9/30 30"0 8 88"9 86"1" 13'3

(200 q- 100) CHA 300 10/30 33.3 6 60-0 49-0 8.3

(150 + 150) CHA 100 11/30 36.7 7 63-6 51.1" 10.4

(50 + 50) TEM + 0-5 8/30 26.7 7 87.5 38 '3 56.4*

tCalculated on the number of females with pre-implantation loss. ++Positive control (triethylenemelamine). Values marked with an asterisk are significantly greater than control values: *P< 0"05.

In wk 2 (Table 3), pre-implantation loss was significantly increased in all groups. The response of females mated to TEM-treated males is typical of that obtained in weekly mating studies by using the conventional dominant lethal assay; that is, the pre-implantation loss in wk 2 was greater than that in wk 1. This response also tends to separate the agents as far as their site of action is concerned. CHA had an immediate effect on pre-implantation, indicating an action on spermatozoa or on mature sperm. TEM, on the other hand, was effective predominantly in wk 2, indicating an action on less mature cells.

To examine other possibilities for the effects seen with CHA, ova were counted and examined under phase-contrast microscopy. Under normal conditions, fertilized eggs should undergo at least two divisions or exhibit two pronuclei in 48 hr. An examination of females mated to CHA- and TEM-treated males is summarized in Table 4. Although the percentage

Table 3. Fertility data from females mated to CHA-treated males: Experiment 11, wk 2 of mating

Com- pound

Total Females with Average Average intra- Females pre-irnplantation pre- post-

peritoneal pregnant/ loss implanta- implanta- dose females Fertility tion losst tion loss

(mg/kg) mated ( ~ ) No. ~o (~o) ( ~ )

- - 0 18/60 30"0 5 27"8 12"6 10"3 CHA 300 8/30 26"7 7 87"5 59"5* 7"4

(200 + 100) CHA 300 7/30 23.3 6 85.7 35.5* 14.8

(150 + 150) CHA 100 9•30 30-0 6 66.7 35"0* 8.3

(50 + 50) TEM + 0"5 7/30 23 "3 7 100.0 97-1 * 100§

tCalculated on the number of females with pre-implantation loss. +Positive control (triethylenemelamine). §Based on two females; insignificant. Values marked with an asterisk are significantly higher than the control value: *P< 0.05.

ANTIFERTIL1TY EFFECTS OF CYCLOHEXYLAMINE

Table 4. Effects of TEM- and CHA-treatment of males on pre-implantation deoelopment

33

Compound

Intra- Females Females with peritoneal pregnant/ non-dividing eggs

dose females Fertility (mg/kg) mated (%) No. %

Non-dividing eggs/female

(%)

- - 0 10/10 I00 6 60 11.0 CHA 300 6/I0 60 5 83 35.16" TEMt 0.5 5/I0 50 4 80 5.2

tPositive control (triethylenemelamine). Value marked with an asterisk is significantly greater than the control value: *P< 0.05.

of females with non-dividing eggs was the same for the TEM- and CHA-treated groups, the average percentage of non-dividing eggs for CHA was seven times higher than that of TEM and three times higher than that of the control (Table 4). As was the case for pre- implantation loss, the average percentage of non-dividing eggs is included for ease of pre- sentation; the sampling unit for the statistical evaluation was the individual female.

DISCUSSION

It is generally assumed that pre-implantation loss indicates a very high incidence of dominant lethal mutations; therefore the embryo or foetus dies earlier (Rohrborn, 1968). If this is true, it should also be possible with decreasing dosages to induce an incidence of mutations that would be compatible with survival to at least the implantation stage. Because the incidence of pre-implantation loss was very high with CHA and lower doses did not result in post-implantation loss, it was questionable whether a true dominant lethal effect was operating.

The results obtained from the study of egg development supported our contention that CHA was operating by a different mechanism. These data (Table 4) clearly differentiate between the action of TEM, which induces true dominant lethality, and that of CHA, which does not. This indicates that the pre-implantation loss seen with TEM is of genetic origin, whereas that seen with CHA is not.

The mechanism by which CHA given to males causes pre-implantation loss in mated females is not known, but there are several possibilities. The stage of spermatogenesis affected is the spermatozoa phase. Spermatozoa spend approximately 15 days in the epididymis. Jackson (1966) suggested that during this time they undergo a maturation process and are also readily accessible to drug action through the blood supply to this organ and the secretion of fluid by the epididymal cells.

Another possibility is that CHA may have an effect on the biochemical processes in- volved at this stage of spermatogenesis. Fox & Fox (1967) have found two enzymes, glycerophosphate dehydrogenase and hyaluronidase, which are operative at the epididymal stage. The former enzyme is involved in glycolysis, whereas the latter catalyses the hydro- lysis of hyaluronate and chondroitin. The possible effects of CHA on hyaluronidase activity warrant investigation, since hyaluronidase is localized in the acrosome of the spermatozoa and may function in the penetration of the egg by the spermatozoa before fertilization (Fox & Fox, 1967). If CHA in some manner renders hyaluronidase incapable of functioning,

34 s. GREEN, K. A. PALMER a n d M. S. LEGATOR

eggs w o u l d n o t be fer t i l ized, as is the case in females m a t e d to C H A - t r e a t e d males . Th i s

poss ibi l i ty , a l o n g wi th o thers , r e m a i n s to be exp lo red be fo re a m e c h a n i s m o f ac t ion can be

ass igned to the an t i - fe r t i l i ty effects o f C H A .

R E F E R E N C E S

Fox, B. W. & Fox, Margaret (1967). Biochemical aspects of the actions of drugs on spermatogenesis. Pharmac. Rev. 19, 21.

Goldstein, A. (1969). Biostatistics, An Introductory Text. p. 55. The Macmillan Company, New York. Green, S., Palmer, K. A. & Legator, M. S. (1970). In vitro cytogenetic investigation of calcium cyclamate,

cyclohexylamine and triflupromazine. Fd Cosmet. Toxicol. 8, 617. Jackson, H. (1966). Antifertility Compounds in the Male and Female. p. 5. Charles C. Thomas, Springfield,

Illinois. Leahy, J. S., Wakefield, M. & Taylor, T. (1967). Urinary excretion of cyclohexylamine following oral

administration of sodium cyclamate to man. Fd Cosmet. Toxicol. 5, 447. Legator, M. S., Palmer, K. A., Green, S. & Petersen, K. W. (1969). Cytogenetic studies in rats of cyclo-

hexylamine, a metabolite of cyclamate. Science, N. Y. 165, 1139. Rohrborn, G. (1968). Mutagenicity tests in mice. I. The dominant lethal method and the control problem.

Hum. Genet. 6, 345. Stoltz, D. R., Khera, K. S., Bendall, R. & Gunner, S. W. (1970). Cytogenetic studies with cyclamate and

related compounds. Science, N. Y. 167, 1501.

Effets de la cyclohexylamine sur la f~condit~ des rats m~les

R~sum~---Afin de d6terminer si la cyclohexylamine (CHA) pouvait provoquer des mutations h[tales dominantes chez les rats m~des, on a accoupl6 des rats femelles avec des mfiles pr6alable- merit trait6s par ce produit, puis on a compt6, chez les femelles, les corps jaunes, le hombre total de f6condations et eelui des f6condations consid6r6es comme pr~cocement avort6es. Les r6sul- tats ont r6v616 un pourcentage significatif de pertes avant f6condation chez les femelles accoup- 16es darts les deux semaines suivant le traitement intrap6riton6al du mille par 100 ou par 300 mg de CHA/kg. Le hombre des morts foetales pr6coces n'a pas 6t6 significatif.

En moyenne 35 % des ovules 6vacua, dans les 48 h suivant l'ins6mination, des oviductes de femelles accoupl6es b. des names trait6s au CHA, n'6taient pas cliv6s et ne pr~sentaient pas deux pronuclei. Ils n'avaient donc pas 6t6 f~cond6s. On en conclut que les pertes avant f6condation, chez les femelles accoupl~es b. des mfiles trait6s au CHA, sont imputables/~ un ph6nom~ne autre que celui de la mutation 16tale dominante.

Einfluss yon Cyclohexylamin auf die Fertilitfit mfinnlicher Ratten

Zusammenfassung--Cyclohexylamin (CHA) wurde auf m/Sgliche dominante letale Mutationen in m~innlichen Ratten gepriift. Weibliche Ratten wurden mit CHA-behandelten m~innlichen Tieren gepaart und dann auf die Zahl der Corpora lutea, die Gesamtzahl der Implantationen und die Zahl der als frtihes Absterben angesehenen Implantationen untersucht. Die Ergebnisse zeigten signifikante Pr~iimplantationsverluste in weiblichen Tieren, die w~ihrend der ersten und zweiten Wochen nach der intraperitonealen Behandlung der m~innlichen Tiere mit 100 oder 300 mg CHA/kg gepaart worden waren. Die Zahl der friihen Absterbef/ille war nicht signifi- kant.

Durchschnittlich 35 Yo der 48 Stunden nach der Insemination aus den Eileitern der weiblichen, mit CHA-behandelten mannlichen Tiere gepaarten Tiere ausgespiilten Ova zeigten keine Furchung und nicht zwei Pronuclei. Dies I/isst darauf schliessen, dass die Befruchtung nicht erfolgt war. Es wird daraus gefolgert, dass der Pr~implantationsverlust bei weiblichen Tieren, die mit CHA-behandelten m~innlichen Tieren gepaart worden waren, auf einen anderen Mechanismus als den dominanter letaler Mutation zuriickzufiihren ist.