Effect of 0.1% ethacridine lactate on the vas deferens and human spermatozoa- (A preliminary report based on animal and in vitro experiments)DS Kamat, EK Bharucha
Departments of Obstetrics & Gynecology and Surgery, B.J. Medical College and Sassoon General Hospitals, Poona 1., India
Ethacridine Lactate (0.1%) has been tried in dogs to show it's safety by clinical and histopathological examination of vas at different time intervals. Also in vitro titration was carried out with normal human semen and serially diluted ethacridine lactate.
One of the limitations of vasectomy is the time taken for the semen to become azoospermic. A world wide search is going on for an effective, nontoxic chemical agent which can be injected into the distal segment of vas at the time of vasectomy so as to produce immediate sterility I - washing out and destroying residual sperms in the distal genital tract.
Various workers have reported their success in this respect by using various solutions for irrigation of distal vas.
Von Friesen  has advocated a method by using 0.1% ethacridine injection into the distal segment of vas. He claimed that ethacridine killed the spermatozoa in the vas deferens and the seminal vesciles.
Craft and McQueen  have described a significant success over plain vasectomy by using "Sterile Water".
Similarly Urquhart-Hay  has reported his success by using 0.1% Euflavin.
None of these workers have noted any major side effects or complications after this irrigation technique.
A question was raised about the safety and efficacy of ethacridine lactate (0.1%) after injection into the distal segment of the vas. An animal experiment using adult male dogs was, therefore, carried out to see the safety of ethacridine lactate by clinical and histopathological examination of vas and genital organs at different time intervals.
Secondly, an in vitro study was conducted on normal human semen to see the efficacy of ethacridine lactate by doing sperm immobilization and lysis with different dilutions of ethacridine lactate.
(A) Male dogs (more than 12 months old) were used for this study. Vasectomies were carried out on 4 dogs under open-drop ether anaesthesia. In each dog, on one side, the vas was exteriorized through a para-scrotal incision-1 cm long and divided and ligated as usual to act as a control. The distal end of the vas on the other side was cannulated with a blunt ended No. 24 needle attached to a 5 ml plastic syringe containing a sterile solution of 0.1% ethacridine lactate. Two ml of the solution were injected slowly down the vas. Both proximal and distal ends of the vas were ligated. The skin was closed with a single suture of thread. Injection benzathine penicillin was given intramuscularly to protect the animal from infection.
Each dog was observed clinically every day till it's utilization. These dogs were killed at different time intervals, i.e. after 48 hours, 1 week, 2 weeks and 3 weeks and the whole genital tract along with the bladder and urethra were subjected to histopathological examination.
(B) The effect on the normal human semen was tested by mixing it with serial dilutions of ethacridine lactate (0.1, 0.09, 0.08, 0.07, 0.06 and 0.05%).
The immediate effect was noted by observing one drop of serially diluted ethacridine lactate and one drop of semen under the microscope. A drop of semen was also added in these test tubes and was observed under microscope at 5, 10 and 15 minutes intervals. The motility and integrity of spermatozoa were noted and compared with the normal semen.
(A) ne dogs appeared healthy after surgery. They were killed at 48 hours, 1 week, 2 weeks and 3 weeks intervals and both the vasa along with the bladder, the prostate and a part of the urethra were dissected out and examined histopathologically. The results are presented in [Table 1].
In the 48 hour specimen, the vas which was irrigated showed mild acute inflammatory exudate in only one section. The opposite vas, the prostatic urethra, the bladder and the prostate showed no histopathological changes.
In the one week specimen, a minimal scanty inflammatory exudate was observed in the prostatic urethra. The rest of the sections were normal.
The two and three week specimens were absolutely normal without any evidence of inflammation or fibrosis.
(B) It was observed that 0.1% ethacridine caused instant immotility of spermatozoa followed by lysis. With the 0.057, solution, a few spermatozoa remained sluggishly motile for 5 minutes but became motionless at the end of 10 minutes and were partially lysed within 15 minutes.
The observations with the intermittent concentrations of ethacridine are shown in [Table 2].
Persistence of sperms in semen after vasectomy is wellknown and many surgeons insist on collecting 2 consecutive azoospermic specimens of semen, 2 to 3 months after operation, before labelling the subject as sterile.
We routinely advise use of condoms for a minimum period of 3 months after vasectomy. Many patients find this onerous and some ignore the advice. Last year, 6 pregnancies were observed to occur after vasectomy operation at our centre. In all these, cases, either ignorance or accidental rupture of condom was the likely cause.
The acridine derivatives, ethacridine, acriflavine, euflavin are slow acting disinfectants. They are bacteriostatic against many Gram positive and a few Gram negative bacteria. They inactivate or inhibit some viruses and their activity is not reduced by tissue fluids or pus. They are used in the treatment of contaminated or suppurative wounds. Lastly, 0.1% ethacridine lactate was used intravenously for the control of infections, in the second world war and is now used as a safe, effective drug for termination of pregnancy in the 2nd trimester. It has been shown not to damage the epithelium of the genital tract and conception occurs without difficulty later in such patients.
It is known that irrigation of the vas during vasectomy flushes the sperms out of the ejaculatory ducts and the semen becomes sperm-free faster. It was, therefore, logical to test the utility of ethacridine lactate (proved harmless to the female genital tract) for carrying out such irrigation. The present study establishes the efficacy and safety of 0.1% ethacridine lactate for this purpose in dogs. The inflammatory exudate observed in one section of the vas in the 48 hours' specimens was minimal and probably due to surgical trauma. Similarly, very scanty inflammatory exudate was observed in sections of the prostatic urethra in the one week specimen. None of the sections showed any significant inflammation or change in the 2 and 3 week specimens. Thus, it would appear possible to perform vasovasostomy, if needed, at a later date. However, this experiment was not performed as a part of this study.
The drug also proved effective in in vitro experiment with normal human semen. It caused instant immobilization and lysis of spermatozoa in 0.1% concentration. Even in the concentration of 0.05% it was effective in immobilizing the sperms within 10 minutes and causing their lysis within 15 minutes. Von Friesen  has rightly claimed that 0 1% solution kills the spermatozoa and dissolves if injected slowly.
We are thankful to The Director of Medical Education and Research, Bombay and Dean, B. J. Medical College, Poona for permitting us to conduct this experiment.
We are very much thankful to Dr. R. D. Kulkarni, Professor of Pharmacology, Dr. Mrs. V. S. Gokhale, Reader in Pharmacology, Dr. Agarwal, Professor of Pathology, Dr. Karve and Dr. Mrs. Phadke, Readers in Pathology for their extended help. We are also thankful to Mr. Pillay, Mr. Abdul and all those who helped us during this trial.
Our thanks are due to Dr. U. D. Sutaria and Dr. S. P. Dani for their guidance and help.
[Table 1], [Table 2]