CRRONIC TOXICITY OF STYRRNX MALXIC ANRYDRIDE, A MALE CONTRACEPTIVR, IN RHESUS RONXRYS (Macaca mulatta)
N. Sethi, M.B.B.S., M.S. (USA), Path. Board (USA) R.K. Srivastava, Ph.D. R.K. Singh, Ph.D. G.S. Bhatia, Ph.D. N. Sinha, Ph.D. Division of Toxicology Central Drug Research Institute Lucknow - 226 001, India Abstract
A newly developed male contraceptive, styrene maleic anhydride (SMA), was injected in the vas deferens of male rhesus monkeys for safety evaluation at the dose of 100 mg (contraceptive dose, CD), 250 mg (CD x 2.5) and 500 mg (CD x 5.0), and the monkeys were kept under observation for one year. The observed behavioural, haematological, biochemical and histopathological parameters in treated monkeys were comparable to controls. The results suggest the polymer SMA to be safe up to 5 times CD in monkeys. Introduction
non-invasive male A new simple occlusive and contraceptive method has been developed (1) by using the polymer, styrene maleic anhydride (SMA). This polymer, when injected into the lumen of the vas deferens, exerts a contraceptive effect by occlusion as well as lowering the pH of the lumen (1). Before a new drug is released for clinical trials in humans, the subacute and chronic safety evaluation studies are done in at least two species of rodents and one species of non-rodents, i.e., monkeys (ICMR 1987 (2)). Earlier, the subacute safety evaluation studies In this communication, chronic were done in rats (3). toxicity data generated in rhesus monkeys are communicated. All correspondence to the first author: Dr. (Mrs) N. Sethi Assistant Director & Head Division of Toxicology Central Drug Research Institute Lucknow - 226 001, India Submitted for publication April 2, 1990 Accepted for publication June 28, 1990
SEPTEMBER 1990 VOL. 42 NO. 3
337
CONTRACEPTION Materials
and Methods
Fourteen healthy mature male rhesus monkeys, which were negative (tuberculosis was tested with KOT tuberculin Tuberculin Diagnostic Kit obtained from Budapest, Hungary) with body weight of 4.6 + 0.42 kg, were purchased from a acclimated to dealer and the laboratory commercial conditions for a period of one month, whilst fed on monkey pellet diet (supplied by Hundustan Lever Limited, Bangalore) Bananas and seasonal green vegetables and water & libitum. were supplemented from time to time. Monkeys were carefully observed for weight loss and Twelve animals were randomly divided untoward symptoms. were used as into 4 groups of 3 each, and 2 monkeys under uniform untreated controls. They were maintained conditions throughout the experiment and were husbandry carefully observed daily for any behavioural change. In the beginning, the polymer styrene maleic anhydride (SMA) dissolved in 0.5 ml DMSO was injected into the vas deferens at the dose levels of 100, 250 and 500 mg per vas deferens in low (group II), high (group III) and highest (group IV) dose groups, respectively, while vehicle and blank control (group I) monkeys received 0.6 ml DMSO and 0.5 ml distilled water per vas deferens per monkey. For injection, monkeys were anaesthetized with 30 mg/kg body weight intraval sodium (M & B, Bombay). The vas deferens just above the scrotum in the inguinal region were bilaterally exposed, care being taken not to injure the SMA dissolved in DMSO was associated blood vessels. injected into the lumen of the vas deferens in the direction pointing distally from the testis. Care was taken so that the injections were made at the same level of the vas deferens in all the animals. The wound was stitched, dressed with neosporin antibiotic powder and merbromin solution NF (2% w/v). After surgery, 0.5 ml teramycin solution (250 mg/5 ml, Pfizer) was injected intramuscularly in each monkey for 5 successive days. All monkeys were kept under observation until healing was completed. A daily record of the behaviour and general health of all monkeys was maintained. Body weight of the monkeys was recorded at monthly intervals, while haemograms-haemoglobin, haematocrit, total erythrocyte count, total leucocyte count, count, differential leucocyte platelet count, mean corpuscular volume, biochemical investigations -and cholesterol, blood urea, blood sugar, serum creatinine, serum alkaline serum phosphatase, glutamate pyruvate transaminase, albumin, total protein, globulin, and bilirubin -- were carried out at 0, 3, 6, 9 and 12 months of the experiments. Food consumption of each animal was regularly checked. Animals were carefully observed for
338
SEPTEMBER 1990 VOL. 42 NO. 3
CONTRACEPTION
changes in gross activity, hair loss, pupil size, ptosis, respiration, general reflexes, muscle tonicity of body and The production of limbs, after implantation of SMA. catalepsy, blanching, reddening, cyanosis, salivation, lachrymation, involuntary micturition and defecation, if any, was routinely checked. All the animals were sacrificed after 12 months of the implant by a high dose (40 mg/kg) of pentathol sodium. The gross observation included absolute and relative weights of liver, lung, spleen, kidney, brain, heart and gonads. The tissues -- liver, heart, kidneys, spleen, adrenals, trachea, lungs, oesophagus, stomach, pancreas, testes, brain, skin, eyes -- were fixed in 10% formal saline, processed and embedded in paraffin wax: serial sections (5 pg thick) were stained with haemotoxylin-eosin for microscopic examination. observations and Results &&y weisht -- In control (blank and vehicle) and low dose monkeys, steady body weights were observed, while in high and highest dose group animals, fluctuating body weights were seen. At the termination of the experiment, body weight in the high dose group remained unchanged and had slightly fallen in the highest dose monkey (Fig. 1). Food consumntion -- Food/water intake in all the animals of treated and control groups were almost the same. Hortalitv -- One monkey each of the control and the low dose group died after 7 and 10 months, respectively, after injection of the vehicle or drug. Initially, these monkeys tuberculin negative, while were after death, the histological examination of the lungs revealed that they were tuberculosis positive. Probably this was due to the husbandary conditions of the primate colony at that time. Gross behaviour -- Within a week, healing in animals was completed, and the monkeys became adapted to the stress of handling and injections. They did not show any symptom of nervousness or hyper/hypoexcitability, irritation/ depression, etc., at the time of drug or thereafter. Haematoloaical studies -- There were variable changes in haematological parameters, but they were within the physiological range (Table I). Biochemical studies -- The biochemical parameters studied did not cross the normal limits of variation in any of the groups. Fluctuations observed within the groups were attributed to intrinsic biological differences (Table II). Autonsy, aross and microsconic observation -- The .animals were found to be healthy and active at the time of
SEPTEMBER 1990VOL. 42 NO. 3
a
340
I
SEPTEMBER 1990 VOL. 42 NO. 3
13200 24170
320333 397500 k149694 + 3535
10800 21352
11250 27721
Platelet count 330000 385500 (per mm3) 228284 + 6363
Totalleucocyq 11700 count (per rrm )-(789
12400 _+2076
10866 &2309 12300 23394
2152752 +38857
396666
3066666
74.3 +2.0
73.0 22.8
72.6 22.7
~37527
361616
10400 2 530
41.6
76.5 43.5
391000 317500 +50921 224748
22600
10000
+2.1
75.0
78.3 21.1
42.1
75.5
74.0 A3.5
76.0 Mean corpuscular vol (cy) lt2.8
24.5
+2.2
+4.9
40.14
41.9
Haematocrit (X1
+ 5ooc
2750000
10500 21913
22.4
74.0
38.6 21.1
34.3
40.2 40.5
39.7 13.4
44.3
42.5
42.5
38.0
37.4 ro.9
35.3
5.23 to.33
5.43 ~0.36
5.01 40.21
5.34 40.79
6.36 20.19
5.34 20.71
5.52 ~0.24
5.53 $0.33
5,26 10.27
Red blood car- 5.22 puscles (million/nm3) k"*28
12.9 LO.2
13.5 20.8
12.7 LO.1
14.3 ~0.6
12.6 20.9
14.0 '0.9
12.6 ,O.l
13.9 20.6
12.9 20.1
13.0 20.2
Haemoglobin (g%)
Final Initial ----_--
Initial
Final
Initial
Final
Initial
Final
Highest dose
Final
(Values are mean + S.D.) -_-_-.Low dose Control High dose
Initial
-_-_ -_ Blank Control Vehicle
Haematological values in control and polymer-implanted monkeys
Measurement (unit)
Table I.
E T1
8 3
Biochemical
26.0 t 0.9
28.0 _ + 0.0
7.6 2 0.
0.4 + 0.0
SGPT (units/ml)
Total protein (q/l00 ml)
Bilirubin (mg/lOO ml)
0.4 t 0.0
7.2 t 0.6
1.4 +1.2
0.20 +o.o
0.30 tO.0 -
Serum creatinine (mg/lOO ml)
Serum alkaline 1.4 phosphatase +0.2 (BL units/ml) -
97.5 t 2.7
95.0 + 4.2
Blood sugar (mg/lOO ml)
0.4 t 0.0
7.5 t 0.3
26.0 t 0.0
1.1 t0.2
0.50 tO.0
92.6 +15.2
28.3 t 7.5
96.6
t 7.6
99.0
27.0 + o-0
control
are
0.3 t 0.0
7.0 ~0.8
25.3 t 9.5
1.0 co,4
0.40 tO.01
92.6 + 3.5
29.9 t 2.1
t 3.5
97.5
Initial Final --------
Vehicle
(Values
monkeys
0.4 t 0.1
7.7 t 0.6
t 4.0
29.0
1.6 to.3
0.30 IO.01
92.0 + 7.6
27.3 t 1.5
+ 1.4
99.0
Initial
0.3 9 0.0
0.4 to.1
7.7 t 0.9
28.3 t 3.0
26.0 t 2.8 7.4 t 0.3
1.2 tO.l
0.40 +0.02
96.6 t 7.9
26.6 t 2.3
t 1.8
95.0
2.7
94.0
0.4 t 0.2
7.4 t 0.4
25.6 t 9.2
1.1 t0.2
0.30 to.01
t 7.6
95.6
25.3 t 1.7
,*
93.3
7.0 2 0.6 0.4 t 0.0
0.4 t 0.1
25.7 * 2.1
1.0 tO.l
0.30 to.03
91.0 t 6.5
26.2 t 1.4
t14.0
7.2 t0. 2
26.3 t 1.1
1.5 $0.2
0.30 to:02
96.6 t 3.3
28.0 t 2.7
t 2.3
94.6
.__.I_-_-.I_I-Highest dose High dose Initial Final Initial Final --II__--
1.4 to.1
0.40 to.03
91.0 t 6.2
23.5 t 1.2
+ 3.5
92.5
Final
mean + S.D.) _-.-Low dose
in control and polymer-implanted
+ 1.4
27.6 t 1.3 -
99.0 + 1.4
Final
COIItrOl
values
(mg/lOO m4
Blood urea
Total cholesterol(mg/lOOml)
Blank Measurement Initial (unit) ----
_--_.--------
Table II.
P
F z s
5
8
CONTRACEPTION nasal mucosae sacrifice. Fur coat, conjunctivae, in the vas deferens normal, sites of implantations normal.
were were
revealed Gross examination of the tissues varying degrees of splenic enlargement in a small percentage of both All other organs and control and drug implanted animal. shape and size (Table viscera were normal in appearance, III). The microscopic examination of the vas deferens in polymer-injected monkeys revealed the polymer was seen lying in the lumen as an amorphous material which was stained pink with eosin (Fig. 2). This occuppied one-fourth of the lumen and did not cause any occlusion (4). It had entered the follicles of the mucosa, but no implant was seen in the muscular layer. The wall of the vas deferens was not infiltrated, and there was no evidence of any inflammatory reaction. The mucosa of the vas deferens was lined by low cuboidal cells which looked like columnar cells in a few places. In control monkeys, the mucosa of the vas deferens was normal (Fig. 3). Incidental batholouv -- Incidental pathology was seen in the lungs, liver, kidney and spleen. In tot0 four animals had pulmonary tuberculosis which was diagsd only at the time of autopsy. Out of 4 monkeys, 2 monkeys had died during the after 7 and 10 experiment months, respectively. These animals were distributed in all the including the control group. In the other 2 groups, monkeys, tuberculosis diagnosed after was histological examination of the lung only at the termination of the experiment. Discussion Many methods of male contraception are presently based only on the blocking of spermatozoa in the vas deferens lumen. Several investigators have reported the blocking of the lumen of vas deferens with the use of injectable and solid plugs (5-10). A new contraceptive, SMA, was selected for study, being a polymer of maleic anhydride and styrene. When SMA comes in contact with water, the acid anhydride The time required for hydrolysis component is hydrolysed. in experimental studies is one week. The polymer, after injection, reacts with cellular secretion and forms a stable precipitate within the lumen and makes the environment such that the spermatozoa passing through are killed. Chronic safety evaluation studies are done with a view to find whether newly invented drugs could affect the target Likewise, the organ as well as other organs of the body. organs in different different new drug reach may proportions/constituent which may likely have/not have side
SEPTEMBER 1990VOL. 42 NO. 3
343
2.55 2.58
3.03
3.02
R
L
Gonad
_I--
0.11 0.11
0.15 0.16
R L
Mrenal
--_“_
2033 2.37
2.79 2.96
Kidney R L
w_--
1,96
2.09
2.27
Spleen
.-
.
2.40
2.40
0.11 0.11
2.31 2.25
11.63
11.96
14.96
Brain
_-
A-1
2.46 2.45 0.10 0.10 2.65 2.66
2.48 2.43 0.13 0.13 2.57 2.58 L__-._I__
1.89
13.05 2.48
13.73
29.60
31.24
26.03
27.58
33.21
Liver
4.06
4.69
3.96
4.13
4.91
Heart
Highest dose
High dose
Vehicle control
Blank control
Low dose
--
Mean relative organ weight (g/kg body weight) of control and polymerimplanted monkeys
Organ
Table III.
CONTRACEPTION
Fig. 2.
Fig. 3.
Lumen of vas deferens SMA (6.8 x 8).
filled with polymer
Lumen of vas deferens before polymer SMA (6.8 x 8).
SEPTEMBER 1990 VOL. 42 NO. 3
injection
of
345
Earlier, the polymer was declared safe in subacute effects. In the present study, when the evaluation in rats (3). polymer SMA remained in the lumen of the vas deferens for The one year, erratic changes in body weight were observed. behavioural, haematological and biochemical parameters were During the experiment, only 2 monkeys died due all normal. to intercurrent pulmonary infection after 7 and 10 months, which was not due to toxicity of the polymer but due to infection prevailing at the CDRI Animal Colony at that time. no pathology was observed which could be Histologically, In light of all related to the effect of the polymer SMA. the above facts, it is concluded that the polymer SMA did not cause any side effects either in the vas deferens or body during following organs of the 12 months other treatment. Acknowledgements Our sincere thanks are due to Dr. B.N. Saxena, M.D., Senior Deputy Director General, ICMR, New Delhi, for timeand financial assistance during the to-time suggestions We wish to thank Prof. S.K. Guha, Head, Department study. IIT, New Engineering, Delhi, for kindly of Biomedical supplying the polymer SMA for our study. Em. Anupma is acknowledged for technical help during the course of the study. References 1.
Guha SK, Ansari S, Anand S, Farooq A, Misro MM, Shanna Contraception in male DN. monkeys by intra-vas deferens injection of a pH-lowering polymer. Contraception 1985;32:109-18.
2.
Standard protocol for preclinical toxicity ICMR 1987. testing of new compound/drug/vaccine, submitted by Dr. Sethi, Scientist-in-Charge, (Mrs) N. Division of Toxicology, CDRI, Lucknow, and approved by the Indian Council of Medical Research (ICMR), New Delhi.
3.
Sethi N, Srivastava RI
N. Sethi, M.B.B.S., M.S. (USA), Path. Board (USA) R.K. Srivastava, Ph.D. R.K. Singh, Ph.D. G.S. Bhatia, Ph.D. N. Sinha, Ph.D. Division of Toxicology Central Drug Research Institute Lucknow - 226 001, India Abstract
A newly developed male contraceptive, styrene maleic anhydride (SMA), was injected in the vas deferens of male rhesus monkeys for safety evaluation at the dose of 100 mg (contraceptive dose, CD), 250 mg (CD x 2.5) and 500 mg (CD x 5.0), and the monkeys were kept under observation for one year. The observed behavioural, haematological, biochemical and histopathological parameters in treated monkeys were comparable to controls. The results suggest the polymer SMA to be safe up to 5 times CD in monkeys. Introduction
non-invasive male A new simple occlusive and contraceptive method has been developed (1) by using the polymer, styrene maleic anhydride (SMA). This polymer, when injected into the lumen of the vas deferens, exerts a contraceptive effect by occlusion as well as lowering the pH of the lumen (1). Before a new drug is released for clinical trials in humans, the subacute and chronic safety evaluation studies are done in at least two species of rodents and one species of non-rodents, i.e., monkeys (ICMR 1987 (2)). Earlier, the subacute safety evaluation studies In this communication, chronic were done in rats (3). toxicity data generated in rhesus monkeys are communicated. All correspondence to the first author: Dr. (Mrs) N. Sethi Assistant Director & Head Division of Toxicology Central Drug Research Institute Lucknow - 226 001, India Submitted for publication April 2, 1990 Accepted for publication June 28, 1990
SEPTEMBER 1990 VOL. 42 NO. 3
337
CONTRACEPTION Materials
and Methods
Fourteen healthy mature male rhesus monkeys, which were negative (tuberculosis was tested with KOT tuberculin Tuberculin Diagnostic Kit obtained from Budapest, Hungary) with body weight of 4.6 + 0.42 kg, were purchased from a acclimated to dealer and the laboratory commercial conditions for a period of one month, whilst fed on monkey pellet diet (supplied by Hundustan Lever Limited, Bangalore) Bananas and seasonal green vegetables and water & libitum. were supplemented from time to time. Monkeys were carefully observed for weight loss and Twelve animals were randomly divided untoward symptoms. were used as into 4 groups of 3 each, and 2 monkeys under uniform untreated controls. They were maintained conditions throughout the experiment and were husbandry carefully observed daily for any behavioural change. In the beginning, the polymer styrene maleic anhydride (SMA) dissolved in 0.5 ml DMSO was injected into the vas deferens at the dose levels of 100, 250 and 500 mg per vas deferens in low (group II), high (group III) and highest (group IV) dose groups, respectively, while vehicle and blank control (group I) monkeys received 0.6 ml DMSO and 0.5 ml distilled water per vas deferens per monkey. For injection, monkeys were anaesthetized with 30 mg/kg body weight intraval sodium (M & B, Bombay). The vas deferens just above the scrotum in the inguinal region were bilaterally exposed, care being taken not to injure the SMA dissolved in DMSO was associated blood vessels. injected into the lumen of the vas deferens in the direction pointing distally from the testis. Care was taken so that the injections were made at the same level of the vas deferens in all the animals. The wound was stitched, dressed with neosporin antibiotic powder and merbromin solution NF (2% w/v). After surgery, 0.5 ml teramycin solution (250 mg/5 ml, Pfizer) was injected intramuscularly in each monkey for 5 successive days. All monkeys were kept under observation until healing was completed. A daily record of the behaviour and general health of all monkeys was maintained. Body weight of the monkeys was recorded at monthly intervals, while haemograms-haemoglobin, haematocrit, total erythrocyte count, total leucocyte count, count, differential leucocyte platelet count, mean corpuscular volume, biochemical investigations -and cholesterol, blood urea, blood sugar, serum creatinine, serum alkaline serum phosphatase, glutamate pyruvate transaminase, albumin, total protein, globulin, and bilirubin -- were carried out at 0, 3, 6, 9 and 12 months of the experiments. Food consumption of each animal was regularly checked. Animals were carefully observed for
338
SEPTEMBER 1990 VOL. 42 NO. 3
CONTRACEPTION
changes in gross activity, hair loss, pupil size, ptosis, respiration, general reflexes, muscle tonicity of body and The production of limbs, after implantation of SMA. catalepsy, blanching, reddening, cyanosis, salivation, lachrymation, involuntary micturition and defecation, if any, was routinely checked. All the animals were sacrificed after 12 months of the implant by a high dose (40 mg/kg) of pentathol sodium. The gross observation included absolute and relative weights of liver, lung, spleen, kidney, brain, heart and gonads. The tissues -- liver, heart, kidneys, spleen, adrenals, trachea, lungs, oesophagus, stomach, pancreas, testes, brain, skin, eyes -- were fixed in 10% formal saline, processed and embedded in paraffin wax: serial sections (5 pg thick) were stained with haemotoxylin-eosin for microscopic examination. observations and Results &&y weisht -- In control (blank and vehicle) and low dose monkeys, steady body weights were observed, while in high and highest dose group animals, fluctuating body weights were seen. At the termination of the experiment, body weight in the high dose group remained unchanged and had slightly fallen in the highest dose monkey (Fig. 1). Food consumntion -- Food/water intake in all the animals of treated and control groups were almost the same. Hortalitv -- One monkey each of the control and the low dose group died after 7 and 10 months, respectively, after injection of the vehicle or drug. Initially, these monkeys tuberculin negative, while were after death, the histological examination of the lungs revealed that they were tuberculosis positive. Probably this was due to the husbandary conditions of the primate colony at that time. Gross behaviour -- Within a week, healing in animals was completed, and the monkeys became adapted to the stress of handling and injections. They did not show any symptom of nervousness or hyper/hypoexcitability, irritation/ depression, etc., at the time of drug or thereafter. Haematoloaical studies -- There were variable changes in haematological parameters, but they were within the physiological range (Table I). Biochemical studies -- The biochemical parameters studied did not cross the normal limits of variation in any of the groups. Fluctuations observed within the groups were attributed to intrinsic biological differences (Table II). Autonsy, aross and microsconic observation -- The .animals were found to be healthy and active at the time of
SEPTEMBER 1990VOL. 42 NO. 3
a
340
I
SEPTEMBER 1990 VOL. 42 NO. 3
13200 24170
320333 397500 k149694 + 3535
10800 21352
11250 27721
Platelet count 330000 385500 (per mm3) 228284 + 6363
Totalleucocyq 11700 count (per rrm )-(789
12400 _+2076
10866 &2309 12300 23394
2152752 +38857
396666
3066666
74.3 +2.0
73.0 22.8
72.6 22.7
~37527
361616
10400 2 530
41.6
76.5 43.5
391000 317500 +50921 224748
22600
10000
+2.1
75.0
78.3 21.1
42.1
75.5
74.0 A3.5
76.0 Mean corpuscular vol (cy) lt2.8
24.5
+2.2
+4.9
40.14
41.9
Haematocrit (X1
+ 5ooc
2750000
10500 21913
22.4
74.0
38.6 21.1
34.3
40.2 40.5
39.7 13.4
44.3
42.5
42.5
38.0
37.4 ro.9
35.3
5.23 to.33
5.43 ~0.36
5.01 40.21
5.34 40.79
6.36 20.19
5.34 20.71
5.52 ~0.24
5.53 $0.33
5,26 10.27
Red blood car- 5.22 puscles (million/nm3) k"*28
12.9 LO.2
13.5 20.8
12.7 LO.1
14.3 ~0.6
12.6 20.9
14.0 '0.9
12.6 ,O.l
13.9 20.6
12.9 20.1
13.0 20.2
Haemoglobin (g%)
Final Initial ----_--
Initial
Final
Initial
Final
Initial
Final
Highest dose
Final
(Values are mean + S.D.) -_-_-.Low dose Control High dose
Initial
-_-_ -_ Blank Control Vehicle
Haematological values in control and polymer-implanted monkeys
Measurement (unit)
Table I.
E T1
8 3
Biochemical
26.0 t 0.9
28.0 _ + 0.0
7.6 2 0.
0.4 + 0.0
SGPT (units/ml)
Total protein (q/l00 ml)
Bilirubin (mg/lOO ml)
0.4 t 0.0
7.2 t 0.6
1.4 +1.2
0.20 +o.o
0.30 tO.0 -
Serum creatinine (mg/lOO ml)
Serum alkaline 1.4 phosphatase +0.2 (BL units/ml) -
97.5 t 2.7
95.0 + 4.2
Blood sugar (mg/lOO ml)
0.4 t 0.0
7.5 t 0.3
26.0 t 0.0
1.1 t0.2
0.50 tO.0
92.6 +15.2
28.3 t 7.5
96.6
t 7.6
99.0
27.0 + o-0
control
are
0.3 t 0.0
7.0 ~0.8
25.3 t 9.5
1.0 co,4
0.40 tO.01
92.6 + 3.5
29.9 t 2.1
t 3.5
97.5
Initial Final --------
Vehicle
(Values
monkeys
0.4 t 0.1
7.7 t 0.6
t 4.0
29.0
1.6 to.3
0.30 IO.01
92.0 + 7.6
27.3 t 1.5
+ 1.4
99.0
Initial
0.3 9 0.0
0.4 to.1
7.7 t 0.9
28.3 t 3.0
26.0 t 2.8 7.4 t 0.3
1.2 tO.l
0.40 +0.02
96.6 t 7.9
26.6 t 2.3
t 1.8
95.0
2.7
94.0
0.4 t 0.2
7.4 t 0.4
25.6 t 9.2
1.1 t0.2
0.30 to.01
t 7.6
95.6
25.3 t 1.7
,*
93.3
7.0 2 0.6 0.4 t 0.0
0.4 t 0.1
25.7 * 2.1
1.0 tO.l
0.30 to.03
91.0 t 6.5
26.2 t 1.4
t14.0
7.2 t0. 2
26.3 t 1.1
1.5 $0.2
0.30 to:02
96.6 t 3.3
28.0 t 2.7
t 2.3
94.6
.__.I_-_-.I_I-Highest dose High dose Initial Final Initial Final --II__--
1.4 to.1
0.40 to.03
91.0 t 6.2
23.5 t 1.2
+ 3.5
92.5
Final
mean + S.D.) _-.-Low dose
in control and polymer-implanted
+ 1.4
27.6 t 1.3 -
99.0 + 1.4
Final
COIItrOl
values
(mg/lOO m4
Blood urea
Total cholesterol(mg/lOOml)
Blank Measurement Initial (unit) ----
_--_.--------
Table II.
P
F z s
5
8
CONTRACEPTION nasal mucosae sacrifice. Fur coat, conjunctivae, in the vas deferens normal, sites of implantations normal.
were were
revealed Gross examination of the tissues varying degrees of splenic enlargement in a small percentage of both All other organs and control and drug implanted animal. shape and size (Table viscera were normal in appearance, III). The microscopic examination of the vas deferens in polymer-injected monkeys revealed the polymer was seen lying in the lumen as an amorphous material which was stained pink with eosin (Fig. 2). This occuppied one-fourth of the lumen and did not cause any occlusion (4). It had entered the follicles of the mucosa, but no implant was seen in the muscular layer. The wall of the vas deferens was not infiltrated, and there was no evidence of any inflammatory reaction. The mucosa of the vas deferens was lined by low cuboidal cells which looked like columnar cells in a few places. In control monkeys, the mucosa of the vas deferens was normal (Fig. 3). Incidental batholouv -- Incidental pathology was seen in the lungs, liver, kidney and spleen. In tot0 four animals had pulmonary tuberculosis which was diagsd only at the time of autopsy. Out of 4 monkeys, 2 monkeys had died during the after 7 and 10 experiment months, respectively. These animals were distributed in all the including the control group. In the other 2 groups, monkeys, tuberculosis diagnosed after was histological examination of the lung only at the termination of the experiment. Discussion Many methods of male contraception are presently based only on the blocking of spermatozoa in the vas deferens lumen. Several investigators have reported the blocking of the lumen of vas deferens with the use of injectable and solid plugs (5-10). A new contraceptive, SMA, was selected for study, being a polymer of maleic anhydride and styrene. When SMA comes in contact with water, the acid anhydride The time required for hydrolysis component is hydrolysed. in experimental studies is one week. The polymer, after injection, reacts with cellular secretion and forms a stable precipitate within the lumen and makes the environment such that the spermatozoa passing through are killed. Chronic safety evaluation studies are done with a view to find whether newly invented drugs could affect the target Likewise, the organ as well as other organs of the body. organs in different different new drug reach may proportions/constituent which may likely have/not have side
SEPTEMBER 1990VOL. 42 NO. 3
343
2.55 2.58
3.03
3.02
R
L
Gonad
_I--
0.11 0.11
0.15 0.16
R L
Mrenal
--_“_
2033 2.37
2.79 2.96
Kidney R L
w_--
1,96
2.09
2.27
Spleen
.-
.
2.40
2.40
0.11 0.11
2.31 2.25
11.63
11.96
14.96
Brain
_-
A-1
2.46 2.45 0.10 0.10 2.65 2.66
2.48 2.43 0.13 0.13 2.57 2.58 L__-._I__
1.89
13.05 2.48
13.73
29.60
31.24
26.03
27.58
33.21
Liver
4.06
4.69
3.96
4.13
4.91
Heart
Highest dose
High dose
Vehicle control
Blank control
Low dose
--
Mean relative organ weight (g/kg body weight) of control and polymerimplanted monkeys
Organ
Table III.
CONTRACEPTION
Fig. 2.
Fig. 3.
Lumen of vas deferens SMA (6.8 x 8).
filled with polymer
Lumen of vas deferens before polymer SMA (6.8 x 8).
SEPTEMBER 1990 VOL. 42 NO. 3
injection
of
345
Earlier, the polymer was declared safe in subacute effects. In the present study, when the evaluation in rats (3). polymer SMA remained in the lumen of the vas deferens for The one year, erratic changes in body weight were observed. behavioural, haematological and biochemical parameters were During the experiment, only 2 monkeys died due all normal. to intercurrent pulmonary infection after 7 and 10 months, which was not due to toxicity of the polymer but due to infection prevailing at the CDRI Animal Colony at that time. no pathology was observed which could be Histologically, In light of all related to the effect of the polymer SMA. the above facts, it is concluded that the polymer SMA did not cause any side effects either in the vas deferens or body during following organs of the 12 months other treatment. Acknowledgements Our sincere thanks are due to Dr. B.N. Saxena, M.D., Senior Deputy Director General, ICMR, New Delhi, for timeand financial assistance during the to-time suggestions We wish to thank Prof. S.K. Guha, Head, Department study. IIT, New Engineering, Delhi, for kindly of Biomedical supplying the polymer SMA for our study. Em. Anupma is acknowledged for technical help during the course of the study. References 1.
Guha SK, Ansari S, Anand S, Farooq A, Misro MM, Shanna Contraception in male DN. monkeys by intra-vas deferens injection of a pH-lowering polymer. Contraception 1985;32:109-18.
2.
Standard protocol for preclinical toxicity ICMR 1987. testing of new compound/drug/vaccine, submitted by Dr. Sethi, Scientist-in-Charge, (Mrs) N. Division of Toxicology, CDRI, Lucknow, and approved by the Indian Council of Medical Research (ICMR), New Delhi.
3.
Sethi N, Srivastava RI
