Safety evaluation of a male injectable antifertility agent, styrene maleic anhydride, in rats

CONTRACEPTION

SAFETY

E AGENT,

TION OF STYRENE

A

LE IC

INJEC B HYDRIDE,

N. S e t h i , M . S . (USA) , P a t h . R.K. Srivastava, . D. R.K Singh, Ph.D.

ANTIFERTILITY IN S

Board

(USA)

Division of Toxicology Central Drug Research Institute Lucknow 2 2 6 001, I n d i a

Abstract Injection o~ a pol er, s t y r e n e maleic anhydride (S ) , into the lumen of the vas deferens of rats, was obsez-ved to have effective and reversible contraception. The safety evaluation of the pol er, f o r 90 d a y s , was carried out in Charles Foster male rats. There have been no significant changes in any of the toxicity parameters in the test animals as compared to the control animals. Hence, it i s t ha~ ~" concluded the compound is n o n - t o x i c to rodents e

Reprint

Submitted Accepted

rec~aests:

for publication for publication

FEBRUARY 1

V O L . 39 NO. 2

Dr. ( M r s . ) N. S e t h i Assistant Director & Head Division of Toxicology Central Drug Research Institute Lucknow - 226 001, India

May Nove

6, 1 9 8 8 e r 14,

1988

217

CONTRACEPTION Introduction Fertility control is v i t a l for our society. Some female methods of contraception a r e in u s e , b u t a s y e t , o t h e r t h a n vasectomy, there are no well established male methods. Vasectomy is e s s e n t 0 i a l l y a t e inal technique and, hence, there is o f t e n a reluctance to adopt the approach, although effective. An impr ement which is non-invasive, would pe it mass scale delivery and also be reversible in character, is re ired. Injection of t h e p o l y m e r styrene maleic anhydride dissolved in dimethyl sulphoxide (DMSO) , into the lumen of vas deferens of rats and rhesus monkeys has been easily carried out (i,2,3) . The pol er has the d u a l f e a t u r e t h a t it c a n o c c l u d e the vas deferen's lumen and also it can inhibit the fertilizing ability of spe atozoa by virtue of its pH lowering effect. The pol er S itself does not degrade in t h e p r o c e s s but can be flushed out to regain fertility. DMSO acts merely as a temporary solvent and has no contraceptive action at all. Preclinical toxicity studies of the out in rodents (Charles Foster rats)

in

this

compound and data

were carried are presented

manuscript. Materials

and

Methods

Adult male Charles Foster rats of average body weight, 1 2 0 +~ 40 gin, w e r e obtained from the C I animal ceding colony and watched for one week. General health condition and mortality, if a n y , w e r e recorded. Forty animals selected for the experiment were randomly divided into 4 groups o f i0 animals each. Pol er styrene maleic anhydride dissolved in 0.03 ml SO was implanted intravasally at the dose levels of i. 0 m g ( c o n t r a c e p t i v e dose) , 2.5 mg (contraceptive dose x 2.5), 5.0 mg (contraceptive dose x 5) p e r v a s d e f e r e n s in low (Gr. I I ) , high (Gr. III) , a n d h i g h e s t (Gr. IV) g r o u p s , respectively, while control (Gr. I) r a t s received 0.03 ml SO per vas deferens. The method o f S i n g h e t al. (i) w a s followed for the injection of S in rats. ~or injection, the rats were anaesthetized by ether and a single cut was made ventrally, just above the urethral opening, to expose the vas deferens on both sides. The ampullary portions of the vas deferens (i.e., distal region) were located beneath the fat bodies. The reason being that there the duct was of greater diameter as compared to other regions, and others have also suggested the same (2,3). Care was taken that the injections were made at the same level of the vas deferens in a l l the animals, the skin wound was stitched, dressed with neosporin antibiotic powder and merbromin solution NF (2% "w;/V). J u s t a f t e r t h e s u r g e r y , a single injection of 0.5 ml terramycin (Pfizer ~imited, Bombay) was given intramuscularly to each rat and the animals were kept under obse ation until needed The animals were kept in

218

FEBRUARY 1

VOL.

NO. 2

CONTRACEPTION identical

environmenta~ conditions and were maintained on animal feed (pellets m a n u f a c t u r e d by M/s Lipton Indian Limited, Bangalore) and water ad I ib. throughout the experimental period.

standard

laboratory

A daily record of the behaviour and general health of all rats was maintained. The body weight of each rat was recorded weekly. Haemogram (haemoglobin, haematocrit, total erythrocyte count, total leucocyte count, differential leucocyte count, platelet count and mean corpuscular volume) was done initially and then at the termination of the experiment, i.e., 90 days. The biochemical parameters (c h o l e s t e r o l , blood urea, blood sugar, serum c r e a t in ine, serum alkaline phosphatase, serum glutamate pyruvate transaminase, total protein, albumin, globulin and bilirubin) were studied at t h e t e r m i n a t i o n of the experiment for different organ function tests. The rats were autopsied under anaesthesia, after 90 d a y s of the implant; gross examination of the different organs was done, and absolute and relative weights of liver, heart, spleen, kidneys, adrenals and gonads were recorded. Pieces of the organs as well as vas deferens, testes, epididymis, prostate, seminal vesicle, eye, skin, trachea, intestine, thyroid and adrenals were fixed in 10% formal saline. Paraffin sections of 5 ~ were stained with haematoxylin and eosin for microscopic examinations. Obsez~Jations

and

Results

Body w e i q h t " The body weight gain a t w e e k l y inte_z-~als of differently treated as well as control groups is shown graphically in Figure 1. There was steady gain in body weight of all animals of all groups. Food and animals intake.

water intake" showed any

None of the treated noticeable deviation

or

control group in food/water

Gross behaviourWithin a week, healing in animals was completed and the rats became adapted to the stress of handling, implants and surgical procedure. None of them showed any undue s~n~ptoms of hypo/hyperexcitability or ne~-~-ousness, at t h e t i m e of i m p l a n t , p o s t - o p e r a t i v e handling or thereafter. four M o r t a l d tyO n e r a t of t h e t o x i c d o s e g r o u p d i e d a f t e r Probably this rat died e i t h e r due to weeks of i m p l a n t . implant or some infection which was surgical stress of the not detected.

F E B R U A R Y 1989 VOL. 39 NO. 2

219

CONTRACEPTION

22C o.--- - - . - o ¢~.. . . .

~o

Contro[ L O w dOSiZ High dose Toxic

20[

dose

!6t

2 ~

161

,.....

E .E "-" ~c

121

101 -D o

E .E

81

1

2

3

4

5

6

7

8

9

10

11

12

13

1/.

:5

WEE'KS

Figure

1.

Weekly body weight gain in different groups of male rats receiving intravas styrene maleic anhyd r ide d i sso ived in d imethy 1 sulphoxide.

22O

F E B R U A R Y 1989 VOL. 3-9 NO. 2

CONTRACEPTION ;~@e a t o l _ o g i c a ! ............ s..... ,~dies: The haematological control and drug-treated rats, both initially ~e inat ion of experiment, were comparabl e ..~'~ysioiogical r a n g e o f n o alcy ( T a b l e I).

values in and at the and within

~/o~h~ i c a ! ...... s...~ t u d ~ e s: (Table II)None of the biochemical 9.arameters showed significant alteration in activity that c~uld be related to toxicity of vasal implant. G~CQS~ ..............9 ...........n..d .............. :mi ....... C r o scoop i c ..........o ....b s ! ~ T h er e w as no significant variation in absolute and relative weights of the organs in any of the groups of test or control animals (Table III) ° Microscopic examination from the site of implant showed the pol er a s a n a m o r p h o u s material lying in t~e lumen of the vas deferens, staining pink with eosin. The wall of the vas deferens was not infiltrated. The mu~sa was lined by a single l a y e r of l o w c u b o i d a l cells and the muscle and serosal layers were no al. There was no evidence of any inflammatory reaction, and the lumen did not have any spe atozoa. Incidental pathology was observed in the lungs, liver, kidneys and spleen. None of the organs showed any lesions that could be drug related (Table IV). Discuss

ion

Vasecto is a well established ~ethod of male contraception. The side ef cts of vasectomy are still under investigation; nevertheless, surgical sterilization is one of the most reliable methods of population control. Male methods of fertility control were at one time based exclusively on blocking passage of spe atozoa in the vas deferens. More recently, other proposals have come into active consideration. The poi er S c a n be u s e d t o b l o c k the lumen of the vas deferens over an extended period of time. The special feature which makes the technique distinctive from earlier vas occlusive techni es (4,5) is that, even when the pol e r is p r e s e n t in t h e l u m e n of the vas deferens in a vol e ich does not prevent spe atozoal passage, contraception is still maintained. A class of pol ers known as hydrogeis has the intrinsic property of lowering pH and thereby inhibiting the functional ability of the spe atozoa to fertilize the ovum. Styrene maleic anhydride was selected as an ideal agent in a series of investigations (6-8) . In the study 0.03 ml D M S / V . D.. w a s used. At this very small .dose, SO i s not reported to cause any toxic effect (9). The pol er, after injection, reacts with cellular secretion and fo s a stable precipitate within the lumen and makes t h e envir, ment such that the spe atozoa passing through are all killed.

FEBRUARY 19

VOL..

NO. 2

I

Z 0

r-

< 0

C > K

m

m

nn

63.40 ± 5.14

Mean Corpus-

(%)

± 6.27

2.22

39.80

10180 ± 4222

762300 ± 104713

63.60 ± 4.19

6.22 ± 1.02

±

36.60

10380 ± 4427

Total Leucocyte Count (per cmm)

HaematOcrit

643600 ± 66645

Platelet Count (million/cmm)

cular Volume (~m)

5~59 ± 0.39

RBC (million/ cram)

12.60 ± 2.04

12.96 ± 1.O7

Haemoglobin

(g%)

Control Initial Final

4.60

±

36.30

11890 ± 4247

649500 ± 82266

63.96 ± 4.17

5.92 ~ 0.88

12.34 ± 0.99

1.30

±

46.10

6830 ± 1037

732100 ± 122299

67.00 ± 1.7

7.24 ± 0.53

13.80 ± 0.8

3.81

±

34.50

12400 ± 4153

644000 ± 100746

64.00 ~ 4.80

5.68 ± 0,88

12,32 ± 0.76

2.56

±

46.47

11730 ± 2302.19

760000 ± 81605.07

67.40 ~ 2.06

3.47

±

34.16

9840 ± 2952

620000 ± 99128.25

64.60 ~ 3.59

5.17 ± 0.53

i.i0

0.67 7.52 ± 0.80

12~06 ±

6.52

±

41.50

13644 ± 1998.87

735111 ± 398151

2.82

63.60

6.49 ± 1.05

1.19

12.40 ±

Highest Dose Initial Final

15.31 ±

Groups Low Dose High Dose initial Final Initial Final

Haematological values in control and polymer implanted rats

Parameter

Table I.

0 Z

C~ m

>

C~ 0 Z -'I

O

Z

r-

< 0

33 -<

>

c

lm

-m m

17.60±3.30

Blood Urea (mg%)

7.08±0.21 4.72±0°42

Total Protein (gm%) 7.10±0.20 4.80±0.21 2.26Z0.25

Albumin ( g m % )

Globulin ( g m % )

2.36±0.40

18,00±4,55

23.30±8.43

1.06±0.31

Serum Alkaline 1.27Z0.22 Phosphatase (BL units)

SGPT (Unit/ml)

0.85±0,24

100.00±10.27

14.11±2.80

73.80~66.79

Low Dose

0.85±0.44

Serum Creatinine (mg%)

Blood Sugar (mg%) 109.70±10.31

76,10±5.91

Control

2.08~0,19

4.71±0.40

6.80±0.57

18.43±6.34

1.25~0.30

0.82±0.36

105.40±10.06

15.90±2.28

78.50~7.30

High Dose

Dose Groups

2.16~0.22

4.67±0°48

6.80±0.65

15.00±3.10

1.58±0,43

1.01±0.30

104.50±5.12

15.80±2.36

76.30~6.76

Highest Dose

Biochemical values in control and polymer implant rats

Cholesterol (mg%)

Parameter

Table II.

O Z

m

nl

O

>

-4

O Z

O

0

Z

< 0 .r-

(0

-<

>

c

m

1.29~0.246 2.68±0.410 i0.15±0.851 0.75±0.158

Heart

Lung

Liver

Spleen

Right Gonads Left 1.28Z0.608

1,31±0.552

1.18±0.214

1.11±0.191

2.08~0.376

Brain

Right Kidney Left

Group I

1.49~0.207

1.2±0,465

1.15±0.227

1.15±0.227

0.73±0,221

i0.i±0.8~3

i

2.39±0.360

2.12~0,373

2.12±0.373

1.18±0.154

1.16±0.154

0.52±0,080

10.50±0,591

2.85±0,343

1,02±0o063

1.08±0,091

~

Group I!I 2.35~0.365

i

2.03~0.254

Group Ii .....

1.22~0,582

1.24~0.675

0.96±0.347

0.96~0.347

0.64~0.327

9.05±3.45

2.31±1.00

1.04~0,394

1.95Z0.761

.....................

Group IV

Summary of absolute organ weights (gm ± SD) of different groups of SMAimplanted rats

Organ

Table III.

Z

O

O m

>

Z

0

0

CONTRACEPTION Table Histopathological different doses

obser~'ations of rats implanted of pol er S dissolved in

(Sub-Acute

GToUp

..................................................

Rat~ NO -

Control

Low

High

Toxic

Dose

Dose

Dose

IV

4

9

..............................

Toxicity

1

1

Study)

orga~n ........................P ....e ..... rtin@nt

fiDdings

Kidney

Mild swelling epithelium

Gonads

No

al

Gonads

No

al

Gonads

5% spermatogenesis maturation arrest

ng

in

Occasional focus c a I c i f ic a t io n

Gonads

No

Gonads

10% non-functional tubules

Gonads

. . . . . . . . . . . . . . . . .

tubular

with

Bronchopneumonia

Gonads

ng

with SO

al

Large walled (acute) No

of

abscess

al

The new pol er c a n a l s o be injected into the vas deferens through e intact scrotal wall, thus avoiding surgery in the initial sterilization .procedure In view of the side effects of vasect and other existing procedures, a newer and safer techni e will be more useful. Thus, with the use of this pol er and this technique, side effects can be mini ized. In our study we have concluded that the compound appears to be safe in rodents up to dose levels of 5.0 m vas deferens.

F E B R U A R Y 1989 VOL.

NO. 2

CONTRACEPTION Acknowledgements We express our sincere thanks to Dr. B.N. Saxena, M.D. , Senior Deputy Director General, !. C.M.R., New Delhi, for sanction of the project "Preclinical toxicity study of new compounds" of Indian Council of Medical Research, New Delhi. Prof. S.K. Guha, Head, Department of Biomedical Engineering, I.I.T., New Delhi, for kindly supplying the pol er S for our study. Miss Neeta Vaishnavi and Miss Anupma Srivastava are acknowledged for technical assistance in this study. References O

Q

O

O

8

O

g

B

O

Singh M, R a y AR, Vasudevan P, V e a K and Guha Potential biosoluable carrier biocompatibil ity biodegradability of oxidised cellulose. Biomater Devices Artif Organs 1979; 7-595-612. Hamilton DW and Cooper TGvariation along the length of Rec 1978; 190:795-810. Kennedy the rat

SW and Heidger vas de~erens.

Setty BS, of vas in

P!4Anat

and histological Gross Anat rat vas deferens.

Fine structural studies c 1979; 194:159-180.

Dasgupta and Kar rats. Contraception

Malaviya B, C h a n d r a by vas by inacrine 1974; 12:560-561.

SKand Med

H and Kar in rhesus

of

AB" Chemical occlusion 1972 ; 6-329--334. AB" Chemical occlusion monkeys. Ind J Exp Biol

Misro , Guha SK, Singh HP, Mahajan S, Ray AR and Vasudevan PInj e c t a b l e non-occlusive che ical contraception i n m a l e - I. Contraception 1979; 20-467473. Guha SK, Ansari, S, A n a n d S, F a r o o q A, M i s r o and Sharma DNContraception in m a l e m o n k e y s by intra-vas de ferens injection of a pH lowering polymer. Contraception 1 9 8 5 ; 32- 1 0 9 - 1 1 8 . Ve a K, M i s r o , S i n g h H, M a h a j a n S, R a y A R a n d G u h a SKHistology of the rat vas deferens after injection of a non-occlusive chemical contraceptive. J Reprod Fert 1981; 63:539-542. Rubin LFAnn NY Acad

Toxicologic Update Sc 1983; 411:6-i0.

of

Dimethyl

F E B R U A R Y 19

Sulphoxide.

V O L . 39 N O . 2