International Journal of Toxicology

1-9
Toxicity and Mutagenicity Evaluation ª The Author(s) 2018
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Following RISUG Contraception DOI: 10.1177/1091581818809473
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Reversal in Rats

Abdul S. Ansari1, Mubarik Hussain2, Sadi Rehan Khan1,

Ayesha Badar1, and N. K. Lohiya1

Abstract
Reestablishment of fertility, after a male contraceptive method, is of great concern. In this context, RISUG (Reversible Inhibition
of Sperm Under Guidance) has been evaluated for its mutagenicity following reversibility with dimethyl sulfoxide (DMSO)/sodium
bicarbonate (NaHCO3) in Wistar albino rats. Animals were divided into 7 groups, namely, sham-operated control, vas occlusion
with RISUG for 90 and 360 days, reversal with DMSO and NaHCO3 after 90 and 360 days, respectively. The testis, cauda
epididymis, cauda epididymal spermatozoa, and serum were evaluated for apoptosis and hormonal status through various assays.
RISUG was subjected to Ames test at dose levels of 10, 50, and 100 mL. Results of terminal deoxynucleotidyl transferase nick end
labeling and caspase-3 assays in testes and cauda epididymis revealed that the percentage of positive cells in the experimental
groups was comparable to sham-operated control. Annexin V assay in cauda epididymal spermatozoa showed slight elevation in
group II (P < 0.05), whereas in the remaining groups, minimum numbers of positive sperms were found. Hormone profile, namely,
testosterone, prolactin, cortisol, prostate-specific antigen, and sperm antibody concentration, remained unchanged. In Ames test,
no significant increase was observed in the number of revertant colonies on plates containing RISUG in the presence and absence
of S9 mix in all 3 strains. Therefore, the reversal of RISUG-induced contraception by solvent vehicle DMSO/NaHCO3 was
successful without any toxicity at the cellular levels.

Keywords
contraception, RISUG, reversal, toxicity, mutagenicity

Introduction rabbits to achieve reversibility.6,7,11 The current research

From a global standpoint, the concept of contraception as a
method for population control is of paramount importance. The
prospects of available contraceptives in male found to be ideal
on the basis of 3 main criteria, namely, one-time application,
long-term effect, and reversal upon need without any side
effects. As the reversal compliance is lacking in most of the
approaches and, if present, with less success rate, most men
choose vasectomy and condoms as a contraceptive option now.
Vasectomy, although long acting, is generally considered per-
manent due to difficulties of reversal, the expense, and devel-
opment of antisperm antibodies (ASAs), which bring failure
rate in restoring fertility.1 At present, this has led to lacunae in
contraceptive choices to men without toxicity after reversal.
However, RISUG that has been already proven to be single-
intervention, long-lasting, vas-based reversible contraceptive
method in rats, rabbits, and monkeys 2-7 has successfully
completed phase I and II clinical trials and is currently under
multicentric phase III clinical trials in India.8,9 Noninvasive
reversal approaches have been studied in langur monkeys.2,10
The dimethyl sulfoxide (DMSO) and sodium bicarbonate
(NaHCO3) solvents were used to dissolve RISUG in rats and
intended to assess the mutagenicity of the drug and solvents
following reversal in rats.
Materials and Methods
Drug
RISUG, a copolymer synthesized through gamma irradiation of
the monomers, styrene, and maleic anhydride, dissolved in the
solvent vehicle DMSO in 1:2 ratios. It was kindly provided by
Prof S. K. Guha, School of Medical Science and Technology,
Indian Institute of Technology, Kharagpur, West Bengal, India.
1
Department of Zoology, Centre for Advanced Studies, University of
Rajasthan, Jaipur, Rajasthan, India
2
S. S. Jain Subodh College of Global Excellence, Sitapura, Jaipur, Rajasthan,
India
Corresponding Author:
Abdul S. Ansari, Department of Zoology, Centre for Advanced Studies,
University of Rajasthan, Jaipur, Rajasthan 302004, India.
Emails: abdulsansari@uniraj.ac.in; abdulsansari@yahoo.com
2 International Journal of Toxicology XX(X)
Animals
Seventy adult Wistar albino rats (Rattus norvegicus), aged
10 to 12 weeks, weighing 150 to 180 g and of proven fertility,
were used in the present study. Animals were maintained in the
Departmental Experimental Animal Facility with the provision
of 12-hour/12-hour light and dark schedule in polypropylene
cages (size 43 cm  27 cm  15 cm). The temperature in
animal house during the study period was maintained at 23 C
+ 2 C, and the relative humidity ranged between 40% and
70%. Animals were fed with rat pellet diet (Ashirwad Indus-
tries Ltd, Chandigarh, India) and water ad libitum. Animals
were maintained under perfect veterinary supervision in accor-
dance to the guidelines of the regulation of scientific experi-
ments on animals. The experimental protocol has approval of
the Institutional Animal Ethical Committee.12
Experimental Protocol
Rats were allocated into the following 7 groups containing
10 animals in each:
Group I: Sham-operated control
Group II: Vas occlusion with RISUG for 90 days
Group III: Vas occlusion with RISUG for 90 days and rever-
sal with DMSO
Group IV: Vas occlusion with RISUG for 90 days and rever-
sal with 5% NaHCO3
Group V: Vas occlusion with RISUG for 360 days
Group VI: Vas occlusion with RISUG for 360 days and
reversal with DMSO
Group VII: Vas occlusion with RISUG for 360 days and
reversal with 5% NaHCO3.
Vas Occlusion
Rats of groups II to VII were subjected to bilateral vas occlu-
sion under sodium thiopentone anesthesia (20 mg/kg body
weight intravenously, THIOSOL Sodium; Neon Laboratories
Ltd, Mumbai, Maharashtra, India). After skin disinfection and
hair shaving of the scrotum region, a single median incision
was made just above the scrotum to expose the vas of inguinal
region. The therapeutic dose of RISUG, that is, 5 to 7 mL, was
injected into each vas deferens sufficient enough to block the
pathway. The polymerization of styrene–maleic acid was cat-
alyzed by application of normal saline and solidified on contact
with fluid medium in the vas. The syringe was slowly with-
drawn. The vas was placed in its original position, and incision
was closed by catgut suture in the inner layer and by silken
suture outside. The vas deferens of group I animals was
exposed in a similar manner; however, no drug was injected.
Postoperative care included antibiotic ceftriaxone (Cefoat; A to
Z Pharmaceuticals Ltd, Gujarat, India) and anti-inflammatory
drug meloxicam (Melonex; Intas Pharmaceuticals Ltd, Gujarat,
India), injected in the gluteal muscles, along with topical appli-
cation of Healex Plus spray (Shreya Life Sciences Pvt Ltd,
Mumbai, India). Proper bedding and feed after half an hour
was provided to the treated animals. All rats were kept under
observation during healing period. The sutures were removed
after complete healing. The vas deferens of group I animals
were exposed in a similar way, but no drug was injected.
Success of vas occlusion was confirmed by cohabitation of
the vas occluded animals with the proven fertile females.5,6
Mating was confirmed by vaginal smears for the appearance
of spermatozoa.
Vas Occlusion Reversal
Reversal was performed following 90 and 360 days of vas
occlusion under sodium thiopentone anesthesia. The vas was
exposed in a similar way as that of occlusion and injected
bilaterally with 250 to 500 mL of DMSO in groups III and
VI and 500 to 700 mL of 5% NaHCO3 in groups IV and VII to
dissolve RISUG. Dissolution of RISUG was ensured by free
flow of liquid droplets from the vas. The vas was returned to
its original position, and the incision was closed with catgut
and silken sutures. Postoperative care was provided as
described previously. Success of reversal was assessed after
15 days of reversal by fertility studies with proven fertile
female at 1:2 ratio.5,6
Sacrification Schedule
Following completion of experimental schedule, all animals
were euthanized with an overdose of sodium thiopentone
anesthesia.
Parameters
Animals of all groups were examined for the following
parameters.
Apoptotic Markers
Terminal deoxynucleotidyl transferase nick end labeling assay. The
terminal deoxynucleotidyl transferase nick end labeling
(TUNEL) assay was performed using the DeadEnd fluoro-
metric TUNEL system (Promega Corporation, Madison, Wis-
consin) following the manufacturer’s instructions with
modifications. In summary, sections of testis and cauda epidi-
dymis were deparaffinized with xylene, dehydrated in a graded
ethanol series, rehydrated, and treated with 3% hydrogen per-
oxide for 20 minutes to reduce nonspecific staining. Sections
were immersed in hot 10 mmol/L citrate buffer solution (pH
6.0) for 5 minutes, then incubated with normal goat serum for
20 minutes. Sections were incubated overnight at 4 C with the
p53 primary antibody, washed with phosphate-buffered saline
(PBS), and exposed to the avidin–biotin peroxidase complex
for 1 hour at room temperature. The chromogenic substrate of
peroxidase was developed using a 0.05% solution of
3,3-diaminobenzidine tetrahydrochloride, 0.03% hydrogen
peroxide, and imidazole in Tris–HCL buffer (pH 7.6). Sections
were counterstained with hematoxylin, and the number of
apoptotic and p53-positive cells in each section was analyzed
Ansari et al
under a nonconfocal fluorescence microscope (Labomed; Labo
America Inc, Fremont, CA) using excitation at 480 nm and
emission at 535 nm barrier filter to view the green fluorescence
of fluorescein and a 525/615 nm excitation/emission spectrum
to view the red fluorescence of propidium iodide (PI). Images
were generated using the DigiPro 4.0 and Image J program
(Image J, Rockville, Maryland). For statistical analysis, 500
cells were counted in randomly selected fields and the numbers
of positive cells were counted.13
Immunofluorescent detection of activated caspase-3. The activity of
caspase-3 was measured by tetrapeptide caspase substrate Asp-
Glu-Val-Asp (DEVD)-7-amino-4-trifluoromethylcourmarin
(DEVD-AFC) caspase-3 kit (Santa Cruz Biotech Ltd, Dallas,
TX). In brief, testis and cauda epididymal sections were
deparaffinized with xylene, rehydrated in a graded ethanol series,
and permeabilized in 0.3% Triton-X 100. DEVD-AFC synthetic
substrate was used for estimation and localization of activated
caspase-3, which corresponds to the upstream amino acid
sequence of the caspase-3 cleavage site in Poly ADP ribose
polymerase and the fluorophore AFC. Caspase-3 hydrolyzes the
peptide bond between D amino acid and AFC, releasing AFC.
Apoptotic cells activate caspase-3, hence emitting high levels of
fluorescence compared to control nonapoptotic cells. Free AFC
levels were measured at 420 nm excitation and a 535 nm emission
barrier filter. Images were generated using the DigiPro 4.0 and
Image J software (Image J). For statistical analysis, 500 cells were
counted in fields selected by systematic random approach and the
numbers of positive cells were counted.14
Annexin V binding assay. The membrane flip-flop was assessed as
described by Shen et al,15 according to the manufacturer’s
protocol (Santa Cruz Biotech Ltd). Briefly, cauda epididymal
sperms were washed twice in PBS at 1,200 rpm for 5 minutes
and resuspended in 1 mL 1 assay buffer. Samples were redis-
tributed in 100 mL aliquots, stained with annexin V–FITC con-
jugate and PI, and observed under nonconfocal fluorescent
microscope at 480 and 525 nm excitation and 535 and
615 nm emission barrier filters. Results were compared with
unstained cauda epididymal cell suspension. Images were gen-
erated using the DigiPro 4.0 and analyzed by Image J program
(Image J). For statistical analysis, 500 cells were counted in
fields selected by systematic random approach and the numbers
of positive cells were calculated.
Serum Clinical Biochemistry
Hormone analyses. At the end of the experiment, animals were
anesthetized and blood samples were obtained from the hearts
of animals and allowed to clot for 20 minutes in laboratory
temperature and then centrifuged at 3,000 rpm for 10 minutes
for serum separation. Circulatory levels of serum testosterone,
cortisol, and prolactin were assayed by enzyme-linked immu-
nosorbent assay (ELISA) kits (DSI, Saronno, Italy). The intra-
and interassay coefficients of variation from routine assays
3
Figure 1. Percentage DNA breakage in experimental groups of testis
and cauda epididymis. DNAase I-treated parallel positive controls
showed significant variation in percentage of DNA damage comparing
to test groups (I-VII). Positive control of testis showed highly signifi-
cant (P < 0.001) variation comparing to positive controls of cauda
epididymis. Images were analyzed by ImageJ and DigiPro software.
were 4.9% and 5.4% for testosterone, 2.95% and 4% for pro-
lactin, 1.9% and 5% for cortisol, respectively.
Prostate-specific antigen. Prostate-specific antigen (PSA) was
assayed in the separated serum with commercially available
ELISA kits (DSI). The intra- and interassay coefficients of
variations from routine assay were 6% and 6.6%, respectively.
Antisperm antibodies. The levels of ASAs were measured in
serum using commercially available ELISA kits (DRG Inter-
national, Inc, Springfield, NJ) in serum. The intra- and inter-
assay coefficients of variations from routine assay were 6.88%
and 6.45%, respectively.
Salmonella typhimurium reverse mutation assay (Ames test). Ames
test was carried out according to the method of Maron and
Ames.16 The S typhimurium tester strains TA97a, TA98, and
TA100 were used in the Ames reversion test purchased from
Institute of Microbial Technology (Chandigarh, India). Stan-
dard plate incorporation assay was used that consisted of
exposing the tester strain(s) to the test substance (RISUG) at
dose levels of 10, 50, and 100 mL directly on a glucose minimal
(GM) agar plate in the presence and absence of a metabolic
activation system. The RISUG doses S9 suspension and bac-
terial suspension were added to sterile test tubes containing
2 mL of molten agar supplemented with limited histidine and
biotin. Temperature of top agar was maintained between 43 C
and 48 C and to minimize prolonged exposure to avoid killing
of the tester strains. Then contents of the tubes were mixed and
poured on GM agar plates. After the top agar has solidified, the
plates were inverted and incubated at 37 C for 48 hours. After
incubation, histidine-revertant colonies were counted on all
plates and the results were expressed as the number of revertant
colonies per plate. Sodium azide and daunomycin (daunorubi-
cin hydrochloride; Himedia Pvt Ltd, Mumbai, Maharashtra,
India) 5.0 mg/plate were used as positive control.
4 International Journal of Toxicology XX(X)

Figure 2. Paraffin sections of (A) testis and (B) cauda epididymis of vas occlusion with RISUG and vas occlusion reversal by dimethyl sulfoxide
(DMSO) and sodium bicarbonate (NaHCO3) animals were treated with recombinant terminal deoxynucleotidyl transferase (TdT) and pro-
pidium iodide. Pointed red arrows indicate terminal deoxynucleotidyl transferase nick end labeling (TUNEL)-positive cells. DNAase I treatment
was used as positive control. Fluorescent images were observed at 400 and analyzed by ImageJ and DigiPro software.

Statistical Analysis in the range of 10% to 12% and 6% to 10%, respectively.

However, DNAase I-treated respective tissues showed
The mean values were compared using respective standard
47.94% (18.16%) and 40.74% (16.45%) of TUNEL-positive
deviation, followed by statistical comparison between sham-
cells. Testis showed slightly higher number of positive cells
operated control, vas occlusion, and vas occlusion reversal for
than cauda epididymis (Figure 1). The TUNEL-positive cells
evaluation of significant changes. Power calculation was per-
were localized to spermatogonial cells and spermatids in testes,
formed, and difference between groups was analyzed by w2 test
whereas principal and basal cells in cauda epididymis. How-
using PS version 3.12 software (Department of Biostatistics,
ever, the TUNEL values of both short- and long-term reversal
Vanderbilt University, Medical Centre, Nashville, Tennessee).
groups were normal compared to sham-operated control
The P values <0.05 were considered as significant.
(Figure 2).

Results Immunofluorescent detection of activated caspase-3. The percen-

tages of caspase-3-positive cells in testes and cauda epididymis
Apoptotic Markers in all the experimental groups were found in the range of 0.68%
The TUNEL assay. The percentage of TUNEL-positive cells in to 1.08% and 0.40% to 0.92%, respectively. A slight insignif-
testes and cauda epididymis of all the studied groups was found icant elevation was observed in groups II and V. Testis showed
Ansari et al
Figure 3. Percentage of positive cells showing activated caspase-3 in
cauda epididymis and testis of vas occlusion with RISUG and vas
occlusion reversal by dimethyl sulfoxide (DMSO) and sodium bicar-
bonate (NaHCO3) animals (groups I-VII). Images were analyzed by
ImageJ and DigiPro software.
slightly higher number of positive cells than cauda epididymis
(Figure 3). Caspase-positive cells were localized to spermato-
gonial cells and spermatids in testes, whereas principal and
basal cells in cauda epididymis. However, the percentage of
caspase-3 activated cells in experimental groups was compa-
rable to sham-operated control (Figure 4).
Annexin V binding assay. The percentage of annexin V-positive
cauda epididymal spermatozoa in groups I to VII was observed
to be minimum, that is, below 1% without much variation,
except group II, that observed with slight elevation in the value
significantly (P < 0.05; Figure 5). Fluorescent images of
annexin V–FITC conjugate and PI-treated samples showed
green fluorescence as the indication of the presence of few
positive cells. However, the results did not show significant
differences in all vas occlusion and vas occlusion reversal
groups, and the values were comparable to sham-operated con-
trol group (Figure 6).
Serum Clinical Biochemistry
Hormone analysis. The range of concentrations of testoster-
one (4.54-5.45 nmol/L), prolactin (37.80-43.15 mIU/L),
and cortisol (101.36-116.53 nmol/L) in all vas occlusion
and reversal groups did not show statistically significant
changes compared with those of control levels (data not
shown).
Prostate-specific antigen. The levels of serum PSA in all experi-
mental groups were noticed in the range of 0.22 to 0.30 ng/mL
with a nonsignificant variation when compared with sham-
operated control group (data not shown).
Antisperm antibody. Determination of ASA with the ELISA
method offered a range of 35.10 to 43.60 U/mL in all studied
groups. The values were comparable to sham-operated control
group (data not shown).
5
Salmonella typhimurium reverse mutation assay (Ames test). Muta-
genicity of RISUG was tested at dose levels of 10, 50, and 100
mL by S typhimurium tester strains TA97a, TA98, and TA100.
Sodium azide and daunomycin (daunorubicin hydrochloride)
at the dose of 5.0 mg/plate were used as positive controls,
whereas DMSO at the doses of 10, 50, and 100 mL were used
as a negative control in the presence and absence of the S9
metabolic activation system.
In positive control, a dramatic increase in the number of
revertant colonies to >500 and >1,000 was observed on GM
agar plates in all 3 strains (TA97a, TA98, and TA100) in the
presence of S9 mix, which was significantly higher. In negative
control (DMSO), the number of revertant colonies at 10 mL was
12 to 15, at 50 mL was 22 to 33, and at 100 mL was 35 to 46 in
the presence of S9 mix, whereas the number of revertant colo-
nies on plates containing RISUG at 10 mL was 17 to 25, at 50
mL was 12 to 62, and at 100 mL was 12 to 68 in the presence of
S9 mix (Table 1).
However, in the absence of S9 mix, the positive control also
showed a remarkable increase in the number of revertant colo-
nies to >500 and >1,000 was observed on GM agar plates in all
3 strains (TA97a, TA98, and TA100), which was considerably
elevated. In negative control (DMSO), the number of revertant
colonies at 10 mL was 11 to 13, at 50 mL was 12 to 20, and at
100 mL was 18 to 40, while the number of revertant colonies on
plates containing RISUG at 10 mL was 12 to 22, at 50 mL was
13 to 58, and at 100 mL was 21 to 67 in the presence of S9 mix
(Table 1). No significant increase was observed in the number
of revertant colonies on plates containing RISUG in the pres-
ence and absence of S9 mix in all 3 strains.
Discussion
In the era of innovations in reproductive health, RISUG is an
emerging reversible male contraception method in terms of
safety, efficacy, and reversibility. The clinical acceptability
based on both reversal approaches for RISUG in higher animals
would be increased, as the predicted results of the present study
following injection as well as its reversal were in support. The
chromatin status and DNA integrity of a cell are critical factors
that may affect individual fertility potential. So it is essential to
evaluate the effects of chronic stress of RISUG reversal, if
occurs, on various reproductive tissues as well as hormone
level along with the mutagenicity of the test material. Further-
more, there is no information available about the copolymer
reversal on hazardous substances database. Therefore, the pres-
ent study aimed to through light toward mutagenicity of
RISUG followed by its nonlethal or harmless reversal in rat
animal model depicting short- and long-term evaluation.
Among all available approaches for men, the vas-based
methods are mostly appreciated, while vasectomy accounts for
more than 20% of the current methods of contraception in
male. After vasectomy reversal, ASAs, abnormal sperm mor-
phology, epididymal dysfunctions, and so on, are the reasons of
lower pregnancy rate.17 Vasectomy with subsequent reversal
may also be associated with detectable alterations in sperm
6 International Journal of Toxicology XX(X)

Figure 4. Fluorescent images of (A) testis and (B) cauda epididymis of vas occlusion with RISUG and vas occlusion reversal by dimethyl sulfoxide
(DMSO) and sodium bicarbonate (NaHCO3) animals were observed at 400 and analyzed by ImageJ software. Pointed red arrows indicate the
release of fluorophore 7-amino-4-trifluoromethylcourmarin (AFC) in caspase-3 activated cells.

DNA integrity.18 For these reasons, vasectomy cannot be rec-
ommended as a truly reversible method of contraception. In the
present study, RISUG has surely created a new concept of
contraception with great feasibility and long-lasting sterility.
As concern with its reversal, RISUG tends to dissolve easily at
higher pH (8-9) solution, mainly unstable its components and
allows it to unbind from the wall of vas deferens. Hence,
DMSO and NaHCO3 are used to flush RISUG plug from the
vas deferens. To evaluate the effects after reversal, toxicity-
related assays were performed.
Apoptosis is a major regulating factor proposed to cause
DNA damage in spermatozoa before and after spermatogen-
esis. As an antagonist of cell proliferation, apoptosis contri-
butes to keeping the cell number in testicular tissue and helps
to remove superfluous and damaged cells, but excessive apop-
tosis could cause destruction of male reproductive function.
Several methods exist for the detection of apoptosis using fea-
tures of the cell as it undergoes the various stages, leading to
the death of the cell. Recently, TUNEL assay, caspase-3, and
annexin V have claimed more specificity. The present findings
of apoptotic markers in testes, cauda epididymis, and cauda
epididymal spermatozoa were pointed out without any evi-
dence of genotoxicity related to drug injection followed by its
reversal. There are a variety of etiologic factors that have
been associated with sperm DNA fragmentation and/or
impaired chromatin integrity. The minimum damage that
observed could be due to individual animal response to
various internal and external factors. In our recent studies of
Ansari et al
Figure 5. Percentage of annexin V-positive cauda epididymal sperms
of groups I to VII. Significant variation was observed in percentage of
annexin V-positive cells of group II cauda epididymal sperm when
compared with other experimental groups.
Figure 6. Annexin V assay of cauda epididymal spermatozoa of experimental groups. Spermatozoa were treated with annexin V-FITC conjugate
and propidium iodide (PI). Few spermatozoa with green fluorescence are showing annexin V-positive cells. Images were observed at 400 and
analyzed by ImageJ and DigiPro software.
7
genotoxicity in rats, the comet assay presented the evidence
that vas occlusion with RISUG and its reversal are highly
unlikely to produce any genotoxic activity in leukocytes and/
or testis, when injected under prescribed dose regimen.5 Simi-
larly, in rabbits, the overall results of genotoxicity and apopto-
tic markers indicated that no serious toxic effects were found
related to any response of RISUG or its reversal.11
In addition to apoptotic proteins, hormones have evident
role in spermatogenesis. The present investigation based on
reversal showed no significant fluctuations in the level of tes-
tosterone, prolactin, and cortisol. The values obtained were
within control range, which reveals that RISUG reversal does
not directly influence the metabolism of reproductive hor-
mones. Prostate-specific antigen and ASA are valuable serum
markers for diagnosing and monitoring infertility. Their levels
were noticed without appreciable changes in all experimental
groups as compared with control animals. Unlike our results,
serum clinical chemistry (testosterone and PSA) in langur
 monkey observed with values fluctuated within control limits,

31.66 (10.41)

21.00 (3.60)

66.66 (7.63)
TA100 S9
TA97a S9

TA98 S9
indicating safety of the procedure at the level of accessory
reproductive organ.10,19,20

100 mL
The Ames test was performed with RISUG to be reported

33.33 (10.40)

68.33 (20.20)
first time in the literature. Presently, the mutagenicity of

12.66 (2.51)
TA100 S9þ
þ

TA98 S9þ
TA97a S9
RISUG as a test compound was examined at different dose
levels, that is, 10, 50, and 100 mL. Hence, it suggests that
RISUG doses tested against the 3 strains did not meet the
30.00 (5.00)

13.33 (1.52)

58.33 (7.63)
TA100 S9


TA98 S9 criteria for a potential mutagen. The overall results depicted
TA97a S9

the successful reversal of the test material without any adverse

RISUG

property in concern of toxicity at the cellular level and hormo-

50 mL

61.66 (12.58)
nal imbalance. Although more detailed work is required to
31.66 (7.63)

12.33 (2.51)
TA100 S9þ
þ

TA98 S9þ

Values are mean (SD); Salmonella typhimurium tester strains: TA97a, TA98, and TA100; S9þ: Metabolic activation system; S9: Without metabolic activation system.
TA97a S9

bridge the association, the important consideration concerns

are the accomplishment of the study in higher animals in future
and findings of molecular mechanism of the intrinsic apoptotic
18.66 (5.50)

12.66 (3.05)

22.66 (6.42)
TA100 S9

pathways including expression and roles of apoptotic proteins



TA98 S9
TA97a S9

that should be highlighted soon. Furthermore, clinical studies

should be performed using above reversal solvents to deter-
10 mL

mine its acceptability in men.

21.00 (3.60)

17.66 (2.51)

25.00 (5.00)
TA100 S9þ
þ

TA98 S9þ
TA97a S9

Conclusion
40.00 (5.00)

18.33 (2.88)

26.66 (7.63)
TA100 S9


TA98 S9

The preclinical toxicity testing after reversal of RISUG on

TA97a S9

various biological samples revealed the dose-specific effects

100 mL

in male albino rats. Conclusively, the present study highlighted

46.66 (11.57)

RISUG as nonmutagenic with the negative response of geno-

45.00 (5.00)

35.00 (5.00)
TA100 S9þ
þ

TA98 S9þ
TA97a S9

toxicity markers linked with its reversal. The normal hormonal

status, PSA, and ASAs revealed safety of the procedure that did
not cause any alteration in the functions of accessory reproduc-
Negative control (DMSO)

11.66 (2.88)

13.33 (2.88)

20.00 (5.00)
TA100 S9


TA98 S9

tive organs. However, RISUG is considered as safe procedure

TA97a S9
Table 1. Number of Revertant Colonies Over Glucose Minimal (GM) Agar Plates.a

to be used with its reversibility upon need, even after long

duration without any toxic effects.
50 mL

21.66 (2.88)

33.33 (5.77)

31.66 (2.88)
TA100 S9þ
þ

TA98 S9þ
TA97a S9

Acknowledgments
The infrastructural facilities provided by the Head of the Department
are gratefully acknowledged. The authors are thankful to Prof Sujoy
11.66 (2.88)

13.33 (5.77)

13.33 (2.88)
TA100 S9


TA98 S9
TA97a S9

K. Guha, School of Medical Science and Technology, Indian Institute

of Technology, Kharagpur, West Bengal, India, for providing RISUG.
10 mL

The award of NASI Senior Scientist’s assignment to NKL is gratefully

12.33 (2.51)

11.66 (2.88)

15.00 (5.00)

acknowledged.
TA100 S9þ
þ

TA98 S9þ
TA97a S9

Author Contributions
Daunomycin (5 mg/plate)

TA100 S9


TA98 S9

Abdul S. Ansari and N. K. Lohiya designed the study. Mubarik Hus-

TA97a S9
>1,000

>1,000

>1,000

sain and Sadi Rehan Khan performed the experiments and analyzed
Abbreviation: DMSO, dimethyl sulfoxide.

the data. Ayesha Badar represented the data and wrote the manuscript.
N. K. Lohiya finally approved the version to be published. Abdul S.
TA100 S9þ
þ

TA98 S9þ
TA97a S9
>1,000

>1,000

>1,000

Ansari substantially contributed to conception or design and critically

revised the manuscript for important intellectual content. Mubarik
Hussain contributed to acquisition, analysis, and interpretation and
critically revised the manuscript. Sadi Rehan Khan contributed to
Sodium azide (5 mg/plate)

TA100 S9


TA98 S9
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>500

>500

acquisition, analysis, and interpretation and critically revised the

manuscript. Ayesha Badar contributed to interpretation and drafted
Positive control

the manuscript. N. K. Lohiya contributed to conception and

TA100 S9þ
þ

TA98 S9þ

design and critically revised the manuscript. All authors gave

TA97a S9

final approval and agree to be accountable for all aspects of work

>500

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ensuring integrity and accuracy.

a

8
Ansari et al
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to
the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support
for the research, authorship, and/or publication of this article: The
study was supported by the Indian Council of Medical Research,
New Delhi.
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