Toxicology Letters, 56 (1991) 207-212

@ 1991 Elsevier Science Publishers B.V. (Biomedical Division) 0378-4274/91/$3.50

ADONIS037842749100067L

TOXLET 02550

Effects of triphenyltin acetate on pregnancy in rats by oral administration

Tsutomu Noda, Shigeru Morita, Tetsuo Yamano, Mitsuru Shimizu and Akio Yamada

Osaka City Institute of Public Health and Environmental Sciences, Osaka (Japan)

(Received 27 July 1990)

(Accepted 6 November 1990)

Key words: Triphenyltin acetate; TPTA; Embryotoxicity; Fetotoxicity

207

SUMMARY

A teratological test was carried out on triphenyltin acetate (TPTA) used as a fungicide and antifouling

agent. Pregnant Wistar rats were treated orally with TPTA at dose levels of 0, 1.5, 3.0, 6.0, 9.0 and 12.0

mg/kg/d during days 7-17 of gestation. Cesarean sections were performed on day 20 of gestation. In the

pregnant rats, 2 of 13 and 2 of 12 dams died at 9.0 and 12.0 mg/kg, respectively. Vaginal bleeding, bloody

mouth and nose, somnolence and depression of body weight gain and food intake were observed at 9.0

and 12.0 mg/kg at late stages of pregnancy. No statistically significant reductions in maternal thymus and

spleen weights were observed on day 20 of gestation. Increase in embryonic and fetal deaths and in dams

with total resorption of fetuses were observed at doses of more than 6.0 mg/kg. The doses of TPTA in

this experiment, however, induced no teratogenic effects in rats.

INTRODUCTION

Triphenyltin compounds have been widely used as biocides in marine antifouling paints and they are still important agricultural fungicides due to their specific activi- ties [l]. Triphenyltin compounds have chemosterile properties in insects, such as house flies (Musea domestica), German cockroaches (Blatella germanica), flour bee- tles (Tribolium confusum) etc. [2]. Triphenyltin acetate (TPTA) and triphenyltin chlo- ride produce degenerative changes in testicular tissues [3] and also produce significant

Address for correspondence: Tsutomu Noda, Osaka City Institute of Public Health and Environmental

Sciences, 8-34 Tojo-cho, Tennoji-ku, Osaka 543, Japan

208

changes in ovarian tissues including a decreased number of mature follicles, and an

increased incidence of atresia in early follicle growth in rats [4].

As regards mammalian teratogenic studies of triphenyltin compounds, two studies

have been published in which TPTA [5] and triphenyltin hydroxide [6] were shown

to have no teratogenicity in rats. But in the study of TPTA, the chemical was admin-

istered to pregnant females on days 5-14 of gestation and maternal body weight on

day 5 of gestation was used for calculation of the doses. Thus the doses became re-

latively low at the end of the treatment period because the animals underwent a more

than 15% increase in body weight during that period. In the present study, we re-

evaluated the teratogenic potential of triphenyltin acetate (TPTA) in rats treated

orally during the organogenetic phase of gestation (days 7-l 7).

MATERIALS AND METHODS

TPTA was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

Four-week-old Wistar rats of both sexes (CLEA Japan, Inc., Tokyo, Japan) were

individually housed in a room with a constant day/night cycle (dark period from 7:00

p.m. to 7:00 a.m.) at 23 f 2°C temperature and 60 f 10 % relative humidity. They were

given feed (NMF, Oriental Yeast Co., Ltd., Tokyo, Japan) and tap water ad libitum.

A 3-month-old female rat was paired overnight with a male of the same age, and

the day on which sperm was observed in the vaginal smears was designated as day

0 of gestation. Mated females were randomly assigned to 6 groups (13-14/group).

TPTA was dissolved in olive oil and administered to females by stomach tube at

doses of 0, 1.5, 3.0, 6.0, 9.0 and 12.0 mg/kg during days 7-17 of gestation. The vol-

ume of vehicle was held constantly at 2 ml/kg of maternal body weight. Control ani-

mals were administered an appropriate volume of vehicle without TPTA.

Every day, body weight and food consumption were measured and general behav-

ior was observed. All females were euthanized under ether anesthesia on day 20 of

gestation. Upon laparotomy, the position and number of live and dead fetuses in-

cluding resorbed fetuses in the uterus were recorded. The number of corpora lutea

and maternal thymus and spleen weights were also recorded. A uterus with total re-

sorption was isolated and stained with 10 % ammonium sulfide to determine the total

number of implantations. Live fetuses were weighted and examined for their sex and

external malformations. One-half of the live fetuses in each litter was fixed in 95%

ethanol and processed for staining of the skeletons for skeletal abnormalities by the

alizarin red S dye method [7]. The other half was fixed in Bouin’s solution and exam-

ined for visceral abnormalities according to the method of Wilson [S].

Data were analyzed by Dunnett’s [9] or Scheffe’s [lo] multiple comparison method

in a parametric or non-parametric manner. Data were analyzed in the groups which

received doses up to 9.0 mg/kg because there was only one pregnant animal with live

fetuses in the highest dose group (12.0 mg/kg).

209

RESULTS

Oral treatment of pregnant rats with TPTA during the organogenetic phase of ges-

tation caused maternal death and reduction of maternal body weight gain and food

intake at 9.0 and 12.0 mg/kg (data not shown). Two pregnant animals with vaginal

bleeding at 9.0 and 12.0 mg/kg, respectively, died during day 9-13 of gestation. Ani-

mals receiving 6.0 mg/kg or more showed piloerection. Vaginal bleeding, somnolence

and bloody mouth and nose were observed in about half of the animals at 9.0 mg/kg

and almost all at 12.0 mg/kg. However, no toxic signs were seen at doses up to 3.0

mg/kg. Maternal thymus weights seemed to be reduced at 9.0 mg/kg, although this

was not statistically significant, whereas maternal spleen weights were not reduced

at the same dose level (Table I).

Total resorption of fetuses was observed in 2/13 litters at 6.0 mg/kg, 6/l 1 at 9.0

mg/kg, and S/l0 at 12.0 mg/kg. Even in the dams with live fetuses, the rate of resorp-

tion increased at late stages of gestation at 9.0 mg/kg. The number of live fetuses

and the mean fetal body weights of both sexes seemed to decrease at 9.0 and 12.0

mg/kg, although the number of implants and the sex ratio were unaffected (Table

II). When data were calculated for all the pregnant animals including those with total

resorption, the rates of resorption in the groups treated with TPTA at 6.0, 9.0 and

12.0 mg/kg were 21.4, 60.8 and 97.3% (P~0.01) at an early stage of gestation, and

1.6, 11,9 and 1.3% at late stages of gestation, respectively, while the mean numbers

of live fetuses were 10.4 f 4.84, 4.2 f 5.74 and 0.2 f 0.63 (P-C 0.0 l), respectively.

From external and visceral observations, the incidence of fetuses with malforma-

tions did not increase in the TPTA-treated groups. Also from the skeletal observa-

tions, no significant increase in malformations was found in the fetuses in the treated

groups. Minor skeletal variations, such as cervical rib and rudimentary lumbar rib

(less than half the length of 13th rib [ 111) did not increase in the fetuses in the treated

groups. The number of sternebrae and proximal and middle phalanges of the hind-

limbs as an index of the degree of ossification decreased in the fetuses from dams that

received 9.0 mg/kg (Table III).

DISCUSSION

Snoeij et al. [12] have reported that triphenyltin chloride induced a dose-related

reduction of thymus and spleen weights in rats fed at dietary concentrations of 15,

50 and 150 ppm for 2 weeks. A reduction in spleen weight was also observed upon

feeding triphenyltin compounds [13]. In the present study, however, the thymus and

spleen weights of pregnant rats treated orally with TPTA at dose levels of 1.5, 3.0,

6.0, 9.0 and 12.0 mg/kg from days 7-17 of gestation were not reduced significantly.

Even when data were calculated for all the pregnant animals including those with

total resorption of fetuses (2 at 6.0 mg/kg, 6 at 9.0 mg/kg and 9 at 12.0 mg/kg), no

significant reductions were observed in these organ weights.

TA

BL

E

I

EFF

EC

TS

OF

TPT

A

ON

MA

TE

RN

AL

SP

LE

EN

A

ND

T

HY

MU

S W

EIG

HT

S 3

_-._

-_

- --

-.

0

Oliv

e oi

l T

PTA

@

g/kg

) (2

ml/k

g)

1.5

3.0

6.0

9.0

12.0

n 14

13

12

II

5

1 B

ody

wt.

(8)

314k

l2.1

32

8 5

24.9

31

4k19

.2

303

+- 2

5.7

288i

38

.0

228

Sple

en w

t. (m

g)

591*

31.5

62

2 f

69.4

56

2k45

.4

546h

67.4

52

3& 1

26.3

405

Thy

mus

w

t. (m

g)

174+

15.7

19

9k37

.1

188

+ 2

7.9

153k

66.7

16

+

372

73

Val

ues

are

the

mea

n +

sta

ndar

d de

viat

ion.

Tw

o da

ms

(6.0

mg/

kg),

6

dam

s (9

.0 m

g/kg

) an

d 9

dam

s (1

2.0

mg/

kg)

with

tot

al

reso

rptio

ns

are

excl

uded

fr

om

thes

e da

ta.

TA

BL

E

II

EFF

EC

TS

OF

TPT

A

ON

TH

E

PRE

GN

AN

T

RA

TS,

L

ITT

ER

S A

ND

FE

TU

SES

AFT

ER

T

RE

AT

ME

NT

O

F PR

EG

NA

NT

R

AT

S

Oliv

e 01

1 fP

TA

(w/W

(2

ml/k

g)

1.5

3.0

6.0

9.0

12.0

-.

No.

of

fem

ales

in

sem

inat

ed

14

13

13

13

13

13

No.

of

preg

nant

fe

mal

es

14

13

12

13

13

12

No.

of

dam

s w

ith l

ive

fetu

ses

14

13

12

11

5**

I**

No.

of

dam

s w

ith t

otal

re

sorp

tion

0 0

0 2

b**

9**

No.

of

dead

dam

s 0

0

0

0

2 2

No.

of

corp

ora

lute

a a.

b 14

.3*0

.73

15.4

+

1.94

14

.7+

1.

97

14.8

22.6

4 l&

2+2.

59*

IS.0

N

o. o

f im

plan

tvb

13.6

rtO

.93

14.2

kO.9

9 13

.5+

2.15

13

.5rt

: 1.

51

15.6

kO.8

9 15

.0

Inci

denc

e of

dea

d or

res

orbe

d fe

tuse

s (s

)~

Ear

ly

stag

e 5.

9 4.

3 7.

5 7.

1 13

.8

73.3

L

ate

stag

e 0

0.5

0.8

1.9

26.3

**

13.3

N

o. o

f liv

e fe

tuse

sa

12.9

k1.2

3 13

.5+

1.2

7 12

.422

.47

12.3

Lt: 1.62

9.2k

4.97

2.

0 Se

x ra

tio

(M/F

) 1.

1(86

/94)

1.

0(83

/93)

1.

2(78

:71)

1.

5(75

/M))

1.

7(25

/21)

I.o

(l:l)

B

ody

wei

ght

of l

ive

fetu

sesa

(g)

M

ale

3.2k

O.2

7 3.

3iO

.18

3.4

+ 0

.49

3.2~

0.11

2.

3 20

.47

1.5

Fem

ale

3.0*

0.17

3.

liO.1

4 3.

2+0.

52

3.0&

0.16

2.

1 io

.52

1.9

~___

__

_-__

a Val

ues

are

the

mea

n f

stan

dard

de

viat

ion.

hT~o

da

ms

(6.0

mgi

kg),

6 d

ams

(9.0

mg/

kg)

and

9 da

ms

(12.

0 m

g/kg

) w

ith t

otal

re

sorp

tions

ar

e ex

clud

ed

from

th

ese

data

.

l S

igni

fica

ntly

di

ffer

ent

from

con

trol

s,

P<

0.

05.

**Si

gnif

ican

tly

diff

eren

t fr

om c

ontr

ols.

P

<O

.Ol.

The

litt

er

was

use

d as

the

sta

tistic

al

unit

for

calc

ulat

ion

of f

etal

va

lues

. T

hus

thes

e va

lues

re

pres

ent

the

mea

ns

of l

itter

m

eans

w

ithin

ea

ch g

roup

.

TA

BL

E

111

EX

TE

RN

AL

, V

ISC

ER

AL

A

ND

SK

EL

ET

AL

O

BSE

RV

AT

ION

S O

F FE

TU

SES

FRO

M

DA

MS

TR

EA

TE

D

OR

AL

LY

W

ITH

T

PTA

Ext

erna

l ob

serv

atto

ns

No.

of

fetu

ses

exam

ined

Inct

denc

e of

fet

uses

with

mal

form

atio

ns

(%)

No.

of

fetu

ses

with

mal

form

atio

ns

Ves

tigia

l ta

il

Vis

cera

l ob

serv

atio

ns

No.

of

fetu

ses

exam

rned

Inci

denc

e of

fet

uses

with

mal

form

atio

ns

(%)

Skel

etal

ob

serv

atio

ns

No.

of

fetu

ses

exam

ined

Inci

denc

e of

fet

uses

w

ith m

alfo

rmat

ions

(%

)

No.

of

fetu

ses

wtth

mal

form

atio

ns

Age

nesi

s of

the

coc

cyge

al

vert

ebra

e In

cide

nce

of f

etus

es w

th

vari

atio

ns

(F)

No.

of

fetu

ses

with

var

iata

ons

Occ

ipita

l hy

popl

asia

Cer

vica

l ri

b

Split

ting

of 1

st c

ervi

cal

vert

ebra

l ar

ch

Shor

teni

ng

of 1

3th

rib

Rud

imen

tary

lu

mba

r ri

bs

Var

iatio

ns

in n

umbe

r of

lum

bar

vert

ebra

e

Deg

ree

of o

ssif

icat

iona

No.

of

ster

nebr

ae

No.

of

prox

imal

an

d m

tddl

e ph

alan

ges

Fore

hmb

Hin

dlim

b

No.

of

sacr

al

and

caud

al

vert

ebra

e

Oliv

e oi

l

(2 m

l/kg)

P

TA

(w

/W

I.5

3.0

6.0

9.0

12.0

I80

176

149

I35

46

2

0 O

.S( I

) 0

0 0

0

0 l(

l)

0 0

0 0

83

81

68

62

22

0

0 0

0 0

0 0

97

95

81

73

24

0 1.

0(l)

0

0 0

0 l(

l)

0 0

0

7.2(

5)

1.1(

l)

6.1(

3)

14.1

(7)

I I .4

(2)

0 0

0 0

0

6(5)

l(

l)

2(2)

8(

5)

3(2)

l(

l)

0 0

2(I)

0

0 0

0 0

l(l)

0

0 0

l(l)

0

0 0

2(2)

0

2(l)

0 50.q

I)

l(l)

0 0 0 0 l(

l)

4.3*

0.41

4.

8&0.

44

5. I

f0.4

2”

4.7k

O.3

5 3.

1 kO

.92’

. 2.

0

4.0

* 0.

84

5.2k

O.6

3 5.

5*

1.00

’ 5.

7*0.

26**

3.

9+

1.03

4.

0 *

0.00

4.

0 *

0.00

4.

3*1.

10

4.0~

0.00

3.

6 k

0.44

.’

6.9+

0.36

7.

1+0.

36

7.3k

O.5

8 7.

1 kO

.23

5.7k

O.7

8

3.0

3.5

5.0

‘Val

ues

are

the

mea

nt

stan

dard

de

wat

ion.

( ) N

o. o

f co

ncei

ved

mot

hers

w

ith c

ase

*Sig

nifi

cant

ly

difT

eren

t fr

om c

ontr

ols,

P

< 0

.05.

**Si

gnif

ican

tly

diff

eren

t fr

om c

ontr

ols.

P

cO.0

1.

The

litt

er

was

use

d as

the

sta

tistic

al

unit

for

calc

ulat

ton

of f

etal

val

ues.

T

hus

thes

e va

lues

re

pres

ent

the

mea

ns

of l

itter

m

eans

w

ithm

ea

ch g

roup

212

No embryotoxic and fetotoxic effects of TPTA were observed at less than 3.0 mg/

kg, although the number of dams with total resorption of fetuses in a dose-dependent

manner at more than 6.0 mg/kg. Moreover, TPTA at 9.0 and 12.0 mg/kg seemed

to increase post-implantation loss and to decrease the body weights of live fetuses

and the incidence of bleeding in the uteri. Giavini et al. [5] have reported that 4 of

10 pregnant females which received TPTA orally at 15 mg/kg on days 5-14 of gesta-

tion showed total resorption of fetuses, but not at up to 10 mg/kg. They used mater-

nal weight on day 5 of gestation to calculate the doses, but since we used daily mater-

nal weight for this, our total dosage of TPTA to a pregnant female was greater than

theirs.

No malformations were found upon external, visceral and skeletal observation in

fetuses from dams treated orally with TPTA during the organogenetic phase of gesta-

tion even at dose levels exhibiting obvious maternal toxicity. The present study sug-

gests that TPTA is not teratogenic, but that it is harmful to pregnant rats.

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