Mutation Research, 124 (1983) 235-240 235 Elsevier

MTR 00821

Evaluation of Propineb, a dithiocarbamate pesticide, in the mouse-sperm morphology assay *

I. Q u i n t o i and E. D e Mar in i s 2

I I I Biological Chemistry Institute, H Medical and Surgical Faculty, University of Naples (Italy); and 2 Toxicology Department, Ente Farmacologico Italiano, Via San Giacomo dei CaprL 66- 80131 Naples (Italy)

(Received 22 April 1983) (Accepted 7 July 1983)

Summary

Propineb, a dithiocarbamate fungicide, was studied by using the sperm morphol- ogy assay in (C57BL 6 ~ x C3H ~ ) F 1 mice. At all dose levels, no statistically significant increase in the percentage of sperm abnormalities was observed. Methyl methanesulfonate (MMS) and 2-acetylaminofluorene (2-AAF), which were tested as positive controls, induced a dose-effect-related increase in teratospermia.

Propineb [zinc propylenebis(dithiocarbamate)] is a fungicide of the class of dithiocarbamates, widely used in agriculture. Despite the possible environmental contamination by this pesticide, its toxic properties are not yet clarified. The teratogenic effects of Propineb in rats (Larsson et al., 1976) and of propylene thiourea, its main metabolite and degradation product (Bleyl and Lewerenz, 1978), have been reported. The results are in agreement with the teratogenic properties shown by other dithiocarbamate pesticides (Petrova Vergieva and Ivanova Chem- ishanska, 1973; Larsson et al., 1976; Marston, 1969) and by ethylenethiourea, their main metabolite and degradation product (Khera, 1973; Tomatis et al., 1978; Teramoto et al., 1981).

The mutagenic properties of various dithiocarbamate compounds have been investigated in several toxicological test systems (Benes and Sram, 1969; Chernov and Kmitsenko, 1969; Pilinskaia, 1970; Seiler, 1973, 1974, 1977; Fahrig, 1974; Zdzienicka et al., 1982). In particular, Propineb was found not to be mutagenic in the Salmonella microsome assay (Chii-Ling and L i Gwo-Chen, 1980) and was

* This work was supported by the CNR (Consiglio Nazionale delle Ricerche) grants RN-18350/A and 82.02146.56.

Abbreviations: 2-AAF, 2-acetylaminofluorene; MMS, methyl methanesulfonate.

0165-1218/83/$03.00 © 1983 Elsevier Science Publishers B.V.

236

reported to be negative in the mouse micronucleus test (Rolandi et al., unpublished data). To get further information on the genetic risks of Propineb, we investigated the effects of this fungicide on the germinal cells by using the sperm morphology test in mice (Wyrobek and Bruce, 1975).

The mouse-sperm morphology assay is considered to be useful to detect the action of chemicals that can interfere with the differentiation process of germinal cells. The genetic basis of this test requires further investigation. However, there is some evidence showing the involvement of genetic mechanisms in the expression of sperm morphology, such as the following.

1. The morphology of sperm and the proportion of teratospermia is constant for each strain.

2. Mutagens can induce sperm abnormalities (Wyrobek and Bruce, 1978) depend- ing on their capability to affect the testes.

3. Induced sperm abnormalities are transmissible to the F 1 male progeny. As this kind of assay in vivo can help in the establishing of the possible damage

induced at the germinal tissue level, the action of Propineb on spermatogonial cells according to Wyrobek's method (Wyrobek and Bruce, 1975) was studied. MMS was used as positive control. Moreover, 2-AAF, a well-known mutagen and carcinogen, which was previously found to be an uncertain negative in this test (Topham, 1980), was further tested.

Materials and methods

Chemicals Propineb (98% purity) was synthesized in the Chemical Department of EFI.

MMS and 2-AAF were supplied by Merck. The vehicles were 0.5% Tween 80 for Propineb, distilled water for MMS and D M S O / H 2 0 (1 : 5) for 2-AAF.

Method Male mice of genotype (C57BL 6 ~ x C3H ~ ) F a, 10-12 week s old, were used.

The parental strains (C57BL 6 and C3H) were obtained from Charles River, Calco, Como, Italy. The mice were given food and water ad libitum. Room temperature (22-24°C), moisture (60%), air changes (10 cycles/h) and lighting (12 h on and 12 h off) were controlled. The test was performed according to Wyrobek's method (Wyrobek and Bruce, 1975).

Chemicals were administered i.p. to groups of 6 mice once a day for 5 consecutive days at a constant volume of 10 ml/kg. The 5th week after the first injection, mice were killed by cervical dislocation, and the testes weight for each mouse was recorded. Sperm suspensions, smears, slides and evaluation of sperm abnormalities were made according to Wyrobek (Wyrobek and Bruce, 1975). 500 spermatozoa for each animal were counted at 400 x , under blue-green filters.

Statistical evaluation of the data was performed by the analysis of variance and Dunnett 's multiple comparison test for testes weight. The average percentage sperm abnormalities were analyzed by the Mann-Whitney U test (Sach, 1974). For active compounds the data were fitted to linear, quadratic, exponential, logarithmic and

237

@I c MI w

m

E

Y-- 2.1 e 0"035x

30

20-

10-

0 1'0 2'0 4'0 8'0

M M s

Fig. 1. Increase of sperm abnormalities in mice treated with methyl methanesulfonate. Dose-effect curve. Mean + S.E.

power regression. The validity of different fittings was compared by the F ratio according to Snedecor and Cochran (1968).

Results and discussion

Negative control animals showed a frequency of sperm abnormalities ranging between 1.5 and 4.1%, independently of the vehicle used. These values are in the range already reported for the same hybrid strain used by Wyrobek (Wyrobek and Bruce, 1975).

Propineb, administered up to the dose of 250 mg/kg day, did not induce any statistically significant increase in sperm abnormalities (Table 1). A higher dose (500 mg/kg /day) was lethal for all animals.

Testes weight, related to the body weight, did not show any significant difference up to the highest dose levels for all tested chemicals (Tables 1 and 2). On the contrary, a statistically significant decrease was observed at the highest dose levels of MMS and 2-AAF, for absolute testes weight of treated animals, compared with that of negative controls (Tables 1 and 2).

Thus, under our experimental conditions, Propineb did not interfere with normal sperm development. In fact, the sperm analysis was performed at a time (5 weeks after the treatment ) when the effects of the administered chemicals on germinal cells at the pre-meiotic stage of spermatogenesis could be evaluated. Under the same experimental conditions, MMS and 2-AAF induced a dose-related increase in sperm abnormalities (Tables 1 and 2).

238

T A B L E 1

E F F E C T O F P R O P I N E B A N D M M S O N W E I G H T O F T E S T E S A N D I N D U C T I O N O F S P E R M

A B N O R M A L I T I E S IN (C57BL6 × C 3 H ) F 1 M I C E (mean_+ S.E.)

C o m p o u n d Dose Testes we igh t S p e r m

( m g / k g / d a y A b s o l u t e W e i g h t / b . w . a b n o r m a l i t i e s

fo r 5 days ) values (mg) ( m g / g ) (%)

T w e e n 80, 0.5%

P r o p i n e b

M M S

- 2 5 7 + 7 9 .04-0 .22 2 . 5 3 + 0 . 4 3

62.5 2474- 7 ns 8 . 6 + 0 . 2 1 ns 2 . 1 5 + 0 . 1 3 ns

125.0 250 + 4 n~ 8.7 + 0.26 n~ 2.53 + 0.22 ns

250.0 235 4-12 ns 8.6 + 0.72 "~ 2.55 + 0.17 ns

10.0 243 4- 6 ~s 8.6 + 0.27 ,s 2.05 ___ 0.23 ns

20.0 2564- 11 ~s 8 . 4 + 0 . 3 6 ~s 5 .484-0 .65 a

40.0 2364- 9 ns 8.1 + 0 . 1 2 ~s 10 .104-0 .86 a

80.0 2194- 5 ~ 8 . 2 + 0 . 2 6 ~s 30 .154-2 .42 ~

ns, no t s igni f icant .

a s ign i f i can t a t 1% level.

As to statistics, the linear regression for 2-AAF and the exponential regression for MMS are the most sensitive instruments of analysis; therefore, the graphic descrip- tion of the data (Figs. 1 and 2) is made with these regressions. For MMS, our data are in good agreement with the results of other authors (Wyrobek and Bruce, 1975; Topham, 1980). In our hands, 2-AAF gave positive results, in contrast with another report (Topham, 1980). However, 2-AAF was previously tested on a different hybrid strain (CBA 8 × Balb/C ~ ) F 1, giving some sporadic non-reproducible effects (Topham, 1980).

In conclusion, even if information is scarce about the mutagenic properties of Propineb, the negative results reported in the Salmonella microsome assay (Chii-Ling and Li Gwo-Chen, 1980) and the mouse micronucleus test (Rolandi et al., unpub-

T A B L E 2

E F F E C T O F 2 - A A F O N T H E T E S T E S W E I G H T A N D I N D U C T I O N O F S P E R M A B N O R M A L I -

T I E S I N (C57BL 6 × C 3 H ) F 1 M I C E ( m e a n 4- S.E.)

C o m p o u n d dose Tes tes we igh t S p e r m

( m g / k g / d a y A b s o l u t e W e i g h t / b . w . a b n o r m a l i t i e s fo r 5 days ) va lues (mg) ( m g / g ) (%)

DMSO-H20 (1:5)

2 - A F F

0 207 + 5 6.6 + 0.22 2.83 + 0.08

125 2 1 8 + 5 ns 6 . 9 + 0 . 1 3 ns 5 . 1 2 + 0 . 1 8 a

250 190 4- 6 ns 6.3 4- 0.12 ns 7.64 + 0.42 a

500 185 + 7 a 6.2 4- 0.27 as 10.30 + 0.30 a

ns , n o t s igni f icant .

a s ign i f i can t a t 1% level.

239

A

~ a

~10

Y " 3.19 + 0 . 0 1 5 x

1~5 2~o s~o

Fig. 2. Increase of sperm abnormalities in mice treated with 2-acetylaminofluorene. Dose-effect curve. Mean + S.E.

lished data), in addition to our negative results in the sperm morphology assay, seem to indicate an absence of mutagenicity of this compound.

Acknowledgements

We thank Dr. C. Mayer for synthesizing samples of Propineb.

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