Mutation Research, 222 (1989) 149-154 149 Elsevier

MTR 01365

Cytogenetic effects of fenvalerate in mammalian in vivo test system

P.C. Pati and S.P. Bhunya Laboratory of Genetic Toxicology, P.G. Department of Zoology, Utkal Unirersity, Bhubaneswar- 751004, Orissa (India)

(Received 15 April 1988) (Revision received 26 July 1988)

(Accepted 24 August 1988)

Keyword~': Synthetic pyrethroid; Fenvalerate; Chromosome aberration; Micronucleus; Sperm abnormality; Swiss mice

Summary

A synthetic pyrethroid insecticide, fenvalerate, was tested for its cytogenetic effects in the mouse in vivo test system at 100, 150 and 200 mg/kg. Bone marrow metaphase analysis revealed significant increases in chromosomal aberrations in the groups treated with 150 and 200 mg/kg by intraperitoneal injection. In the micronucleus test the occurrence of PCEs with MN marginally increased with dose. Induction of PCEs with MN was significant over control again with the higher two doses. Incidence of sperm abnormalities slightly increased with dose but a significant increase was noted in all treated series over control.

In recent years, synthetic pyrethroids are gain- ing wide acceptance in agricultural usage for their high insect knock-down activity but low roam- marian toxicity (Papadopoulou-Mourkidou, 1983). Fenvalerate (a-cyano-3-phenoxybenzyl 2-(4-chlo- rophenyl) isovalerate) (Fig. 1), a potent pyrethroid is used in wide areas of plant protection such as in cotton, fruits, vegetables and in cultivation. It is destined for increased use against agricultural, poultry, dairy and household pests (Mumtaz and Menzer, 1986).

Records on the mutagenic effect of other syn- thetic pyrethroids include negative results with

Correspondence: Dr. S.P. Bhunya, Laboratory of Genetic Toxicology, P.G. Department of Zoology, Utkal University, Bhubaneswar-751004, Orissa (India).

Abbreciations: i.p., intraperitoneal; p.o., oral; s.c., subcuta- neous; DMSO, dimethyl sulphoxide; MN, micronucleus; PCE, polychromatic erythrocytes; NCE, normochromatic erythro- cytes.

deltamethrin, permethrin (Bartsch et al., 1980; Pluijmen et al., 1984), bloresmethrin, cismethrin, resmethrin, deltamethrin and cypermethrin (Pluij- men et al., 1984) using S. typhimurium strains, and V79 Chinese hamster cells with or without meta- bolic activation. Cypermethrin, however, has also been reported to induce micronuclei in bone mar- row cells of mice (Amer and Aboul Ela, 1985) and to increase the frequency of sex-rinked recessive lethals in Drosophila (Batiste-Alentorn et al., 1986). Again, deltamethrin was reported to in- crease the number of chromosome aberrations and micronuclei, and decrease the mitotic index in a dose-dependent manner in plants (Chouhan et al., 1986), but reports on the mutagenicity of fenvalerate are scanty (Pluijmen et al., 1984). In

O C 4 \CH 3

Fig. 1. Fenvalerate.

0165-1218/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

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view of this, an attempt has now been made to study the genotoxic effect of fenvalerate in the mouse in vivo test system utilizing bone marrow chromosome aberration, micronucleus and sperm abnormality assays.

Materials and methods

Test animal and chemical 10-12-week-old inbred Swiss mice with an

average body weight of 25 g, provided with stan- dard food and water ad iibitum, were utilised as the test animals. For bone marrow chromosome aberration and micronucleus assays, mice of either sex were used. Technical grade (94%) fenvalerate donated by Rallis (India) served as the test chem- ical. Analytical grade DMSO (Sisco) was used as the solvent. Controls received an equivalent amount (0.1 ml/individual) of DMSO.

Bone marrow chromosome aberration assay For metaphase analysis 3 doses of the test

chemical (100, 150 and 200 m g / k g b.w.) were used. With 200 m g / k g 3 routes of administration intraperitoneal (i.p.); oral (p.o.) and subcutaneous (s.c.), and 3 post-treatment fixation times (6, 24 and 48 h) were employed.

For chronic treatment, 5 equal subdivisions of the highest acute dose were injected i.p. (5 × 40 m g / k g b.w.) at intervals of 24 h (i.e. at 0, 24, 48, 72 and 96 h) and the mice were killed 120 h after the first injection. Cytological slides were prepared following the usual colchicine air-drying Giemsa schedule. 100 metaphase spreads of adequate qual- ity were scored per animal.

Micronucleus test Each of the 3 doses of the test chemical was

injected i.p. twice at an interval of 24 h (i.e., a total of 200, 300 and 400 mg/kg) and the animals were killed 6 h after the second injection. Bone marrow smears and staining were done following Schmid (1976) with modifications (Das and Kar, 1980). 1000 each of polychromatic erythrocytes (PCEs), normochromatic erythrocytes (NCEs) and immature nucleated cells were scored per animal. The ratio of PCEs to NCEs was also calculated.

Sperm abnormality assay In this assay, 5 equal subdivisions of each dose

were injected successively at intervals of 24 h and the animals were sacrificed 35 days after the first injection. Sperm collection was done from caudae epididymides in physiological saline and stained in 0.1% aqueous eosin-Y. 500 sperms were scored per animal and abnormalities were categorised, wherever possible, following Wyrobek and Bruce (1975) and Bhunya and Behera (1987).

Moreover, in the present study different treat- ment regimens for 3 endpoints were chosen in order to get the maximum reproducible results from these assays.

Statistical analysis All scorings were done from slides under code.

The percentage frequencies of chromosome aber- ra t ions /micronucle i /abnormal sperm were calcu- lated for each animal and then the mean per- centage and standard error for each group were calculated.

The data were admitted to the equality of pro- portion test (normal test) to calculate the signifi- cance of treated values over control using the following formula:

Z = IP,- t"21

}/P(1 - P ) (1 /Na + 1/N2)

Where P1 and P2 are the frequencies of anoma- lies/cell in control and treated series, respectively, N 1 and N 2 are the number of cells studied in control and treated series respectively, and P = (P1N1 + P2N2)/(N1 + N2). The result is consid- ered significant at the 5% level when Z >/1.96.

For dose-response analyses, linear regression equations were derived between the doses of fenvalerate used and the percentage of anomalies induced.

In addition, a one-way analysis of variance (ANOVA) test was conducted when more than 2 means were compared, with the level of signifi- cance chosen as 0.05. Further comparison was made between the calculated CD (critical dif- ference) value with the difference of 2 treatment means.

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152

Result~

Analysis of bone marrow metaphases revealed chromatid-type gaps including isochromatid gaps; chromatid-type deletions including chromatid breaks, isochromatid breaks and fragments, and ch romosome- type deletions yielding double minutes. The spectrum of aberrations also in- cludes a single case of chromatid-type exchange leading to a dicentric and 2 cases of chromosome- type exchanges forming metacentrics. The results have been summarised in Table 1. In all test cases, the gaps outnumbered the deletions. Because of their controversial genetic significance gaps have not been considered for statistical analysis of the data (WHO, 1985). A tentative assessment of the distribution of gaps and breaks revealed that the long chromosomes and distal regions were more vulnerable to the chemical. Occurrence of chro- matid separations has also been observed in the treated series.

A dose-response study (Fig. 2) revealed a lin- ear increase in the frequency of chromosome aber- rations with the corresponding increase in dose (b = 0.009, r = 0.95, P < 0.05). With the different fixation times, a marginal difference was noted among the different chromosome aberration yields. However, a significant variation in the aberration frequency was noted among the 3 routes of chem- ical administration. The s.c. treatment gave signifi- cantly lower response than the i.p. route. Again the chronic treatment produced significantly lower induction of chromosome aberration than the cor- responding acute treatment. Of course, the possi- bility of the impact of difference in fixation time

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Fig. 2. Linear dose-response plot for fenvalerate-induced mi- cronuclci (z,), chromosome aberrations (o ) and sperm abnormalities (O) in mice; b = slope of the regression line

(Y=a+bx).

(24 vs. 120 h) cannot be ruled out. Nevertheless, most of the treated results significantly differed from the corresponding controls.

Significant increase in micronucleated PCEs over control was noticed with the administration of the higher two doses of the chemic'd, but the lowest dose exhibited no positive effect. Moreover, there is clear evidence for a linear relationship (Fig. 2) between the number of micronucleated PCEs and the different doses of fenvalerate used (b = 0.002, r = 0.99, P < 0.01). Induction of MN in NCEs, however, was insignificant in compari- son to control at all the 3 dose levels tested. Frequency of nucleated cells with MN was signifi- cantly different from the control value only with the highest dose of the chemical (Table 2). Other

TABI.E 2

FENVALERATE-INDUCED MICRONUCLEI AND N U CL EAR ANOMAI.IF~S IN BONE MARROW CELLS OF MICE ~

(3000 cells were scored per animal)

Dose PCEs with MN NCEs with MN PCPLs + NCI-Ls Nucleated Nuclear P C F / N C E (mg/kg) with MN cells with MN anomalies

2 x 1 0 0 0.404-0.04 0.204-0.04 0.30 z 0.04 0.204-0.07 0.10-_.0.02 0.94+_0.06 2 × 150 0.50 4- 0.09 b 0.30 + 0.04 0.40 _+ 0.04 0.20 4- 0.09 0.00 0.89 + 0.10 2X200 0.63+0.07 b 0.33+0.07 0.48_+0.02 0.504-0.04 b 0.10+0.02 0.784-0.18

Control 0.20 4- 0.09 0.10 + 0.00 0.15 + 0.09 0.10 -- 0.04 0.00 1.09 4- 0.13

Values are mean % 4-SE for 3 mice/group. h Significantly different from control at 5% level ( Z >~ 1.96).

TABLE 3

INDUCTION OF SPERM ABNORMALITY IN MICE BY FENVALERATE

Dose Number of sperm Number of Percentage (mg/kg) studied/animals abnormal of aberrant

sperm sperm ___ SE ~

100 (5 x 20) 1500/3 50 3.33__+0.98 b.c.~ 150 (5 × 30) 1500/3 88 5.86 _+ 0.54 b.d 200 (5X40) 1500/3 95 6.33_+ 1.18 bx

Control 3000/6 68 2.26 _.- 0.92

Values are mean % =SE for the number of animals men- tioned in each group.

h Significantly different from control at 5% level (Z >i 1.96). c.d Groups with the same letters are significantly different

from each other at 5% level (ANOVA).

nuclear anomalies induced by fenvalerate were trinucleated cells and chromatin bridges at anaphase. The P C E / N C E ratio did not reveal any significant effect on the turnover of PCEs and NCEs.

The results of the sperm head morphology analysis (Table 3) indicate significant increase over control with "all the 3 doses of the chemical. Fur- ther analysis also revealed significant differences in the yield of abnormal sperm between the dose levels 100 vs. 150 and 100 vs. 200 mg/kg. How- ever, there is no linearity (Fig. 2) between the dose of fenvalerate used and the frequency (%) of abnormal sperm (b = 0.021, r = 0.94). Sperms with abnormal head morphology were readily recog- nisable as triangular, rhomboid, hookless, banana shaped, dwarf and giant.

Comparison of the 3 assay results revealed the lowest effect of fenvalerate in the micronucleus test and the highest effect in the sperm abnormal- ity test (Fig. 2).

Discussion

Fenvalerate has been reported to be non-muta- genie in (a) S. typhimurium TAI00 or TA98 in the presence or absence of rat liver activation system using the plate incorporation and fluctuation tests, and (b) in V79 Chinese hamster cells in the pres- ence or absence of hepatocytes (Pluijmen et al., 1984). The present study, however, revealed a

153

variety of cytogenetic effects of fenvalerate in mice in vivo.

FenvMerate has been reported to be rapidly absorbed, distributed to tissues, metabolised and excreted from treated mice (see Mumtaz and Menzer, 1986). The half-life of fenvalerate follow- ing treatment of mice at 30 m g / k g has been noted to be 12-14 h (Mumtaz and Menzer, 1986). This may account for the small and insignificant in- crease in the frequency of chromosome aberra- tions with dose and for the occurrence of maxi- mum aberrations after 24 h of chemicM treatment. Slow rate of chemical absorption from dcrmal tissue might be the reason for the insignificant increase in chromosomal aberrations over control in the s.c. route of chemical administration. The higher incidence of chromatid separations in most of the test cases over corresponding control values suggests that fenvalerate has the ability to induce malsegregation. Moreover, the occurrence of chro- matid separations may be an important observa- tion as it is related to aneuploidy.

The frequency of micronucleated cells was greater with higher doses of fenvalerate. Micro- nuclei are the results of acentric chromosome frag- ments as well as the failure of normal spindle function (Schmid, 1982). Induction of MN in bone marrow cells of mice by synthetic pyrethroids such as cypermethrin (Amer and Aboul Ela, 1985) and deltamethrin (unpublished data), and in plants by deltamethrin (Chouhan et M., 1986) is on record. The induction of chromosome- and chromatid-type deletions, exchanges and MN in the bone marrow cells of mice indicates clastogenic as well as spin- dle poisoning actions of fenvalerate. The P C E / N C E ratio, however, illustrated a lack of cytotoxic effect of the chemical.

As several kinds of mutations can lead to abnormal sperm morphology, this test is con- sidered more sensitive in detecting germ cell mutagens than other germinal mutagenicity assays (Wyrobek et al., 1983). Sperm morphology assay is also said to provide a quantitative method for locating genetic damage in male germline cells (Lock and Soares, 1980). In the present investiga- tion, the higher incidence of sperm abnormality induced by fenvalerate may be a measure of the genetic damage caused at the spermatogonial stage of the mouse germ cells.

154

In summary, the positive results obtained in the present study warrant further investigation in- volving other test systems to evaluate the gent- toxic potency of the chemical.

Acknowledgements

We thank Prof. P. Mohanty-Hejmadi, Head, P.G. Department of Zoology for providing labora- tory facilities and the Education Department, Government of Orissa for granting study leave to one of us (PCP).

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