ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 24, 13- 16 ( 1992)

Investigations of the Acute Toxic, Cytogenetic, and Embryotoxic

Activity of Buminafos

M. NEH~z,*,’ G. W. FISCHER,? H. SCHEUFLER,#

R. SCHMIDT,+ T. SIPOS,* AND I. DBsI*

*Department of Hygiene and Epidemiology, Albert Szent-Gytirgyi University Medical School, D6m tk IO.
H-6720 Szeged, Hungary; iInstitute of Chemical Toxicology, Leipzig9 Germany; and $Department of
Medicine, Institute of Biology, Martin Luther University Halle- Wittenberg, Hal/e, Germany

Received June 17, 1991

The organophosphorus herbicide buminafos (O,Odibutyl-( l-butylaminocyclohexyl)-phospho-

nate) was tested for its acute toxic, cytogenetic, and embryotoxic activity on different strains of
mice. The oral LD% value for male NMRl mice was determined to be 3500 mg/kg. Single oral
doses of 175, 1000, and 2000 mgjkg did not cause any sign&ant enhancement in the percentage
of chromosome aberrations in bone marrow cells of male NMRI mice. After oral administration
of 500 and 1000 mg/kg buminafos to pregnant Halle:DBA and Halle:AB mice at Days 6-l 5 of
gestation no embryotoxic effects were observed. The cytogenetic inactivity of buminafos in the
bone marrow chromosome assay corresponds to negative findings in other mutagenicity tests.
0 1992 Academic Press, Inc.

INTRODUCTION

Although organophosphorus compounds are used in agriculture mainly as insec-

ticides, there are also some representatives of this class with herbicidal activity. One
of these organophosphorus herbicides is the 0,Odibutyl ester of 1-butylaminocy-
clohexyl phosphonic acid (formula I), buminafos, which is available as a 40% emul-
sifiable concentrate.2

Buminafos (I) is applied for the control of annual dicotyledonous and monocoty-
ledonous weeds emerging from seed in agricultural, horticultural, and foresty crops
(e.g., onions, cucumbers, sugar beets, medicinal, and spice plants), for pinching out
suckers and pruning of hops, and for killing onion tops. Furthermore, it lends itself
to the desiccation of legumes and seed plants of special crops as well as for potato
haulm destruction (Kramer et al., 1983).
The herbicide I is distinguished by low toxicity to man and animals. Its examination
for genotoxic effects gave negative findings in the Ames test (Barariski, 1987), in tests
on Drosophila melanogaster (Miiller and Friede, 1986, 1989) and in the chromosome

’ To whom requests for reprints should be addressed.

* Manufactured by Chemie AG Bitterfeld-Wolfen under the trade name Trakephon.

0147-6513192 $5.00 13
Copyright 0 1992 by Academic Press, Inc.
All rights of reproduction in any form reserved.
14 NEHfiZ ET AL.

assay on mice bone marrow cells (Landmann et al., 1986), whereas the SCE assay on
mice showed a dose-dependent low genotoxic activity (Baranski and Bajerska, 1988).
The aim of the present study was to ensure the cytogenetic inactivity of buminafos
toward mice bone marrow cells and furthermore to test it for possible embryotoxic
effects. To select suitable dosages for the cytogenetic and embryotoxicity studies, the
acute oral toxicity (LD& of buminafos on mice was determined.

MATERIAL AND METHODS

Buminafos (I) was obtained from Chemie AG, Bitterfeld-Wolfen, and freshly destilled
in vucuo before use (boiling point, 78-8O”C/O.35 mm). The determination of the acute
oral LDsO was performed with male NMRI mice. The observation of the animals after
treatment lasted 14 days. The LDsO value was calculated by probit analysis.
For the bone marrow chromosome analysis 10 male NMRI mice weighing ap-
proximately 30 g were used in each experimental group. The animals were given single
oral doses of 175 and 1000 mg/kg buminafos in pharmaceutically pure sunflower oil
(oleum helianthi) and 1000 and 2000 mg/kg buminafos without oleum helianthi,
respectively; parallel to these experiments the solvent (0.1 ml oleum helianthi per
mouse), the positive control ( 100 mg/kg cyclophosphamide), and the untreated control
groups were studied. The bone marrow preparation was carried out 24 and 48 hr after
treatment. Following band technique staining, 20 mitoses per mouse were evaluated
(Datta et al., 1970; Wurster, 1972). Significance calculations were made by the Fisher
probe (Delaunois, 1973).
Embryotoxic effects were studied following oral administration of 500 and 1000
mg/kg, respectively, buminafos in olive oil daily to pregnant Halle:DBA and Halle:
AB mice at Days 6-l 5 of gestation. On Day 18 of gestation the mice were sacrificed
and the reproductive status was determined (number of corpora lutea and dead and
live fetuses). The data were analyzed statistically using the X2 test.

RESULTS AND DISCUSSION

The acute oral LDsO value of buminafos for male NMRI mice was determined to
be 3500 mg/kg (95% CL = 3007-4095 mg/kg). Mice are obviously more sensitive to
buminafos than rats and hamsters, for which the oral LD50 values were determined
to be 7000 and 10000 mg/kg, respectively (Kramer et al., 1983).
The results of the cytogenetic investigation of buminafos on bone marrow cells of
NMRI mice are presented in Table 1. No significant increase in the number of cells
showing alterations as well as in the frequency of numerical and structural chromosome
aberrations could be observed, neither 24 nor 48 hr after treatment with the herbicide.
Exchange, isobreak, dicentric, and ring chromosomes, which contribute to the highly
significant enhancement in the percentage of chromosome aberrations after treatment
with cyclophosphamide (positive control), were not found among the structural al-
terations. The cytogenetic inactivity of buminafos in the bone marrow chromosome
assay on mice in the dose range of 175-2000 mg/kg corresponds to negative results
obtained in the same assay on mice with doses of 500 and 2500 mg/kg, respectively
(Landmann et al., 1986), as well as to negative findings in other mutagenicity tests
(Baranski, 1987; Miiller and Friede, 1986, 1989).
The results of the embryotoxicity test with pregnant mice of strains Halle:DBA and
Halle:AB are given in Table 2. In neither strain did buminafos have a marked influence
 TABLE 1
CHROMOSOMEABERRATIONSINBONEMARROW CELLS OFNMRI MICE 24 ANDRE~~AF~ERORALADMINISTRATIONOFBUMINAFOS

No. chromosomeaberrations
No. cells
Dose No. cells with Acentric
Experimentalgroup (mg/kg) examined aberrations Numerical Gap Break Isogap fragment Deletion Translocation Other” Total
24 hr after treatment 3
B
I in oleum 175 200 20 18 2- L - - 22 z
helianthi 1000 200 20 15 31 2- I -
- 20
I without oleum 1000 200 21 17 48 - - 21 g
v)
helianthi 2ooo 200 25 3- I 2- 1
- -
Cyclophosphamide 100 200 83*** :;** 36*** 5yc** s** 39*** 1 19*** $*** $
Controlb 200 20 15 4-l - i - - 20
48 hr after treatment i
I in oleum 175 200 21 17 3 -- I - 21 2
helianthi 1000 200 16 12 2 -- 2- z - - 16
I without oleum 1000 200 23 19 4-l - - - - 24 %
helianthi 2000 200 22 18 1 - 1 - - 24
Cyclophosphamide 100 200 67*** 44** A* 20*** 1 16*** 3 2 2- 101***
Control b 200 4 ij
15 -1 1 - - - 21
Untreatedcontrol 200 ;: 17 4 -- 1 1 - - 23 =i
4
y Exchange, isobreak, dicentric, and/or ring chromosome.
b Control animals received 0.1 ml oleum helianthi.
* P < 0.05.
** P < 0.01.
*** P < 0.001.

8, 88 ,, ,,,,, ,,, ,/, ,,, ,,,, ,,,,,,,, ,, ,,,

16 NEH& ET AL.

TABLE 2
EMBRYOTOXIC EFFECTS OF BUMINAFOS AFTER DAILY ADMINISTRATION OF 500
AND 1000 mg/kg, RESPECTIVELY, AT DAYS 6- 15 OF GESTATION
TO PREGNANT HALLE: DBA AND HALLE: AB MICE

Total
implantation
Experimental Preimplantation Postimplantation losses
group (strain, Pregnant corpora Total Living k&S 1OS.W (?6corpora
dose) females lutea implants implants (96corpora lutea) (5%total implants) lutea)

Halle: DBA
5OO&kg 24 220 186 153 34(15.5) 33 (17.7) 67 (30.5)
loo0 wdk 16 156 133 121 23(14.7) 12 (9.0) 35 (22.4)
Control” 30 281 252 215 29(10.3) 37(14.7) 66 (23.5)
Uotreated
control 24 235 206 183 29 (12.3) 23(11.2) 52(22.1)
Halle: AB
5OOwVk 26 300 261 223 39(13.0) 38(14.6) 77(25.7)
1OOClmgkg 21 239 218 179 21 (8.8) 39(17.9) aO(25.1)
Control” 25 275 252 221 23(8.4) 31 (12.3) 54 (19.6)
Untreated
control 28 325 300 239 25 (7.7) 61 (20.3) 86 (26.5)

0 Control animals received 0.3 ml olive oil.

on the total implantation losses, neither at daily doses of 500 mg/kg, nor at doses of
1000 mg/kg.

CONCLUSION
Under the experimental conditions described the organophosphorus herbicide
buminafos failed to produce any cytogenetic and embryotoxic effects on mice.

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