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Toxicoba Letters, 69 (1993) 155161 d 1993 Elsevier Se&rice Publishers B.V. All rights reserved U378~27~93~ 6.00 155 TDXLET 02937 Behavioural effects of piperonyl butoxide in male mice Toyohito Tanaka Department of Tox~eology* Tokyo Metr~polifan ~ese~~c~ ~or~tory of Public Health, Tokyo (Japunj (Received 10 December 1992) (Accepted 3 February 1993) Key wor*dr:Piperonyl butoxide; Behavioural toxicity; Maze learning; Exploratory behaviour; Male mice SUMMARY Piperonyt .butoxide was ad~~~~~ to male mice from 5 to 12 weeks of age in the diet at levels of 0 (control), 0.15,0.30, and 0.6095, and some behavioural parameters were measured. The animals performed three trials in multiple water T-maze at 10 weeks of age, and the number of errors was signhicantly decreased in treatment groups on the 3rd trial, while there was no biologically significant effect of piperonyl butoxi~.on maze learning. The motor activity of the exploratory behaviour was measured by ANIMATE AT-420 at 8 and 11 weeks of age. At 8 weeks of age, some parameters were increased in the 0.30% group, while there was no consistent compound- or dose-related effect. At 11 weeks of age, some parameters were different in treatment groups, and there were biologically consistent significant effects; i.e., number of movements, movement time, total distance, average speed, and number of turnings increased. From these results, piperonyl butoxide showed adverse effects on the motor activity of the exploratory behaviour in male mice. INTRQDUCTION Pi~ronyl butoxide (a?-[2-f2-butoxyethoxy)ethoxy]~,5-methy~e~~ioxy-2-propyl- toluene) is a pesticide synergist used to enhance the effect of pyrethrins, rotenone, tetramethrin, and other insecticides in oil solutions; aerosols, dusts, wettable powders and slurries, and is registered for use on a variety of fruit, vegetable, forage, and grain crops, as well as on livestock and agricultural premises [1,2]. Piperonyl butoxide is used as a food additive in Japan, and its maximum allowable use level is 0.024 g/kg Correspondence to: Toyohito Tanaka, Department of Toxicology, Tokyo Metropolitan Research Labora- tory of Public Health, 24-1, Hyakunincho 3 chome, Shinjnku-ku, Tokyo 169, Japan.
Transcript

Toxicoba Letters, 69 (1993) 155161 d 1993 Elsevier Se&rice Publishers B.V. All rights reserved U378~27~93~ 6.00

155

TDXLET 02937

Behavioural effects of piperonyl butoxide in male mice

Toyohito Tanaka

Department of Tox~eology* Tokyo Metr~polifan ~ese~~c~ ~or~tory of Public Health, Tokyo (Japunj

(Received 10 December 1992) (Accepted 3 February 1993)

Key wor*dr: Piperonyl butoxide; Behavioural toxicity; Maze learning; Exploratory behaviour; Male mice

SUMMARY

Piperonyt .butoxide was ad~~~~~ to male mice from 5 to 12 weeks of age in the diet at levels of 0 (control), 0.15,0.30, and 0.6095, and some behavioural parameters were measured. The animals performed three trials in multiple water T-maze at 10 weeks of age, and the number of errors was signhicantly decreased in treatment groups on the 3rd trial, while there was no biologically significant effect of piperonyl butoxi~.on maze learning. The motor activity of the exploratory behaviour was measured by ANIMATE AT-420 at 8 and 11 weeks of age. At 8 weeks of age, some parameters were increased in the 0.30% group, while there was no consistent compound- or dose-related effect. At 11 weeks of age, some parameters were different in treatment groups, and there were biologically consistent significant effects; i.e., number of movements, movement time, total distance, average speed, and number of turnings increased. From these results, piperonyl butoxide showed adverse effects on the motor activity of the exploratory behaviour in male mice.

INTRQDUCTION

Pi~ronyl butoxide (a?-[2-f2-butoxyethoxy)ethoxy]~,5-methy~e~~ioxy-2-propyl- toluene) is a pesticide synergist used to enhance the effect of pyrethrins, rotenone, tetramethrin, and other insecticides in oil solutions; aerosols, dusts, wettable powders and slurries, and is registered for use on a variety of fruit, vegetable, forage, and grain crops, as well as on livestock and agricultural premises [1,2]. Piperonyl butoxide is used as a food additive in Japan, and its maximum allowable use level is 0.024 g/kg

Correspondence to: Toyohito Tanaka, Department of Toxicology, Tokyo Metropolitan Research Labora- tory of Public Health, 24-1, Hyakunincho 3 chome, Shinjnku-ku, Tokyo 169, Japan.

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(24 ppm) in raw cereals, while the acceptable daily intake (ADI) is 0.03 mg/kg body wt.

Several carcinogenicity studies of the chemical were reported. However, in those studies, there was no consistent significant effect of piperonyl butoxide in the rat [ 1,3]. Several teratogenic studies of piperonyl butoxide were reported, and there was no consistent significant effect observed in those studies [2,4]. However, piperonyl butox- ide showed adverse effects on reproductive and neurobehavioural parameters in mice in a two generation toxicity study [S]. In that study, open field activity of F0 genera- tion male mice was reduced in the treatment groups. Piperonyl butoxide was also reported to be neurotoxic in male mice [6].

While there is little study of piperonyl butoxide in the behavioural toxicity in any animals, this study was designed to evaluate the behavioural effects of piperonyl butoxide in male mice.

MATERIALS AND METHODS

Materials Piperonyl butoxide was obtained from Tokyo Kasei Co., Ltd., Tokyo, Japan,

~n~rna~~ and maintenance Male mice (Crj:CD-1, 4 weeks of age) were obtained from Charles River Japan

Inc., Kanagawa, Japan. Mice were individually housed in polycarbonate solid- floored cages with wood flakes in an air-conditioned room maintained 24 rt l°C, with a humidity of 55 ? 5%. They were given control or experimental diets and water ad libitum.

Experimental design Piperonyl butoxide was administered in the diet to 60 male mice (20/group) at

dietary levels of 0.15, 0.30, and 0.60%. The 20 mice in the control group were given the basal diet {Nihon Clea, CE-2) for the corresponding period for time. The animals were 5 weeks of age at start of the study. Individual food intake was measured every week from 5 to 12 weeks of age. The behavioural tests were performed as follows.

Maze learning (at 10 weeks of age). The animals performed three trials in multiple water T-mazes of the Biel’s type adapted for mice [7,8]. Water temperature was main- tained at 18 ? 1°C. The time taken and number of errors were measured from the start to finish for a maximum of 120 s. If the time taken was greater than 120 s, it was recorded as 120 s [8].

Exploratory behaviour (at 8 and 11 weeks of age). The motor activity of the exploratory behaviour was measured by ANIMATE AT-420 (Toyo Sangyo Co., Ltd., Toyama, Japan) for animal movement analyzing systems [9,10]. The system consisted of a doughnut-shaped cage with 36 units of detectors for measuring sponta- neous motor activity. Scanning of each detector unit was carried out three-dimension- ally, and then scanning data were fed into a personal computer. These steps were

157

completed within 0.1 s and repeated. The behavioural parameters were recorded at every 1 min for 10 min. The measurement parameters were number of movements, movement time (s), number of horizontal activities, total distance (cm), number of vertical activities, vertical time (s), number of turnings, average distance (cm), average speed (cm/s), and defecation.

~tutistical analysis Average food intake was assessed with Bonferroni multiple comparison test after

Kruskal-Wallis test. The behavioural parameters were assessed with Mann-Whitney U-test.

RESULTS

Food and chemical intake Average food intake of the higher treatment groups was decreased in the first week

(Table I). While there was no adverse effect of food intake in any treatment group from 6 to 11 weeks of age. Therefore, piperonyl butoxide was taken in proportion to dietary levels except in the first week (Table I).

TABLE I

AVERAGE FOOD AND CHEMICAL INTAKES OF MALE MICE ADMINISTERED PIPERONYL BUTOXIDE IN BEHAVIOURAL TOXICITY STUDY

Dose level (%) 0 0.15 0.30 0.60

Food intake (g/kg/day) Weeks of age

5 182.28 2 2.28 6 155.58 f 2.80 7 152.65 & 2.08 8 146.03 + 3.04 9 139.32 f 3.44

10 135.10 f 2.84 11 132.78 + 3.30

Chemical intake (m~k~day) Weeks of age

5 6 7 8 9 -

10 11 -

172.30 i: 4.25 156.56 f 2.19** 139.38 z!z 2.70’* 165.24 zlz 3.89 155.24 & 2.39 157.58 It 1.74 159.84 + 4.29 155.61 f. 2.83 158.94 & 2.58 155.88 + 5.45 148.05 & 2.54 147.44 * 2.82 161.52 ?J 6.67* 149.32 f 3.95 143.62 + 2.78 145.72 5 4.90 143.89 + 2.57 141.66 It 3.35 139.98 + 3.61 135.62 ? 2.27 137.89 + 3.08

258.45 5 6.38 469.68 f 6.57 836.28 + 16.20 247.86 + 5.84 465.72 f 7.17 945.48 + 10.44 239.76 + 6.44 466.83 ?r 8.49 953.64 It 15.48 233.82 2 8.18 444.15 f 7.62 884.64 4 16.92 242.28 + 10.01 447.96 + 11.85 861.72 4 16.68 218.58 z!z 7.35 431.67 z! 7.71 849.96 ? 20.10 209.97 4 5.42 406.86 2 6.81 827.34 It 18.48

Each value represents the mean + SE. Significantly different from controls: *P c 0.05, **P <: 0.01 (food intake only).

158

Maze leurning

There were some significant effects for multiple water T-maze performance in treat- ment groups on the 3rd trial (Table II). The aumber of errors was decreased in all treatment groups as compared to controls, and the time taken was decreased in the 0.30% group. In treatment groups, each parameter was decreased through trials, but it was greater on the 3rd trial than 2nd trial in the control group. Therefore, piperonyl butoxide showed no consistent adverse effect on maze learning.

At 8 weeks of age, some parameters were increased in the 0.30% group, and defeca- tion was increased in all treatment groups (Table III). However, there was no biolog- ically consistent compound- or dose-related effect of piperonyl butoxide for the mo- tor activity of the exploratory behaviour.

At 11 weeks of age, some parameters were signi~cantly different in the treatment groups (Table III). In significantly different parameters, number of movements and movement time were increased in the 0.30% group. The number of horizontal activi- ties was decreased in the 0.60% group. Total distance was significantly increased in all treatment groups all over for 10 min (Fig. l), and these effects were consistent com- pound- or dose-related. Average distance was increased in all treatment groups. Aver- age speed was significantly increased in the 0.30% group. The number of turnings was increased in the higher dosed groups.

DISCUSSION

In this study, there were some signi~cant effects of piperonyl butoxide in the behav- ioural tests. The animals performed maze learning, and the number of errors and time taken were significantly decreased in treatment groups on the 3rd trial. While it

TABLE II

MULTIPLE WATER T-MAZE PERFORMANCE OF MALE MICE ADMINISTERED PIPERONYL BUTOXIDE AT 10 WEEKS OF AGE

Dose level (%) 0 0.15 0.30 0.60

No. of examined 20 19 20 20 Time taken (s)

on 1st day 76.3 k 7.59 69.7 k 7.48 71.7 + 8.04 71.8 f 7.46 2nd day 46.6 + 6.40 36.2 k 5.55 43.4 f 7.26 39.4 k 6.74 3rd day 61.7 k 8.79 34.1 k 6.08** 33.6 -t 6.34* 38.2 I 7.68*

Errors (number) on 1st day 5.9 + 0.65 5.4 * 0.91 6.0 + 0.79 5.0 rt 0.79

2nd day 3.7 + 0.52 3.3 It 0.65 4.5 + 1.00 3.6 rt 0.94 3rd day 5.6 rtr 0.99 2.9 k 0.75 2.4 + 0.53* 2.6 + 0.35

Each value represents the mean + SE. Significantly different from controls: *P < 0.05 **P <: 0.01.

159

seemed that those were caused by the increase of errors on the 3rd trial compared to the 2nd trial in the control group, there was no biologically consistent significant effect of piperonyi butoxide in this change. Therefore, the effects of maze learning in this study were not caused by treatment of piperonyi butoxide.

In the motor activity of the exploratory behaviour, there was no consistent signifi- cant compound- or dose-related effect at 8 weeks of age, while some parameters were increased in the 0.30% group. At 1 I weeks of age, mice of the treatment groups moved long distances rapidly, and there were biologically consistent significant effects of piperonyi butoxide in some parameters. Therefore, it seems that piperonyi butoxide has an influence on the motor activity of the exploratory behaviour at ii weeks of age. Those changes might be caused by the effects of piperonyi butoxide on the central nervous system, because piperonyl butoxide was concentrated in the brain after the oral and intravenous administration in the rat [l 11.

TABLE III

MOTOR ACTIVITY OF EXPLORATORY BEHAVIOUR IN BEHAVIOURAL TOXICITY STUDY OF PIPERONYL BUTOXIDE ADMINISTERED TO MALE MICE

Dose level (56) 0 0.15 0.30 0.60

At 8 weeks of age No. of examined 20 19 20 20 No. of movements 2010.1 5 39.43 2095.1 z!z 44.27 2146.2 + 66.18 2005.0 t 67.31 Movement time (s) 492.0 * 7.65 510.8 + 5.26 514.7 * 8.81* 504.3 ?r 8.53 No. of H. activities 55.6 + 2.82 56.1 + 2.59 52.2+ 3.18 53.4 i 2.93 Total distance (cm) 2146.6 f 109.32 2506.7 + 110.63 267611 + 139.09 2579.4 z!z 159.91 No. of V. activities 138.2 + 4.87 148.7 ?1: 6.03 161.1 + 6.84* 143.4 + 6.64 Vertical times (s) 200.8 f 8.18 221.9 jI 8.62 228.5 f 8.69 208.6 i: 10.49 No. of turnings 47.5 + 2:96 46.9 + 2.69 54.5 f 4.67 51.2+ 4.04 Ave. distance (cm) 46.5 + 4.64 47.1 t 3.77 56.7 + 8.26 54.8 2 7.12 Ave. speed (cmts) 4.69 + 0.177 4.88 f 0.189 5.15 * 0.194 5.05 + 0.245 Defecation 5.4 + 0.60 8.1 z!z Q.W* 7.7 f 0.61* 7.6 It 0.63*

At 11 weeks of age No. of examined No. of movements Movement time (s) No. of H. activities Total distance (cm) No. of V. activities Vertical time (s) No. of turnings Ave. distance (cm) Ave. speed (cm/s) Defecation

20

1727.5 + 74.06 453.6 f 12.40

6&O? 2.16 2063.0 + 157.10

115.6 f 8.83 179.0 f 14.52 36.22 2.11 37.0 + 4.51 4.47 + 0.246

6.1 ? 0.71

19 1909.7 + 63.43 478.4 It 11.52

53.1 rt 2.33 2393.4 z?z 136.31*

127.1 z!z 8.36 203.9 + 14.02 41.4ir 2.31 49.0 f 6.07* 4.96 + 0.191

7.9 + 0.86

20 I97Wf 69.19* 488.9 1 13.30*

54.5 + 2.50 2494.2 + 149.12*

133.7 + 7.92 208.5 f 13.92

50.2 f 2.84** 49.9 2 5.73* 5.03 ? 0.199* 7.4 k 0.65

20 1922.3 z? 104.86 482.2 + is.01

52.0 fi 2.85* 2635.3 rf: 225.84*

135.2 + 10.43 203.8 + 15.69

50.2 i: 3.71** 58.4 + 8.64* 5.29 rt 0.3I7 7.4 rt 8.50

Each value represents the incidence of behaviour per IO min {mean + SE). Si~ificantly different from controls: *PC O.M, **P c 0.01.

MEAN&SE *p<0.05. **p<o.o1

~ CONTROL 0, 15 %

--- 0.30 %

--- 0.60 %

I 1 I , I I 1 I

1 2 3 4 5 6 7 8 9 10

TIME INTERVAL (min)

Fig. 1. Total distance (cm) per 10 min in male mice administered piperonyl butoxide at 11 weeks of age.

Those results were different from the effects of piperonyl butoxide observed in the reproductive and neurobehavioural toxicity study [53. While in that study the motor activity was measured by an open field test, and the sample size was smaller than in this study. Also, the motor activity had only been measured at 8 weeks of age in that study, while in this study the motor activity was measured at 8 and 11 weeks of age. No consistent effect was seen at 8 weeks of age. Moreover, as the methods of the measurement of the motor activity in this study have been different from those in the previous study, it seems that the result of this study may have not been contradictory to the result of the previous study. Piperonyl butoxide has shown the behavioural change of the exploratory beha~oural at only I1 weeks of age in this study. It there-

161

fore seems that the effects of the motor activity of the exploratory behaviour are more reliable in this study.

From the results, the dose levels of piperonyl butoxide in this study produced adverse effects on the motor activity of the exploratory behaviour. However, the presumed dietary intake of piperonyl butoxide in Japan has been reported to be 1.77 pg per person (0.0354&kg), which is less than the AD1 (0.03 mg/kg) of about 1.5 mg per person [12]. It, therefore, seems that the level of the actual dietary intake of piperonyl butoxide has only a limited effect.

REFERENCES

1 Cardy, R.H., Renne, R.A., Warner, J.W. and Cypher, R.L. (1979) Carcinogenesis bioassay of techni- cal-grade piperonyl butoxide in F344 rats. J. Natl. Cancer Inst. 62, 569-578.

2 Kennedy Jr. G.L., Smith, S.H., Kinoshita, F.K. and Carandra, J.C. (1977) Teratogenic evaluation of piperonyl butoxide in the rat. Fd. Cosmet. Toxicol. 15, 337-339.

3 Maekawa, A., Onodera, H., Furuta, K., Tanigawa, H., Ogiu, T. and Hayashi, Y. (1985) Lack of evidence of carcinogenicity of technical-grade piperonyl butoxide in F344 rats: Selective induction of ileocaecal ulcers. Fd. Chem. Toxicol. 23, 6755682.

4 Khera, K.S., Whalen, C., Angers, G. and Trivett, G. (1979) Assessment of the teratogenic potential of piperonyl butoxide, biphenyl, and phosalone in the rat. Toxicol. Appl. Pharmacol. 47, 353-358.

5 Tanaka, T. (1992) Effects of piperonyl butoxide on F, generation mice. Toxicol. Lett. 60, 83-90. 6 Ater, S.B. Swinyard, E.A., Tolman, K.G. and Franklin, M.R. (1984) Anticonvulsant activity and

neurotoxicity of piperonyl butoxide in mice. Epilepsia 25, 551-555. 7 Biel, W.C. (1940) Early age differences in maze performance in the albino rat. J. Gen. Psychol. 56,

439453. 8 Kitatani, T., Akaike, M., Takayama, K. and Kobayashi, T. (1988) Teratological study of cefodizime

sodium in mice - Intravenous administration during period of organogenesis. J. Toxicol. Sci. 13 (Suppl. I) 191-214 (in Japanese).

9 Matsumoto, K., Cai, B., Nakamura, S. and Watanabe, H. (1990) A new system for the measurement and analysis of motor activity in mice. Folia. Pharmacol. Japon. 96, 31-39 (in Japanese).

10 Matsumoto, K., Cai, B., Sasaki, K. and Watanabe, H. (1990) Metamphetamine- and apomorphine- induced changes in spontaneous motor activity using a new system to detect and analyze motor activity in mice. J. Pharmacol. Methods 24, 11 l-l 19.

11 Kimura, R., Deguchi, H. and Murata, T. (1983) Absorption distribution and excretion of piperonyl butoxide in rats. J. Food Hyg. Sot. Jpn. 24, 319-323 (in Japanese).

12 Isshiki, K., Tsumura, S. and Watanabe, T. (1983) Dietary intake of fungicides and piperonyl butoxide. J. Food Hyg. Sot. Jpn. 24, 344349 (in Japanese).


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