L.I. Medved's Research Center of Preventive Toxicology, Food and Chemical Safety, Ministry of Health of Ukraine, Kyiv, Ukraine

Abstract. The offspring’s Wistar Hannover rats behavior during in pre- and postnatal period threated by different doses (0; 17,5; 35; 70 mg/kg bw) of сypemethrin were investigated. Obtained data presents the differences in behavior reactions of males andfemales pups. The male pups were more sensitive to the test-substance. Treatment by сypemethrin at the dose of 70 mg/kg bw in the pre- and postnatal period influenced on the behavioral responses of male rats in the "open field", which is manifested by an increase of latency to the first step and decrease of horizontal activity during all periods of the study. Treatment by сypermethrin in low doses in the pre- and postnatal period didn’t induce any changes in the behavior of male and female pups.
Key words: synthetic pyrethroids, сypermethrin, prenatal period, postnatal period, neurobehavior reactions.

The widespread use of insecticides is an integral part of agricultural production in the modern world. Test-substances of insecticide are represented by different classes of chemical compounds (organochlorine and organophosphorous, carbamates, neonicothyneids, synthetic pyrethroids (SP)). A special place among them is occupied by SP [1,2]. Pyrethroid insecticides are the fourth generation of pesticides with more pronounced insecticidal effectiveness. This is due to their widespread uses in protection from eradicating pest, not only in agriculture, but also in household.  The main target organ is the nervous system (NC) in period of influence of SP [3]. Neurotoxic effects are observed not only in insects, but also noted in  laboratory animals during toxicological studies, and  in acute human poisoning [3,4]. Neurotoxic effect in experiments on rats of different ages is manifested by changes in behavioral reactions as a result of a possible disruption of the animals nerve system functioning [5,6]. Scientific articles show the results of the study of behavioral reactions in experiments on adult rats of both sexes after the SP influence [7]. The authors were determined that SP exhibited dissimilar sexual sensitivity in experiments: males were more sensitive. The influence of SP independently of the structure induces to decrease motor activity in males. Threshold values for motor activity were lower doses that induce toxic effects [5,6].

Cypermethrin is the most common active ingredient among insecticidal pesticides used for agricultural and household purposes [3]. It is well studied in toxicological area, including well studied its neurotoxicity in adult animals [8, 9]. Recently in the scientific literature appeared data on the manifestation of neurotoxicity in young animals [10,11,12], which exposed with cypermethrin in the lactation period. Research presents the effect of cypermethrin in the neonatal period that provokes an increase in the time that male rats spend in the center of the "open field" [13].

In addition to neurotoxicity studies of cypermethrin in young laboratory animals, epidemiological data of neurotoxic effects in children born from mothers who were exposed by insecticide indoor or in agricultural practice  during pregnancy and lactation period  [14,15,16].

However the effects of cypermethrin on animals of different ages were presented, we can not ignore its effects on the development offsprings. The purpose of the study was to assess the influence of cypermethrin in the prenatal and postnatal period on behavioral reactions by using the "open field" method, proposed by Holl [17].

The "open field" test is the most common in toxicology in the study of the specificity of  rats nerve activity, namely, behavioral reactions. The method is based on assessment of animals behavior in a novel surrounding. The test allows to evaluating a manifestation of separate behavioral elements, motor and exploratory activity, emotionality of animals [6, 18].

Materials and methods. Wistar Hannover female rats (18 female weighing 200-220 g) were obtained from the SPF vivarium of L.I. Medveds Reasearch Center of Preventive Toxicilogy, Food and Chemical Safety, Ministry of Health Ukraine and randomized according doses of cypermethrin: group 1 - control (distilled water with an emulsifier OP-10 in equivalent quantities), group 2 – 17,5 mg/kg bw, group 3 - 35 mg/kg bw, group 4 - 70 mg/kg bw. Animals were kept under standard vivarium conditions, fееd and water was offered ad libitum.

Cypermethrin (according to the Certificate of Quality - Cypermethrin 97%, Technical) was administered orally with a metal probe from  gestation day 6 (GD) to  lactation day 21 (LD) in the morning at the same time.

All pregnant females were examined daily for possible clinical sings of intoxication. Pups from the moment of birth and to LD 21 were examined for detection of neurological disorders, such as: behavioral abnormalities, сhanges in gait, disorder of muscle tone [19].

Sixteen selected pups of each group (8 males and 8 females) at  postnatal days 13 and 21 (PND) were subjected to assess the functional state of the central nervous system (CNS) using "open field" test [20]. "Open field" device is a uniformly illuminated arena of 44 cm x 44 cm made of opaque black plastic, delineated into 36 squares and surrounded by walls 30 cm high. Pups were placed into the center of an arena and within 3 minutes their behavior was evaluated. Behavioral parameters were noted: latency to the first step (the time taken to leave the starting square), number of crossed squares, number of rearings, duration and number of grooming.

Testing of animals was carried out in the morning at the same time.

Study was carried out in accordance with requirements of OECD 426 (Guideline for Testing of Chemicals. Developmental Neurotoxicity Study) [21] and approved by the Medical and Biological Research Ethics Commission of Research Center.

The statistical processing results were performed using Student's t-test (the critical level of significance of statistical data was taken equal to p <0,05). For the corresponding calculations, the standard software package of Microsoft Excel 2010 statistical analysis was used. Data are represented as means (M) and its standard error (m).

3. Results and conclusions.  No visible signs of intoxication and mortality of treated and control females in period of gestation and lactation in all doses of cypermethrin were observed. No mortality of newborns was observed. No treatment-related visible signs intoxication, gross neurologic and behavioral abnormalities in pups were observed.

Study conducted on PND 13 showed treatment-related different changes in offsprings behavioral reactions. No treatment-related changes in behavioral reactions females were observed. Changes in motor activity of male pups were noted in the high-dose group. In this group, significant increase of latency to the first step and significant decrease of crossed squares were observed. It is known, latency to the first step is a parameter that characterizes animals ability to adaptation to the new environment [9,22]. In our study, significant increase of latency to the first step in 63% males in the high-dose group was noted, while in the control group, this parameter was recorded in 25% of pups only. There was a significant decrease of crossed squares number in 31% of males in the same group compared with the number of crossed squares to the control group. Reducing of pups motor activity in our study was a consequence of increased stress of the offsprings. No changes in behavioral parameters of males in doses 17.5 and 35 mg/kg bw were observed (see table. 1)

Table 1. Data from the investigation of pups behavioral reactions on PND 13.

The similar data of sexual sensitivity and dose dependence was observed during the study on PND 21. No significant changes of behavioral landmarks females (latency to the first, number of crossed squares, rearings, grooming and self grooming duration) in any of the studied doses were observed. While, in the high-dose group of male pups there were some significant changes in parameters - latency to the first step and number of crossed squares. The significant increase of latency to the first step was noted more frequently compared to PND 13 and compared to control group (PND 21 - 75% pups, PND 13 - 63%, control - 25%). Amount of square crosses remained at the same level as on PND 13. Other behavioral landmarks (rearings, grooming and self grooming duration) remained unchanged. Treatment of males by cypermethrin in doses 17.5 and 35 mg/kg bw did not induce significant changes of their behavioral reactions (see tabl. 2)

Table 2. Data from the investigation of pups behavioral reactions on PND 21.

Distortion of the motor component of behavior was noted mainly in males in comparison to females, which corresponds to literature data. Significant increase in number of males with prolonged latency to the first step and a significant decrease in number of crossed squares on  PND 21 compared with PND 13 were presented in Tab. 2. This data may indicate a further increase in suppression of the CNS functional activity at dose 70 mg/kg bw on  PND 21, as one of the signs of prenatal stress syndrome  of the fetus development central nervous system, which can be realized in changing of behavioral reactions during postnatal period. Increase in suppression of CNS functional activity of pups in high-dose was related to offsprings cypermethrin exposure during prenatal development and during lactation period.

Based on the results of behavioral responses study, the following conclusions can be drawn up for the effects of cypermethrin different doses:

1. The pups sexual sensitivity for cypermethrin influence was established. Male rats were more susceptible to cypermethrin exposure.

2. Administration of cypermethrin in dose 70 mg/kg during the pre- and postnatal period affected the neurobehavioral reactions (an increase of latency to the first step and decrease the number of crossed squares) of male pups using «open field» test. Data of motor activity indicated a decrease of adaptation of the animals to the new environment. Cypermethrin did not affect of pups emotionality.

3. Received data may indicate an increase of suppression of central nervous system functional activity of male pups in the high-dose group during prenatal development and lactation period.

4. Administration of cypermethrin from GD 6 to  LD 21 in doses 17.5 and 35 mg/kg bw did not induce abnormalities in behavioral reactions of male and female rats.

REFERENCES (MLA)

1. Saillenfait, Anne-Marie, Dieynaba Ndiaye, and Jean-Philippe Sabaté. "Pyrethroids: exposure and health effects–an update." International journal of hygiene and environmental health 218.3 (2015): 281-292.

2. Thatheyus, A. J., and A. Deborah Gnana Selvam. "Synthetic pyrethroids: toxicity and biodegradation." Applied Ecology and Environmental Sciences 1.3 (2013): 33-36.

3. Ray, David E., and Jeffrey R. Fry. "A reassessment of the neurotoxicity of pyrethroid insecticides." Pharmacology & therapeutics 111.1 (2006): 174-193.

4. Harchenko О. А., Balan G. М., Bubalo N. М. "Synthetic pyrethroids: mechanism of action, acute poisoning and long-term consequences." Food Problems 1 (2013): 29-39.

5. Shafer, Timothy J., Douglas A. Meyer, and Kevin M. Crofton. "Developmental neurotoxicity of pyrethroid insecticides: critical review and future research needs." Environmental health perspectives 113.2 (2005): 123.

6. Umryukhin, P. E., and O. S. Grigorchuk. "Behavior of Rats in an Open Field Test as a Prognostic Indicator of Corticosterone Levels Before and After Stress." Neuroscience and Behavioral Physiology (2017): 1-3.

7. Wolansky, M. J., and J. A. Harrill. "Neurobehavioral toxicology of pyrethroid insecticides in adult animals: a critical review." Neurotoxicology and teratology 30.2 (2008): 55-78.

8. Kanbur, Murat, et al. "The toxic effect of cypermethrin, amitraz and combinations of cypermethrin-amitraz in rats." Environmental Science and Pollution Research23.6 (2016): 5232-5242.

9. Gould, Todd D., David T. Dao, and Colleen E. Kovacsics. "The open field test." Mood and anxiety related phenotypes in mice: Characterization using behavioral tests (2009): 1-20.

10. Lee, Iwa, et al. "Developmental neurotoxic effects of two pesticides: Behavior and neuroprotein studies on endosulfan and cypermethrin." Toxicology 335 (2015): 1-10.

11. Fluegge, Kyle R., Marcia Nishioka, and J. R. Wilkins III. "Effects of simultaneous prenatal exposures to organophosphate and synthetic pyrethroid insecticides on infant neurodevelopment at three months of age." Journal of environmental toxiology and public health 1 (2016): 60.

12. Godinho, Antonio, Fabio Anselmo, and Daniel França Horta. "Perinatal exposure to type I and type II pyrethroids provoke persistent behavioral effects during rat offspring development." Medical Research Archives5.1 (2017).

13. Nasuti, Cinzia, et al. "Dopaminergic system modulation, behavioral changes, and oxidative stress after neonatal administration of pyrethroids." Toxicology 229.3 (2007): 194-205.

14. Saillenfait, Anne-Marie, Dieynaba Ndiaye, and Jean-Philippe Sabaté. "Pyrethroids: exposure and health effects–an update." International journal of hygiene and environmental health 218.3 (2015): 281-292.

15. Raymer, J. H., et al. "Pesticide exposures to migrant farmworkers in Eastern NC: detection of metabolites in farmworker urine associated with housing violations and camp characteristics." American journal of industrial medicine 57.3 (2014): 323-337.

16. Ding, Guodong, et al. "Prenatal exposure to pyrethroid insecticides and birth outcomes in Rural Northern China." Journal of Exposure Science and Environmental Epidemiology 25.3 (2015): 264-270.

17. Hall C. S. "Emotional behavior in the rat. III. The relationship between emotionality and ambulatory activity." Journal of Comparative Psychology 22.3 (1936): 345.

18. Stanford, S. Clare. "The open field test: reinventing the wheel." Journal of psychopharmacology 21.2 (2007): 134-136.

19. Moser, Virginia C. "The functional observational battery in adult and developing rats." Neurotoxicology 21.6 (2000): 989-996.

20. Millan, Mark J. "The neurobiology and control of anxious states." Progress in neurobiology 70.2 (2003): 83-244.

21. OECD Guideline for Testing of Chemicals; Guideline 426: Developmental Neurotoxicity Study. – 2007.

22. Permyakov А.А, Eliseeva E.V., Yuditskiy A.D. "Behavioral reactions of experimental animals with different prognostic resistance to stress in the "open field" test." Bulletin of Udmurt University. Series "Biology. Earth Sciences. 3 (2013): 83-90.

REFERENCES (APA)

1. Saillenfait, A. M., Ndiaye, D., & Sabaté, J. P. (2015). Pyrethroids: exposure and health effects–an update. International journal of hygiene and environmental health, 218(3), 281-292.

2. Thatheyus, A. J., & Selvam, A. D. G. (2013). Synthetic pyrethroids: toxicity and biodegradation. Applied Ecology and Environmental Sciences, 1(3), 33-36.

3. Ray, D. E., & Fry, J. R. (2006). A reassessment of the neurotoxicity of pyrethroid insecticides. Pharmacology & therapeutics, 111(1), 174-193.

4. Harchenko О. А., Balan G. М., Bubalo N. М. (2013). Synthetic pyrethroids: mechanism of action, acute poisoning and long-term consequences. Food Problems 1, 29-39.

5. Shafer, T. J., Meyer, D. A., & Crofton, K. M. (2005). Developmental neurotoxicity of pyrethroid insecticides: critical review and future research needs. Environmental health perspectives, 113(2), 123.

6. Umryukhin, P. E., & Grigorchuk, O. S. (2017). Behavior of Rats in an Open Field Test as a Prognostic Indicator of Corticosterone Levels Before and After Stress. Neuroscience and Behavioral Physiology, 1-3.

7. Wolansky, M. J., & Harrill, J. A. (2008). Neurobehavioral toxicology of pyrethroid insecticides in adult animals: a critical review. Neurotoxicology and teratology, 30(2), 55-78.

8. Kanbur, M., Siliğ, Y., Eraslan, G., Karabacak, M., Sarıca, Z. S., & Şahin, S. (2016). The toxic effect of cypermethrin, amitraz and combinations of cypermethrin-amitraz in rats. Environmental Science and Pollution Research, 23(6), 5232-5242.

9. Gould, T. D., Dao, D. T., & Kovacsics, C. E. (2009). The open field test. Mood and anxiety related phenotypes in mice: Characterization using behavioral tests, 1-20.

10. Lee, I., Eriksson, P., Fredriksson, A., Buratovic, S., & Viberg, H. (2015). Developmental neurotoxic effects of two pesticides: Behavior and neuroprotein studies on endosulfan and cypermethrin. Toxicology, 335, 1-10.

11. Fluegge, K. R., Nishioka, M., & Wilkins III, J. R. (2016). Effects of simultaneous prenatal exposures to organophosphate and synthetic pyrethroid insecticides on infant neurodevelopment at three months of age. Journal of environmental toxiology and public health, 1, 60.

12. Godinho, A., Anselmo, F., & Horta, D. F. (2017). Perinatal exposure to type I and type II pyrethroids provoke persistent behavioral effects during rat offspring development. Medical Research Archives, 5(1).

13. Nasuti, C., Gabbianelli, R., Falcioni, M. L., Di Stefano, A., Sozio, P., & Cantalamessa, F. (2007). Dopaminergic system modulation, behavioral changes, and oxidative stress after neonatal administration of pyrethroids. Toxicology, 229(3), 194-205.

14. Saillenfait, A. M., Ndiaye, D., & Sabaté, J. P. (2015). Pyrethroids: exposure and health effects–an update. International journal of hygiene and environmental health, 218(3), 281-292.

15. Raymer, J. H., Studabaker, W. B., Gardner, M., Talton, J., Quandt, S. A., Chen, H., ... & Arcury, T. A. (2014). Pesticide exposures to migrant farmworkers in Eastern NC: detection of metabolites in farmworker urine associated with housing violations and camp characteristics. American journal of industrial medicine, 57(3), 323-337.

16. Ding, G., Cui, C., Chen, L., Gao, Y., Zhou, Y., Shi, R., & Tian, Y. (2015). Prenatal exposure to pyrethroid insecticides and birth outcomes in Rural Northern China. Journal of Exposure Science and Environmental Epidemiology, 25(3), 264-270.

17. Hall C. S. (1936). Emotional behavior in the rat. III. The relationship between emotionality and ambulatory activity. Journal of Comparative Psychology, 22(3), 345.

18. Stanford, S. C. (2007). The open field test: reinventing the wheel. Journal of psychopharmacology, 21(2), 134-136.

19. Moser, V. C. (2000). The functional observational battery in adult and developing rats. Neurotoxicology, 21(6), 989-996.

20. Millan, M. J. (2003). The neurobiology and control of anxious states. Progress in neurobiology, 70(2), 83-244.

21. OECD Guideline for Testing of Chemicals; Guideline 426: Developmental Neurotoxicity Study. – 2007.

22. Permyakov А.А, Eliseeva E.V., Yuditskiy A.D. (2013). Behavioral reactions of experimental animals with different prognostic resistance to stress in the "open field" test. Bulletin of Udmurt University. Series "Biology. Earth Sciences, 3 (2013): 83-90.

Надійшла до редакції 24.11.17