State Enterprise “L. I. Medved’s Research Center of Preventive Toxicology, Food and Chemical Safety of the Ministry of Health of Ukraine”, Kyiv, Ukraine

ABSTRACT. Objective. Identification of hazard and assessment of the risk of gonado- and reproductive toxicity of two generic pesticides of technical-grade carbendazim of different purity bin male and female Wistar Han rats.

Materials and Methods. Carbendazim with the purity of 98 % (C1) and 98.1 % (C2) was intragastrically administered on the daily basis, excluding Saturday and Sunday, at the doses of 2.5 and 25 mg/kg body weight in three groups of animals (20 males and female rats in each) during 10 weeks (forfemales) and 11 weeks (for males). Control animals received an equivalent amount of the solvent. Intact animals intended for breeding were managed in parallel with the control and experimental animals. After the end of the exposure, functional parameters of the condition of gonads and reproductive ability of the animals were investigated. Females were used to investigate the condition of the oestral cycle, duration and frequency of each stage of it. The condition of the reproductive function in the female rats was considered at Day 20 of pregnancy. Indices of breeding, conception, fertility, pregnancy were established, and the duration of the pre-coitalperiod was considered.

Results and Discussion. The data obtained suggest that exposure to two generic substances of carbendazim at the dose of 25 mg/kg body weight during 11 weeks is manifested in general toxic effect (body weight reduction) only in males upon exposure to C2. All test substances in the maximum dose have reproductive toxicity and show significant anti-androgenic action that is manifested as changes in the morphological and functional parameters of the condition of gonads in the experimental groups of male rats. Furthermore, the negative effect on the oestral cycle in experimental female rats exposed to C2 was noted.

Conclusion. Based on the obtained results it can be concluded that all test samples of carbendazim have reproductive toxicity at the dose of 25 mg/kg body weight. No-observed-effect level (NOEL) for all test compounds is 2.5 mg/kg body weight. Dose-effect dependence was observed in the range of studies doses.

Key Words: gonado- and reproductive toxicity, Wistar Han rats, carbendazim.

Carbendazim is one of the wide used systemic fungicides of the benzimidazole class designed for plant protection against different fungal diseases [1]. Recently, many studies were conducted on the investigation of carbendazim effect on non-target objects. As per the literature data, it is known that is pesticide is an endocrine disruptor with reproductive toxicity that manifests as changes in the levels of various hormones. It has a negative effect on steroidogenesis in testicles and antiandrogenic effect [2-6].

This study was conducted to assess gonado- and reproductive toxicity of two toxic samples of fungicide carbendazim of different manufacturers and purity in male and female Wistar Han rats.

Materials and methods. The animal study was conducted according to the requirements and provisions of the European Convention on the Protection of Veterinary Animals Used for Experimental and other Scientific Purposes (Strasbourg, 19.03.1986) ETS No. 123, Guide for the Care and Use of Laboratory Animals (National Academies Press, USA, 2011) [7, 8].

Gonado- and reproductive toxicity of two carbendazim samples from different manufacturers was studied. The purity of the test compounds of carbendazim was 98 % (C1) and 98.1 % (C2). Experiments were conducted in male and female Wistar Han rats from SPF nursery State Enterprise “L. I. Medved’s Research Center of Toxicology of the MoH of Ukraine” at the age of 5–6 weeks and body weight of 80–100 g. Adaptation period lasted for five days.

Animals were placed in the convection vivarium. Room was equipped with forced ventilation (12 volumes per hour) that excluded air recirculation. Temperature and relative air humidity was recorded on a daily basis, temperature fluctuations were 19 to 24 °С, humidity — 30 to 70 %. Lightning was natural. Animals received deionised, UV decontaminated and reverse osmosis purified filtered drinking water and balanced pelleted feed with a reduced content of natural phyto-oestrogens manufactured by Altromin (Germany) ad libitum.

The test substances were administered ex tempore on a daily basis, excluding Saturday and Sunday, intragastrically using a gastric tube in two groups of animals (20 males and females in each) at the dose of 2.5 and 25 mg/kg body weight for 10 weeks for females and 11 weeks for males. Control group of males (20 animals) received distilled water with an emulsifier (OP-10) in equivalent amounts. Intact animals intended for breeding were managed in parallel with the control and experimental animals.

After the end of the exposure, functional parameters of the condition of gonads and reproductive ability of the animals were investigated. Females were used to investigating the condition of the oestral cycle, duration and frequency of each stage of it. Males were used for assessment of the number of mobile sperm cells, their total number, as well as the number of abnormally changed forms of germ cells. Morphometric parameters of testicles and appendages were registered

The condition of the reproductive function was considered at Day 20 of pregnancy in experimental females became pregnant from intact males and in intact females coupled with experimental males. This included registration of yellow bodies in ovaries, number of live, dead and resorbed foetuses and embryos, the body weight of foetuses, the total weight of fall, the presence of coarse developmental abnormalities. Indices of breeding, conception, fertility, pregnancy were established, and duration of the pre-coital period was considered. All males rates after necropsy underwent gross examination. Animals were weighed every week throughout the exposure, and pregnant female rats were weighed at Day 0, 6, 13 and 20 роst соitum [9, 10].

Statistical significance of intergroup differences (Р < 0.05) was assessed by Student’s t-test. Studies were conducted in accordance with the recommendations and requirements of Good Laboratory Practice (GLP).

Results and discussion. Technical-grade carbendazim at the tested doses had no visible effect on the general condition of experimental male and female rats and did not result in the death of animals. However, systemic toxicity was found only in male rats during exposure to the test substance C2 at the dose of 25 mg/kg that was manifested as significant body weight reduction in experimental animals (Fig. 1).

Fig. 1. Changes in body weight of male rats over time during C2 exposure (* – P ≤ 0.01; ** – P ≤ 0.001).

Upon investigation of the morphological and functional parameters of the condition of genital glands in experimental groups of males on C1 and C2 at the dose of 25 mg/kg, statistical significant reduction of the total number of sperm cells, number and per cent of mobile sperm cells compared to the control group, as well as significant increase in the per cent of abnormal forms of sperm cells was found.

Upon gross examination of testicles and appendages, no visible abnormality of these organs was found, however, the group of males on maximum dose showed a statistically significant reduction in absolute and relative weight of testicles under the action of both test samples, as well as a reduction in the weight of appendages, was registered in C1. Statistically significant changes in other studied parameters have not been registered at this dose (Table 1, 2).

Table 1

Morphological and functional condition of gonads in male rats after the period of exposure to carbendazim (C1).

Table 2

Morphological and functional condition of gonads in male rats after the period of exposure to carbendazim (C2).

Upon investigation of male fertilizing ability and fertility receiving C2 at the dose of 25 mg/kg assessed by the parameters from intact females, a tendency towards the decrease in conception and fertility indices by 15 % was registered.

In male studies, C1 and C2 at both doses had no negative effect on male fertility that was assessed by the parameters of the reproductive function in conceived intact females. A number of live foetuses in experimental groups, number of yellow bodies in ovaries, number and percentage of foetuses died before and after implantation, as well as the average body weight of foetuses and the total weight of fall did not significantly differ from the control level.

The test substances of carbendazim upon study in females did not show a negative effect on their general condition. Registration of body weight of experimental and control females during exposure showed that the test substances do not induce a significant reduction in body weight during exposure and pregnancy.

Indices of coupling, conception, fertility and pregnancy in both groups of animals on minimum and maximum C1 and C2 doses had no statistically significant changes compared with control, however, there was a tendency towards the reduction of pregnancy index at the maximum test dose of C2 (two females had no live foetuses).

During a 2-week follow-up period over the oestral cycle in females on C2 at the dose of 25 mg/kg, statistically significant increase in the duration of the cycle and its separate stages (oestrus and di-oestrus) compared to the control group was registered (Fig. 2).

Fig. 2. Oestral cycle and duration of its individual stages in females exposed to C2 (* – P ≤ 0.05).

Conclusion.

1. Following intragastric administration of C2 at the dose of 25 mg/kg body weight during 11 weeks, general toxic effect, namely body weight reduction in males during the period of exposure was manifested.

2. All test substances at a maximum dose have reproductive toxicity that is reflected in possible changes of morphological and functional parameters of the condition of genital glands upon analysis of sperm in experimental males, such as: reduction of the total number of sperm cells, number and per cent of mobile sperm cells, as well as the increase in the per cent of abnormal forms of sperm cells. C1 and C2 also resulted in the severe antiandrogenic activity of these active substances: reduction of absolute and relative weight of testicles, as well as the reduction of weight of appendages under exposure to C1 only.

3. During a 2-week follow-up period over the oestral cycle in females on C2 at the dose of 25 mg/kg, statistically significant increase in the duration of the cycle and its separate stages (oestrus and di-oestrus) was registered.

4. Impairment of the oestral cycle in females and antiandrogenic activity in males is a highly sensitive parameter of imbalance in sex hormones that has been identified in this experiment. This is confirmed by the presence of properties of an endocrine disruptor in carbendazim.

Dose-effect dependence was observed in the range of studies doses. It has been established that the no-observed-effect level (NOEL) for all test compounds of carbendazim in terms of gonads and reproductive function in male and female Wistar Han rats is the dose of 2.5 mg/kg body weight.

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