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

ABSTRACT. Introduction. Carbendazim is a systemic benzimidazole fungicide rated as a possible human carcinogen.

In Ukraine, over 20 carbendazim-based preparative forms of fungicides, predominantly generics, are registered, therefore, oncogenicity study of carbendazim from different manufacturers is of immediate interest.

Objective of the paper is to identify carcinogenicity of 98 % technical carbendazim under conditions of chronic experiment in mice

Methods. A total of 280 male and 280 female CBA mice were included in the study. Carbendazim was intragastrically administered in these animals for 18 months 5 times per week via a gastric tube at the doses of 0, 5, 25 and 75 mg/kg body weight as 0, 0.1, 0.5 and 1.5 % ex tempore prepared a suspension. Dead as well as sacrificed animals according to the experimental schedule were subjected to autopsy. Carcinogenicity was assessed using direct effect parameters — tumour ratio and their latent period.

Results and Discussion. Clinical parameters of the condition of experimental animals, survival and incidence of premalignancy did not differ from those in the control animals. The main cause of death in animals throughout the study were non-specific intercurrent diseases developed with approximately the same rate in the experimental and control group. Tumour incidence (n = 37) in experimental and control groups did not statistically differ and complied with the spontaneous level in CBA mice.

Conclusion. Incidence of tumours, their histological structure, location and latent period in experimental animals on carbendazim at the dose of 5, 25 and 75 mg/kg did not differ from those in the control group. Thus, the experimental study of carbendazim in CBA mice after intragastric administration for 78 weeks did not reveal the carcinogenic potential of the substance.

Key Words: pesticides, carbendazim, carcinogenicity, mice.

Carbendazim (methyl-1Н-benzimidazole-2-yl-carbamate) is enzimidazole systemic fungicide designed to protect plants from pathogenic fungi, and it is also used as the preservative for vegetables, fruit and cereals, in varnish-and-paint, leather and paper industry [1]. Carbendazim has experimentally proved reproductive toxicity, its use is prohibited in the USA [2] and EU countries [3]. Already in 1989, EPA experts have classified carbendazim as a possible human carcinogen (group C) [4]. It is well-known that carbendazim results in hepatic tumours in CD-1 and Swiss mice [5, 6]. In Ukraine, over 20 carbendazim-based preparative forms of fungicides, predominantly generics, are registered, [7] therefore, oncogenicity study of carbendazim from different manufacturers is of immediate interest.

Objective of the paper is to identify carcinogenicity of 98% technical carbendazim under conditions of chronic experiment in mice.

Materials and methods

The study was performed at the State Enterprise “L. I. Medved’s Research Center of Preventive Toxicology, Food and Chemical Safety, Ministry of Health of Ukraine” (Kyiv).

Experiments included 560 sexually mature СВА mice — 280 males and 280 females. The average weight of males was 21.6 ± 2.5 g, females — 19.0 ± 3.1 g, the initial age of the animals was 2–2.5 months. Mice were allocated into 4 groups, 140 animals in each (males and females in equal portions), and they were identified using personal marks.

Experimental and control laboratory animals were under similar standardized conditions on a balanced diet. Acclimatization in the setting of vivarium took 2 weeks from the date of receipt. After quarantine, all animals underwent a veterinary exam, and they were randomized, labelled, and placed into cages.

Carbendazim doses were selected based on the analysis of previous studies of its chronic toxicity and carcinogenicity in mice. Carbendazim was administered in animals for 18 months 5 times a week in the stomach using the non-traumatic metal gastric tube at the doses of 5, 25 and 75 mg/kg body weight (group 1, 2 and 3, respectively) as 0.1, 0.5, 1.5% ex tempore prepared a suspension. Control animals received water at the same volume and via the same route of administration as experimental ones.

At the end of every week, animals were weighed, and average weight and bodyweight gain were calculated. Clinical check-up of animals was performed daily for detection of any abnormalities as a result of exposure to the study substance. Behaviour, mobility, condition of the skin and coverings, mucous membranes were evaluated. Animals were palpated to detect superficial and subcutaneous neoplasia.

Dead during the experiment as well as animals who were sacrificed according to the experimental schedule were subjected to autopsy. For the establishment of the histological diagnosis of the detected tumours in mice, classification of tumours of the laboratory animals in IARC Scientific Publications was followed [8].

Statistical analysis of the obtained results was performed using common methods of statistics [9] and methods of analysis of the results of chronic experiments for carcinogenic properties recommended by the International Agency for Research on Cancer [10, 11]. Carcinogenicity was assessed using direct effect parameters — tumour ratio and their latent period. Paired comparison of the parameters in each group with control in respect of actual/expected figures using concomitant mortality adjusted log-rank test was performed via a combination of contingency tables 2×2 by the terms of tumour detection and χ². Significance of histopathological changes of non-tumour origin in tissues was evaluated using exact one-sided Fischer test [9]. The carcinogenic effect was analysed as per the recommendations by R. Petro et al. [10, 11]. Association between exposure of the substance to the body and development of tumour was also established using one-sided analysis of variance [9].

Results and discussion

Clinical check-up that was performed throughout the experiment did not reveal alterations in the physiological condition of experimental mice compared to the control group: there were no differences in behaviour, motor activity, appetite, respiration, condition of skin and coverings, mucous membranes. Bodyweight of males and females who received the product in different doses did not reduce both upon a comparison between experimental groups and in relation to the control animals.

Survival of the experimental animals also did not significantly differ from control mice. Mortality in males on low to medium doses of the product (group 1 and 2, respectively) in the second half of the experiment (10–14 months) was somewhat higher compared to the control group and those who were on the maximum dose. In females, high doses resulted in the increased mortality at approximately the same terms.

Statistical analysis of the obtained data did not reveal a significant association between the dose and average life expectancy (ALE) of animals. ALE in males on low to medium doses was lower compared to control and animals on a high dose. Decrease of the ALE in females was only in high dose group.

The main cause of death of animals throughout the study were intercurrent diseases that had non-specific nature and developed with about the same rate in experimental and control animals. These predominantly were inflammatory processes (pneumonia, isolated abscesses in different organs), metabolic disorders (systemic amyloidosis, dysproteinosis of different severity, calcium petrificates), as well as age-related polycystic devolution of the renal cortex, congestive cysts in genital organs typical for mice.

In isolated cases, hyperplasia that is considered as premalignancy was reported in mice, namely: in the liver in the form of focal hyperplasia of hepatocytes (one case in males per each group 1 and 2) and hyperplasia of vascular endothelium (in group 2 males), in the bladder in the form of papillomatosis (in group 1 male) of urothelium and formation of Brunn nests (in group 3 male). Cases of papillary hyperplasia in seminal vesicles (in 1 animal from group 1) and glandular uterine hyperplasia (one case per group 3 and control) were reported. Since these findings were isolated and developed in both experimental and control animals, it can be considered that premalignancies were occasional and not associated with carbendazim.

Autopsy of tissues of dead and sacrificed animals has found 37 tumours localised in the liver (15), lungs (8), breast (5), uterus (3), ovaries (3), haemopoietic system (3) (Table 1). Histomorphological spectrum of the neoplasms in mice of both control and experimental groups was typical for this species and line of animals [8, 12-14]. Among tumours of the liver and lungs, adenomas and adenocarcinomas were found; as for breast — adenocarcinoma, sarcoma; uterus — adenoma, adenocarcinoma, sarcoma. Ovaries contained granulosa-cell tumours and thecoma, as for haemopoietic system — lymphosarcoma (Fig. 1).

Fig. 1 Tumours of organs and tissues of laboratory CBA mice in the settings of chronic intoxication with carbendazim.

A — pulmonary adenoma in male, 25 mg/kg dose; х100. B — pulmonary adenocarcinoma in male, 5 mg/kg dose; х200.  C — hepatic hepatocellulary adenoma in male, 25 mg/kg dose; х200. D — hepatic adenocarcinoma in male with accumulation of calcium, 75 mg/kg dose; х200. E — breast adenocarcinoma, 25 mg/kg dose; х200.  F — uterine adenocarcinoma, 5 mg/kg dose; х400. G — thecoma, 0 mg/kg dose; х200. H — splenic lymphosarcoma in female, 75 mg/kg dose; х100. Hematoxylin & eosin.

Table 1

Location and histological type of malignant tumours in mice

The direct criterion for the assessment of carcinogenic effect in experimental studies is the tumour rate calculated in respect of the effective number (number of mice who have survived to the first tumour in experimental groups). Statistical analysis did not find an increase in the total rate of tumours in mice of both genders in the experimental groups compared to the control.

There was also no statistically significant difference between the rate of malignant tumours in experimental and control animals. Metastatic and multiple tumours were not reported in this experiment. A number of benign tumours prevailed over malignant in males. By contrast, females had more malignant tumours than benign ones. Their ration in the control group (benign/malignant) was 0.6, and in the experimental group 1 and 2 — 1.0 and 1.0, respectively. Group 3 had only 2 malignant tumours of a different location, while there were no benign tumours.

The overall rate of tumours in the experimental and control groups considering intercurrent mortality of animals corresponded to the oncological characteristics of CBA mice [12-14]. The most noticeable differences between groups were reported in terms of the rate of pulmonary tumours in group 1 males compared to the control, however, more detailed analysis with low allowance, as well as with the use of exact one-sided Fischer test did not find the statistical difference by this parameter (р = 0.0921).

Evaluation of the latent period of tumour onset was performed based on the clinical observations and results of histological exams in animals sacrificed 52 weeks after initiation of the experiment. As a result, several neoplasms of the breast in females were found, and terms of their detection in the control and experimental groups did not statistically differ.

In animals on carbendazim, no premalignancy was detected that suggest carcinogenesis in a certain organ or tissue, increase in the number in further terms of the experiment or may be used for determination of the latent period of the appropriate tumours.

Time of detection of the first tumour in males of the control group was significantly lower compared to mice from experimental groups. In the control males, the first tumour was detected in dead animal 196 days after the experiment (28 weeks), and this was a hepatic adenoma. Among the animals of experimental groups, the first tumour — pulmonary adenoma — was registered at Day 281 (41 weeks) in group 1 male; at Day 298 (43 weeks) hepatic adenoma was registered in group 2 animal; in the group 3, hepatic adenocarcinoma was registered at Day 480 (69 weeks).

In females, the first tumour (breast adenocarcinoma) was detected in a dead animal at Day 307 of the experiment (44 weeks) in group 1. In the control group, the first tumour (hepatic adenoma) was registered in near terms, at Day 353 (51 weeks). In other experimental groups (2 and 3), the first tumours were registered later — at Day 406 (58 weeks, hepatic adenoma) and at Day 455 (65 weeks, lymphosarcoma), respectively.

Therefore, the first tumours, in general, were hepatic neoplasms. Reduction of their latent period did not observe since they are all formed in experimental animals later than in the control one.

Duration of the average latent period of tumour in males on carbendazim was higher compared to the control animals (р < 0.05). In females on carbendazim and in the control animals, the average duration of the latent period did not significantly differ. No reduction in ALE of mice with tumours on carbendazim was reported compared to the control animals. Inversely, ALE of males with tumours was higher compared to this parameter in males from the general population. In females from group 1 and 2, the difference in ALE in animals with tumours and mice from the general population was not registered. Only animals of group 3 had statistically significant (р < 0.05) increase in ALE in mice with tumours compared to all animals in the group.

Analysis with allowance in terms of concomitant mortality did not detect a statistically significant reduction in the duration of the average latent period of hepatic and pulmonary tumour in experimental animals compared to the control. Other tumours were occasionally registered in the group.

Analysis of mortality of animals with tumours depending on the type of neoplasm has shown that the majority of tumours was detected during terminal sacrification.

According to the conclusion of the Pesticides Action Network Europe (PAN Europe), carbendazim is a dangerous chemical toxin that may cause malformation in foetus, impair “unwrapping” of chromosomes, negatively affect the reproductive system, show genotoxicity, and cause infertility and cancer [15]. Currently, carbendazim (as well as benomyl that is metabolised to carbendazim) is prohibited in EU countries, Australia, the USA, Canada, although, its use continues in many other countries including Ukraine.

Conducted study in mice in the settings of the chronic 18-month experiment did not find the carcinogenic activity of this carbendazim. However, considering its mechanism of action (binding to tubulin with further inhibition of microtubule assembly), mutagenicity and reproductive toxicity, as well as the uncertainty of direct data extrapolation from the laboratory animals to human, the danger of carbendazim carcinogenicity cannot be completely excluded in a man.

On the other side, studies of antiproliferative and antitumour properties of carbendazim have recently appeared. Carbendazim inhibits proliferation of tumour tissues of mammals, in particular, multiresistant and p53-deficient cellular lines of melanoma, cancer of the breast, ovaries and large intestine [1]. Antiproliferative action of carbendazim is based on its ability to stop cell cycle in G2/M phase with further induction of apoptosis.

Conclusion

Under conditions of the conducted experiment, the rate of detected tumours, their histological structure, location and latent period in experimental mice on carbendazim doses 5, 25 or 75 mg/kg did not differ from the control animals.

Therefore, the results of the study of carcinogenic activity of carbendazim in CBA mice after intragastric administration for 78 weeks did not reveal the carcinogenic potential of the substance.

Надійшла до редакції 08.09.2019 р.