Abstract

BACKGROUND:

Prior to implementing a positive list system (PLS), there is a need to establish acceptable daily intake (ADI) and maximum residue limit (MRL) for veterinary drugs that have been approved a few decades ago in South Korea. On top of that, chronic dietary exposure assessment of veterinary drug residues should be performed to determine whether the use of these veterinary drugs would cause health concerns or not.

METHODS AND RESULTS:

To establish the ADI, the relevant toxicological data were collected from evaluation reports issued by international organizations. A slightly modified global estimate of chronic dietary exposure (GECDE) model was employed in the exposure assessment owing to the limited residual data. Therefore, only the ADI of ephedrine was established due to insufficient data for the other veterinary drugs. Thus, instead of ADI, the threshold of toxicological concern (TTC) value was used for the other drugs. Lastly, the hazard index (HI) was calculated, except for etizazole hydrochloride, due to the potential of mutagenicity.

CONCLUSION(S):

The HI values of ephedrine, menichlopholan, and anacolin were found to be as high as 6.4%, suggesting that chronic dietary exposure to the residues from these uses was unlikely to be a public health concern. Further research for exposure assessment of veterinary drug residues should be performed using up-todate Korean national health and nutrition examination survey (KNHANES) food consumption data. In addition, all relevant available data sources should be utilized for identifying the potentials of toxicity.

Keyword

Anacolin,Ephedrine,Etisazole hydrochloride,Menichlopholan,Risk assessment

Introduction

Veterinary drugs have been used to prevent disease-outbreak from animals and enhance performance [1]. Additionally, the sales of veterinary drugs have been annually rising in worldwide along with an increase in supply and consumption of livestock products (i.e. meats and eggs) [2,3].

However, abuse or misuse of veterinary drug to animals could lead to human health concern because their residues might present in food. For example, the contaminated food issue called as ‘fipronil-case’ would be well-known [4]. Fipronil-case is caused by a use of illegal pesticide to egg and egg products in Europe. Fipronil is authorized to be used as veterinary drug to treat mites and ticks in pets like dogs and cats, although it is not permitted to be intended for food producing animals such as chicken in Europe [4].

To enhance the regulation of pesticide or veterinary drug residues in food, the positive list system (PLS) has been already implementing in many countries such as Europe, USA and Japan. PLS indicates that pesticide, feed additives or veterinary drugs, which have been not permitted in domestics, should be applied to 0.01 mg/kg, close to the value for limit of quantitation (LOQ).

In South Korea, several veterinary drugs (i.e. ephedrine, menichlopholan, anacolin and etisazole hydrochloride) have been approved in the past without setting MRLs and HBGVs [i.e., acceptable daily intake (ADI)].

Ephedrine has long been used in humans to prevent and treat both bronchitis and asthma [5]. According to the FDA document, its effects have been known to include increased blood pressure, by activating alphaand beta-adrenergic receptors, and increased cardiac contractility.

Menichlopholan, one of the halogen phenols, is used for the treatment of Fasciola hepatica in ruminants, which noted in the specification of bilebon injection. Fasociola hepatica is a common species of medically important trematodes in Korean cattle [6].

Anacolin is an anticholinergic drug used for the prevention and treatment of acute indigestion in cattle, pigs, and horses, as described in veterinary drug specifications. A mode of action of anticholinergics is to compete with acetylcholine for cholinergic receptors and act mainly through the muscarinic acetylcholine receptors in the parasympathetic nervous system [7].

In the Ectimar^® specification, etisazole hydrochloride is a broad-spectrum fungicide used to control trichophytosis and microsporosis in cattle, swine, and horses. Since some fungicides could have induced the hazardous effects such as genotoxic or teratogenic effects [8,9], it is required to review the other toxicological aspects of etisazole hydrochloride, thoroughly.

To respond the PLS of veterinary drugs, effective to be January 1^st 2024, there is a need to evaluate the most appropriate ADI and MRL for four veterinary drugs as mentioned above. Therefore, the aims of this study were to establish the acceptable daily intake of these compounds based on risk assessment by reviewing their safety evaluation documents and calculating the hazard index (HI).

MaterialsandMethods

Hazard identification

Toxicological data were collected from evaluation reports issued by several international organizations, including the Center for Drug Evaluation Research, European Food Safety Agency.

Determination of point of departure (POD)

The most sensitive endpoint was determined by comparing the toxicological/pharmacological data.

Exposure assessment

The Joint FAO/WHO Expert Committee on Food Additives (JECFA) has used the GECDE model, which it first proposed in 2009, for dietary exposure assessment of veterinary drug residue evaluation since 2017. This model may be more practical than the previous model diet, the theoretical maximum daily intake (TMDI), since it allows simultaneous consideration of both high and general consumers. The global estimate of chronic dietary exposure (GECDE) model calculates the sum of the highest dietary exposure for a food category, based on high (97.5th percentile) consumption levels, plus the mean dietary exposure for all other food categories, using individual countries’ food consumption data for the general population. This study used the estimate of chronic dietary exposure (ECDE) model because no median residue-level data were available. The original GECDE model (a) and ECDE model (b) calculation equations are as follow:

Threshold of Toxicological Concern (TTC) concept

The TTC approach was first introduced in 2016 by the European Food Safety Agency (EFSA) and World Health Organization (WHO) [10]. The TTC approach allows the prioritization of chemicals in the regulatory context to be determined, as a screening tool, because it provides toxicological reference values depending on the chemical’s specific hazardous potential. Accordingly, this study superseded TTC values where data to evaluate the ADI were insufficient.

Food consumption data and Proposed MRLs

This study’s exposure assessment incorporated the 2010-2016 KNHANES food consumption data provided by the Korean Disease Control and Prevention Agency (KDCA) and MRLs proposed by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) and the Ministry of Food and Drug Safety (MFDS).

Risk characterization

The HI was calculated using the following formula, where exposures below the threshold values, veterinary drug residues are unlikely to be a public health concern.

Hazard identification of the target chemicals

To establish the ADI for the target chemicals, pharmacokinetic data (laboratory animals or humans); single- and repeated-dose toxicity data, and reproductive/developmental toxicity data; genotoxicity, carcinogenicity, clinical, and pharmacological (cardiovascular system, respiratory system, and central nervous system) data; and residue depletion trials data were investigated through Pubmed, PubChem and EPA comptox. From the investigation, the toxicological and pharmacological information of each chemical were summarized. In case of the chemical that had insufficient toxicological data to evaluate the ADI, TTC value determined to Cramer’s decision tree was assigned.

Exposure and Risk assessment

Chronic dietary exposure to the target chemicals was estimated in this study. Finally, the HI was calculated by dividing the estimates into ADI or TTC values.

ResultsandDiscussion

Hazard identification

The structures of all target chemicals, structural identifiers, and physicochemical characteristics are summarized in Fig. 1 and Tables 1 and 2.

Hazard identification of Ephedrine

Pharmacokinetic profiles are likely to differ depending on species. When various species are orally administered ephedrine, the parent compound is metabolized into norephedrine, hydroxy-norephedrine, and hydroxy-norephedrine [11]. When rabbits or humans are orally exposed to norephedrine, a hydroxy-norephedrine is produced [12]. These metabolites are produced via various metabolic pathways including aromatic hydroxylation, N-dealkylation, and deamination [13]. Little fecal excretion has been reported of these metabolites and ephedrine; therefore, the major route of excretion is likely urine [12]. In rats and humans, predominantly ephedrine is found in the urine, while norephedrine has mostly been noted in the urine of rabbits, dogs, and guinea pigs [13].

The lethal doses (LD₅₀) were investigated for multiple species and routes pertaining to ephedrine, ephedrine sulfate, or hydrochloride. Detailed general toxicity studies are provided in SI Table1.

A study of ephedrine sulfate in mice, carried out by the National Toxicological Program (NTP), reported an LD₅₀ of 1072 mg/kg bw for females and 812 mg/kg bw for males, after a single oral administration. For intravenous administration of ephedrine hydrochloride, the LD₅₀ was 74 mg/kg bw for males [14].

The LD₅₀ values of ephedrine sulfate were evaluated in rats. A single-dose toxicity study carried out by the NTP in the 1980s revealed that all exposure groups died, making it impossible to calculate the lethal dose. A later single-dose toxicity study, using intravenous administration, resulted in a male LD₅₀ of 102 mg/kg bw [15]. Another study reported an LD₅₀ greater than 135 mg/kg bw via intravenous administration in rats [16].

The LD₅₀ values for rabbits have been reported as 60 or greater than 63 mg/kg bw [16,17]. In dogs, the LD₅₀ for a single intravenous administration of ephedrine sulfate was greater than 70 mg/kg bw [16]. A cross these animals, clinical signs after ephedrine administration included convulsions and changes in respiration.

Repeated-dose toxicity

The 13-week repeated-dose toxicity studies in rodents (rats and mice) conducted by the European Chemical Harmonization Agency (ECHA) and NTP reported no compound-related pathological changes; however, decreased weight gain was observed in all rats or mice treated with 50 or 75 mg/kg bw/day, respectively. Hyperactivity and excitement due to drug administration were observed in the groups treated with more than 100 or 150 mg/kg bw/day, rats and mice, respectively. Therefore, the NOAEL of ephedrine in the 13-week repeated-dose toxicity study in rats and mice was determined to be 22 and 35 mg/kg bw/day, respectively.

Local tolerance test

In a local tolerance test using rabbits, the administration of ephedrine sulfate (5% solution, 50 mg/ml) induced thrombosis. However, there is a lack of clinical relevance regarding this effect due to the absence of a clinical report [16]. Detailed study information is presented in SI Table 1.

Reproductive and developmental toxicity

Through the available reproductive and developmental toxicity documents pertaining to Ephedrine and its analogues, the no-adverse effect level (NOAEL) of Ephedrine was considered 4 mg/kg bw/day, equivalent to 10 mg/kg bw/day of ephedrine sulfate. In this paragraph, we briefly introduce reproductive and developmental toxicity data from the FDA REZIPRES^® approval (2021). Other studies discussing the reproductive and developmental toxicity of ephedrine are summarized in SI Table 2.

The effect of fertility and early developmental stage was not observed by ephedrine sulfate in rats. From available data, no effects were reported when male rats were exposed to 0, 2, 10, or 60 mg/kg ephedrine sulfate for both 28 days prior to mating and through gestation, and females were treated for 14 days prior to mating through gestational day 7. However, decreased fetal body weights were observed when pregnant rats were administered intravenous bolus doses of 60 mg/kg ephedrine sulfate from gestational days 6 to 17. The dose was also related to evidence of maternal toxicity, such as decreased body weight and abnormal head movements. However, fetal body weight was unaffected at 10 mg/kg. Additionally, in a study in which pregnant rabbits were administered an intravenous bolus dose of up to 20 mg/kg ephedrine sulfate daily from gestational day 6 to lactation day 20, there was no evidence of malformations or embryo/fetal toxicity. However, the high dose of 20 mg/kg ephedrine sulfate was considered related to pharmacological maternal effects, such as increased respiration rate, dilated pupils, and piloerection. Additionally, when juvenile rats were intravenously exposed to 2, 10, or 60 mg/kg bw/day ephedrine sulfate from postnatal day (PND) 35 to 56, adverse effects—an increased mortality incidence—were only reported for the 60 mg/kg bw/day dose. Therefore, the no-adverse-effect level was considered to be 10 mg/kg bw/day ephedrine sulfate. Lastly, in a study in which pregnant rats were administered an intravenous bolus dose of up to 60 mg/kg ephedrine sulfate daily from gestational day 6 to lactation day 20, decreased fetal survival and body weight, linked with maternal toxicity, was noted at a 60 mg/kg. No adverse effects were observed at a dose of 10 mg/kg.

Genotoxicity

A variety of in vitro and in vivo genotoxicity studies have been conducted. The in vitro bacterial reverse mutation test (Ames test); in vitro DNA damage tests using human lymphocytes, Chinese hamster ovary cells (CHO), or rat hepatocytes; and in vitro mutation tests using mouse lymphoma [17-22]. From the in vivo genotoxicity test, the FDA concluded that ephedrine seems to be a non-genotoxic chemical, as all ephedrine sulfate-treated groups were reported to be negative in the micronuclei test. Detailed explanations of the in vitro/in vivo genotoxicity studies are given in SI Table 3.

Carcinogenicity

The NTP investigated the carcinogenicity of ephedrine by administering 0, 19 or 37.5 mg/kg bw/day of ephedrine to mice for 103 weeks. The results showed decreased average body weight in each sex, but no evidence of potential carcinogenic effects. Thus, the NOAEL was determined to 37.5 mg/kg bw/day. A similar study in rats administered 0, 6.25, or 12.5 mg/kg bw/day of ephedrine for 103 weeks. Again, NOAEL was designated as the highest concentration. Similarly, according to the FDA document (2021), when rats and mice were exposed to ephedrine sulfate up to 10 and 27 mg/kg bw/day, respectively, ephedrine did not induce tumors. Therefore, the FDA authors concluded that it would not be a carcinogen, and no adverse effects were determined at 10 mg/kg and 27 mg/kg bw/day in rats and mice, respectively. Accounting for all of the available data, shown in SI Table 4, it was concluded that there was no evidence of carcinogenicity in ephedrine.

Clinical observation

A summary of clinical studies is shown in SI Table 5. These results indicate that ephedrine and norephedrine were likely to be associated with cardiovascular effects. Specifically, norephedrine induced a change in the diastolic and systolic blood pressure in six healthy males who took 37.5 mg ephedrine; thus, the lowest observed adverse effect level (LOAEL) was determined to be 37.5 mg/person/day [27]. A cross-sectional study also reported an increase in diastolic/systolic blood pressure associated with ephedrine administration [23].

Pharmacology

Similarly, cardiovascular effects have also been observed in laboratory animals. For instance, a study of L-ephedrine in SD male rats reported changes in both systemic and pulmonary blood pressure, concluding that ephedrine-related changes are mediated by the stimulation of direct alpha-adrenergic receptors and controlled by beta-adrenergic receptors [24]. The effects of ephedrine on the central nervous system (CNS) were investigated in mice and rats. In particular, when mice were exposed to ephedrine, motor activity markedly increased after 3 h [25]. Regarding respiratory effects, the increased respiration rate per minute is remarkable in male dogs [15]. The pharmacological aspects of ephedrine are presented in SI Table 6.

POD for Ephedrine

To determine the most sensitive point of departure for ephedrine, the relevant points of departure were summarized (SI Table 7). In order to establish HBGV, human data are preferred as the uncertainties resulted from animal data could be eliminated [26]. Therefore, the clinical data for norephedrine could be a candidate for point of departure of ephedrine [27]. The authors figured out that the lowest observed adverse effect level (LOAEL) of norephedrine for cardiovascular effects was 37.5 mg/person/day [27]. However, ephedrine has considered to be transformed to norephedrine approximately 13.2% in human [28]. Due to the fact that this clinical study has several disadvantages(i.e., a limited number of subjects, single-administration), it is reasonable that animal data for ephedrine hydrochloride or sulfate would be good candidates. Consequently, the appropriate NOAEL was determined to be 4 mg/kg bw/day ephedrine based on fetal and maternal toxicity in rats intravenously exposed to ephedrine sulfate. The observed developmental toxicity is considered to the most sensitive effect resulted from the exposure to ephedrine and its analogues this is because the adverse effect was shown during early life stages. A default uncertainty factor of 100 was applied to the NOAEL to adjust for the intra/inter species difference, resulting in a final ADI of 0.04 mg/kg bw/day (Table 3).

Hazard identification of Menichlopholan

Menichlopholan belongs to the halogen phenols, which include nitroxynyl, disophenol, and bithionol [29]. Menichlopholan has been reported to have moderate-to-severe acute toxicity. For example, the oral LD₅₀ for menichlopholan in rats and hamsters has been reported to be 10 and 50 mg/kg bw, respectively, according to PubChem. Contrastingly, the LD₅₀ range for bithionol was noted as between 7-760 mg/kg bw when rats and mice were exposed through multiple routes (oral, intraperitoneal, and intravenous), as reported in PubChem. However, the LD₅₀ for nitroxynil was 125 mg/kg bw in mammals and ranged from 170 to 450 mg/kg bw in rats, mice, and dogs, as outlined in the evaluation report of the European Medicine Agency (EMA). The lethal dose values for hexachlorphene has been established in rats via a variety of routes: 56 mg/kg bw, 22 mg/kg bw, 7.5 mg/kg bw, and 340 mg/m3 for oral, intraperitoneal, intravenous, and inhalation, respectively, as mentioned in the MSD (Merck&Co.) and TCI America Inc. hexachlorphene safety data sheets (SDS). The acute toxicity data for the halogen phenols are shown in SI Table 8. Based on these data, the acute toxicity of menichlopholan was higher than that of other halogen phenols. Except for the acute toxicity studies, no other toxicity data were available.

Hazard identification of Anacolin

Although the acute toxicity data of anacolin are not available in detail (SI Table 8), compound-related effects—such as parasympathetic blockade—were reported in the Polish Journal of Pharmacology and Pharmacy at 1978. However, no other toxicity information (i.e., mutagenesis, carcinogenesis, and reproductive/developmental toxicity) was available.

POD for menichlopholan and anacolin

Due to the absence of adequate toxicity data for establishing the ADI of menichlopholan and anacolin, the TTC concept was used to evaluate the toxicological reference dose, which provides toxicological threshold values for the structural class (Table 4). In this study, menichlopholan and anacolin were assigned as 0.0015 mg/kg bw/day of Cramer’s class III, as both chemicals have more than one phenyl or benzene ring.

Hazard identification of etisazole hydrochloride

To establish the ADI for this drug, safety documents were investigated for information such as ADME, general toxicity, and reproductive and developmental toxicity, but relevant data were not available.

However, clinical findings have been reported in humans. When Ectimar^® containing 10% etisazole was applied to a mid-fifties farmer's wrist at least twice a day, severe contact dermatitis was observed after a few days [30]. Moreover, the signs persisted for three weeks despite corticosteroid therapy. The patch test, performed after the rash was resolved, was positive for etizazole 48 and 96 h later. Another similar clinical case reported an allergy to Ectimar^® [31]. Presumably, the fragile S-N binding in etisazole could be easily broken in contact with the skin, indicating that metabolites could not cause hypersensitive reactions [30]. This hypothesis, however, seems limited by the lack of relevant published pharmacokinetic research. However, the genotoxic potential of this drug was confirmed by the QSAR database (ECHA); etisazole has been predicted to be a mutagen in both CAESAR mutagenicity model and SARPY mutagenicity model in VEGA (Q)SAR platform as a good reliability. On top of that, it has been also predicted as non-easily biodegradable in Danish QSAR database, which indicating that etisazole has the potentials of persistency in the environment. Therefore, further studies on ADME, genotoxicity and residue depletion trials are warranted to ensure the safety of this chemical.

POD for etisazole hydrochloride

Using the TTC concept, the TTC value assigned to etisazole hydrochloride (0.0025 μg/kg bw/day) was assigned as presented in Table 4, until there was no evidence of mutagen, because etizazole was predicted to be a ‘suspected mutagen’.

Exposure and risk assessment

This study estimated chronic dietary exposure to all the tested chemicals. Detailed information regarding the exposure assessment for each drug is provided in SI Tables 9 to 11. Finally, the HI was calculated by dividing the estimates into ADI or TTC values. The exposure, toxicological reference doses, and risks are summarized in Table 5; risk ranged from 0.2 to 6.4%, except for etizazole hydrochloride. It is essential that further studies ensure the overall safety of etizazole hydrochloride. Taking everything into account, ephedrine, menichlopholan, and anacolin do not present any risk to the consumer, and the proposed MRLs are likely to be appropriate to protect public health.

Note

The authors declare no conflict of interest.

ACKNOWLEDGEMENT

This study was supported by a grant (No. 21161 MFDS361) from the Ministry of Food and Drug Safety of Korea for 2021.

Tables & Figures

Fig. 1.

Structures of ephedrine and its analogues, mechlopholan, anacolin, and etisazole hydrochloride

Table 1.

Physicochemical characteristics of ephedrine and its analogues

Table 2.

Physicochemical characteristics of menichlopholan, anacolin, and etisazole hydrochloride

SI Table 1.

Summary of the general toxicology of ephedrine

SI Table 2.

Summary of reproductive and developmental toxicology of ephedrine

SI Table 3.

Summary of genetic toxicology of ephedrine

SI Table 4.

Summary of carcinogenicity of ephedrine

SI Table 5.

Summary of clinical studies of ephedrine

SI Table 6.

Summary of pharmacology of ephedrine

SI Table 7.

Summary of point of departures (PODs) of ephedrine

Table 3.

Rationales for setting acceptable daily intake (ADI) of ephedrine

SI Table 8.

Summary of acute toxicity data of halogen phenols and anacolin

Table 4.

Rationales for allocating threshold of toxicological concern (TTC) values of menichlopholan, anacolin, and etisazole Hydrochloride

SI Table 9.

Chronic dietary exposure estimates of ephedrine

SI Table 10.

Chronic dietary exposure estimates of menichlopholan

SI Table 11.

Chronic dietary exposure estimates of anacolin

Table 5.

Summary of risk assessment

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