There is growing concern about the negative effects on human health, environment and non-target organisms due to the widespread and indiscriminate use of conventional insecticides [1] . For example, certain insecticides have negative effects on the nervous, renal, respiratory, and reproductive systems of men and women. Epidemiological studies have shown the association between exposure to pesticides and the risk and incidence of cancer [2] [3] [4] [5] . In addition to the detrimental impact of synthetic insecticides on human health, their excessive or inappropriate use is also linked to contamination of water, soil, and air, resistance of some pests to pesticides, and the reduction of biodiversity, among other factors [5] [6] [7] . The potential adverse effects associated with the use of chemical pesticides have also stimulated the research of new alternatives of pesticides from natural sources. In this sense, plants are a safe and efficient source of compounds with less toxicity for mammals, low persistence in the environment, and biodegradability [8] [9] . Currently, several studies have confirmed many of the beneficial biological activities attributed to them, such as insecticide, fungicide and repellent [10] [11] [12] [13] . However, the potential toxic or deleterious effects that their use could induce in short, medium or long term in human health and environment must be evaluated taking into account the potential routes of exposure for human beings [14] .

Hysterionica pinifolia (Poir.) Baker (Asteraceae) is a perennial subshrub, native to the Argentinian flora. It grows in hilly areas of the province of Buenos Aires, between 0 and 800 meters above of sea level and in other South American countries such as Brazil and Uruguay [15] . In previous work the CH₂Cl₂ (dichloromethane) extract of this species showed insecticidal activity against Tribolium castaneum, a stored grain pest [16] . Regarding the chemical composition of H. pinifolia, acetylenic compounds were reported [16] [17] . Cis lachnophyllum methyl ester and the acetylenic alcohol (E)-undec-3-en-5,7-diyne-1-ol were identified as the major compounds in the active CH₂Cl₂ extract [16] .

Polyacetylenes are widely distributed among Asteraceae, Araliaceae, and Umbelliferae families [18] . Some of the polyacetylenes isolated from food plants such as carrots, celery and parsley have various biological activities, like anti-inflammatory, antibacterial, antifungal and anticancer. According to their chemical structures, other polyacetylene compounds have shown toxic effects on humans and animals [19] . Some of these polyacetylenes are potent skin sensitizers and have shown cytotoxic and neurotoxic activities [20] [21] [22] .

Considering the presence of compounds with possible toxic effects in the active CH₂Cl₂ extract from H. pinifolia, the development of a new insecticidal formulation using this extract as raw material requires a study of its safety.

The principal aim of evaluating the safety of any medicinal plant is to identify the nature and significance of adverse effect and to establish the exposure level at which this effect is observed. In the search for safe insecticides for human health and the environment, H pinifolia was studied. In previous work, the CH₂Cl₂ extract of this species has demonstrated promising insecticidal activity and acetylenic compounds were identified [16] . The purpose of evaluating safety is not only to identify a potential adverse effect but also the significance of this effect. Considering the lack of toxicity reports of this species and the compounds identified in it, the safety profile of the CH₂Cl₂ extract must be studied in order to develop a safety insecticidal formulation. In this sense, dermal and ocular irritation, oral acute toxicity and repeated dose safety were evaluated.

Skin and eyes are high risk of exposure to insecticides during their manufacture but also in their use. The hazards may be meaningful not only in case of a single accidental exposure but also in many short and smaller exposures during a continued period of time, so information related to potential skin and eye irritation is a fundamental part to identify the potential risk of the products [28] . In this sense, it is important to determine if a substance or formulation for insecticide use could induce skin and eye irritation to be properly classified and labeled as recommended by Good Agricultural Practices and, if it is necessary, guarantee the appropriate use of personal protective equipment (protective glasses, face shields) [29] [30] .

In the study of primary dermal irritation, the CH₂Cl₂ extract of H. pinifolia presented a slight dermal irritation with barely perceptible erythema formation and no edema formation. Due to the CH₂Cl₂ extract of H. pinifolia did not produce corrosion or severe skin irritation, eye irritation was evaluated, showing that the extract did not produce signs of irritation during observation period.

Besides considering that exposure could occur when toxic substances get into de body through mouth, the oral route represents a potential route of accidental human exposure. Therefore, the determination of acute and repeated dose toxicity allows the hazard classification of test substances [31] . In this sense, acute oral toxicity was determined for the CH₂Cl₂ extract of H. pinifolia at 5000 mg/kg and 2000 mg/kg. The initial dose (5000 mg/kg) was selected adopting the guidelines of the World Health Organization (WHO) (2019) [32] for the toxicological classification of pesticides according to risk and LD₅₀. In this investigation, the acute oral toxicity of the CH₂Cl₂ extract of H. pinifolia was greater than 2000 mg/Kg and less than 5000 mg/Kg. Therefore, according to the WHO toxicological classification (2019), it is considered slightly hazardous (Category III).

The toxic effects observed in the acute oral toxicity at the concentration of 5000 mg/Kg and the slight dermal irritation could be attributed to the presence of acetylenic compounds in the CH₂Cl₂ extract. In this sense, cis lachnophyllum methyl ester and the acetylenic alcohol (E)-undec-3-en-5,7-diyne-1-ol, were identified as major compounds in the CH₂Cl₂ extract of H. pinifolia [16] . Although there are no reports of toxicity of these identified acetylenes, other compounds with analogous structure showed acute toxic effects, contact dermatitis and irritant skin reactions for humans and animals. For example, cicutoxin and oeantotoxin isolated from Cicuta virosa L. (Apiaceae) and Oenanthe crocata L. (Apiaceae) respectively, showed acute toxic effects in humans and domestic animals [22] ; the acetylenic alcohol falcarinol is responsible for most allergic skin reactions caused by plants of the Apiaceae and Araliaceae families, while structurally related polyacetylenes such as falcarindiol and falcarinone had no irritating effect on the skin [18] [22] .

In view of repeated exposure, toxicological evaluations are vital to characterize the toxicological profile of xenobiotics [33] . In this work, the analyzed parameters showed promising results.

Changes in body weight as well as organ weight have been used as an indicator of potential adverse effects of chemical compounds, such as tubular hypertrophy, progressive nephropathy or hepatocellular hypertrophy related to changes in kidney or liver [34] [35] . The administration of repeated doses of the CH₂Cl₂ extract of H. pinifolia did not produce changes in the body weight of the animals, neither in the weight of the organs evaluated.

Histopathological analysis of organs and tissues represents a way to evaluate possible pathological changes related to the administration of repeated doses of a certain substance. The observation of cellular structures allows to determine pathological alterations with greater precision [33] . In the studied organs, a normal cellular morphology was observed, without evidence of necrosis and inflammation between the groups that received daily the CH₂Cl₂ extract of H. pinifolia compared to the corresponding control groups.

On the other hand, considering that level of glucose and lipids can detect alterations in the metabolism [36] , these parameters were measured. The values of blood glucose and cholesterol did not present significant differences between the treated and control groups.

Creatinine, uremia, GOT, GTP and AP are used as biomarkers of renal or hepatic injuries [33] . In this study, no statistical differences in biochemical parameters were observed between the treated and control groups. Only, uremia in males treated with the CH₂Cl₂ extract showed a statistically significant decrease compared to the control group, nevertheless the observed level of uremia (29.20 ± 1.114 mg/dL) lacks biological significance since it is not below 20 mg/dL, a value that indicates hypo-azotemia.

Also, changes in hematological parameters have been used to predict toxic effects [37] but the administration of repeated doses of the CH₂Cl₂ extract of H. pinifolia did not affect the evaluated hematological parameters.

The results obtained in the repeated dose safety test in rats, showed that the administration of the CH₂Cl₂ extract of H. pinifolia did not produce significant adverse effects. The employed dose (100 mg/kg/day) is six times greater than the EC₅₀ of the extract obtained in the insecticide activity bioassay on T. castaneum [16] . Therefore, it could be expected that the use of the CH₂Cl₂ extract of H. pinifolia at the EC₅₀ dose would be safe for human health.

In this study, the CH₂Cl₂ extract of H. pinifolia presented a slight dermal irritation, no ocular irritation, dose of acute oral toxicity is between 2000 mg/Kg 5000 mg/Kg (slightly hazardous) and no signs of toxicity at repeated doses of 100 mg/kg body weight.

The promising results of insecticidal activity obtained in previous works and the safety results obtained in this study with the CH₂Cl₂ extract of H. pinifolia, allow us to consider this extract as a potential raw material for the development of an insecticide of natural origin, safe for human health and the environment.

This work was supported financially by grants UBACYT (University of Buenos Aires, Science, and Technology) 20020170100752BA and UBATCYT 20020130-100705BA of the University of Buenos Aires.

The authors are grateful to University of Buenos Aires for financial support UBACyT. We are also grateful to Prof. Dr. Gustavo Giberti for identification of the plant.

The authors declare no conflicts of interest regarding the publication of this paper.

Cufre, I.M., Broussalis, A.M. and Gorzalczany, S.B. (2023) Safety Evaluation of the Dichloromethane Extract of Hysterionica pinifolia: A Potential Raw Material for the Development of Biopesticides. Journal of Biosciences and Medicines, 11, 66-78. https://doi.org/10.4236/jbm.2023.117007