Introduction

Safety evaluations of herbal medicine, pharmaceuticals, food, chemicals, and cosmetics provide a vital justification for approval for human utilization [1].

While in-vitro and in silico approaches seem to be the leading edge of repeated toxicity testing, animal model cannot be completely overlooked because data generated from such studies could easily be translated (extrapolated) to humans [2]. Based on the postulation that animals respond to drugs in a similar way as humans when a test substance is administered using a similar route, the involvement of experimental animals in toxicity testing have been initiated long ago by Trevan J.W. For instance, asbestos produces lung cancer, while plastic solvent causes liver cancer in both human and animal species [2].

Besides acute toxicity testing, repeated toxicity testing is vital for substances that are used over time [3]. Thus repeated systemic toxicity test is an aspect of toxicity which assesses the potential of substances to produce deleterious effects on an organism following repeated exposure. Routes of 

exposure include oral, intraperitoneal, intravenous, subcutaneous, and implantation. Among these, the oral route is the most common and the route of testing is usually based on the therapeutic use or intended route of exposure of such test substance in human [2]. 

Laid down testing guidelines have been established for repeated systemic safety assessment of herbal products, pharmaceuticals, chemicals, food, and cosmetics [1, 2]. Terminologies such as “subacute”, “subchronic”, and “chronic” in relation to the duration of exposure of test substance in the course of repeated toxicity tests have led to debates in conferences, meetings, workshops, and seminars. To corroborate this fact, the titles of repeated toxicity studies published in several journals are not consistent with the exposure duration protocols. For instance, some repeated toxicity studies that lasted for 28 days were wrongly entitled as “chronic or subchronic”, while some that lasted for 90 days were wrongly titled “chronic” (Table 1). These misconstructions were proved wrongs by highlighting some studies that followed the proposed guidelines (Table 2).

These inconsistencies and deviations from the established guideline have fueled this article to reaffirm the appropriate duration terminology, pinpoint errors that have been made and also bring to light the need for researchers to realize their errors and adhere strictly to the standard duration guidelines. 

Methods

Recently published articles on repeated toxicity studies were searched using Pubmed, Google Scholars, and Scopus database. Keywords used include “guideline on subacute, subchronic, and chronic toxicity testing”, “subacute toxicity study”, “subchronic toxicity studies”, “repeated toxicity studies on plant extracts”, and “6 months chronic toxicity test”. Among the obtained results, a number of articles whose titles harmonized with the standard duration guideline were selected (Table 2). Similar selection was done for articles whose titles deviated from the laid down study duration guideline (Table 1).

Definition of Repeated Dose Toxicity Testing

As the name implies, test substances are administered repeatedly or continuously via a known route over a long period of time [2, 4]. Repeated toxicity test incorporates subacute, subchronic, and chronic toxicity tests [5].

Subacute Toxicity Test

In this test, experimental animals are subjected to graded doses (at least three doses) of the test substance for a duration of 14 – 28 days, 2 - 4 weeks [6]. The term “subacute” does not really mean that the exposure duration will be less than acute (24- hours), but it connotes that the exposure doses have to be below the estimated LD₅₀ value. This accounts for the selection of doses below the LD₅₀ for repeated toxicity test. This study helps to evaluate the systemic side effects of substances on targets organs based on repeated administration of doses below the LD₅₀. The results of this study serve as basis for classification and labeling. It also provides information on the mode of toxic action of a substance. Furthermore, it offers a guideline for designing subsequent studies for longer durations. Hence, subacute toxicity test helps to establish doses for subchronic studies [2]. The exposure duration of 14 – 28 days is consistent with international regulatory guidelines and is considered to represent a reasonable approach [7].

Subchronic Toxicity Test

The goal of this test is to determine the effects that may occur following repeated exposures of animal species to a test substance for a period of three months (90-days) according to the OECD No 408 guidelines for testing of chemicals [8, 9]. This is also referred to as 90-day repeated dose toxicity test [10]. It helps to predict a rational and suitable dose for chronic exposure studies. At least three doses are employed: a high dose that produces toxicity but does not cause more than 10% fatalities, a low dose that produces no apparent toxic effect and an intermediate dose [11].

Chronic Toxicity Test

This test provides insights about the long-term (cumulative) effect of a test substance on experimental animals, usually lasting between 6 months and two years in rodents according to the OECD No 452 test guideline for testing chemicals [3, 4]. It is also applicable in assessing carcinogenic potential of test substances as well as drugs used in the management of terminal diseases such as diabetes, hypertension, arthritis, and rheumatism among others. Study durations of 6 months for rodents and 9 months for non-rodents were considered acceptable for chronic toxicity by the regulatory authorities [3].

The outcome of chronic toxicity test is useful in the establishment of no observed adverse effect levels (NOAEL), the highest dose where no toxicity effect occurs. It also helps in the establishment of safety criteria for human exposure to new drug entities undergoing clinical trials [3]. The major difference between chronic toxicity testing and subacute or subchronic is the duration of exposure [2]. The exposure period usually cover post-weaning maturation and development into adulthood of animals. A group is usually included to monitor reversibility in toxicity for a period of four weeks (28 –days) [11]. 

Biomarkers for Repeated Toxicity Test

Having considered the duration of exposure for subacute, subchronic, and chronic toxicity tests, relevant parameters including body weight, hematological, biochemical, cardiovascular, as well as behavioral parameters could be assessed when necessary before (pre-treatment/pre-exposures/baseline), during (treatment), and after exposure (post-treatment/recovery) of animals to the test substance [2]. Body weight is measured weekly and at the end of the study, samples are collected for biochemical, hematological, and histopathological evaluations [2, 3].

In the course of exposure, during subchronic and chronic toxicity tests, it is essential to carry out periodic (monthly) sample collection and monitoring of hematological, biochemical, cardiovascular, histopathological, and body weight parameters. This would help in observing the onset of toxicity (i.e. to understand whether the toxic effect occurred in the 1^st, 2^nd, 3^rd month for subchronic test as well as 1^st, 2^nd, 3^rd, 4^th, 5^th and 6^th months for chronic test) [4].

Results, Discussion, and Future Directions

The titles of studies in Table 1 revealed a discrepancy in the standard testing duration of exposure. For example, Wang et al. [12] conducted a 28 –day study and titled it “subchronic”, Ntchapda et al. [13] carried out a study that lasted for 42 days and titled it “subchronic” while Quaye et al. [14] did a study that lasted for 42 days and titled it “subchronic”. Following the approved protocol, the titles of typical studies presented in Table 1 did not cohere to the laid down duration guidelines.

On the other hand, the titles of studies depicted in Table 2 are succinctly in consonance with the laid down study duration guidelines. As a case in point, Balogun and Ashafa [15] administered aqueous root extract of Dicoma anomala Sond to Wistar rats for 90 days and titled the study “subchronic”. Ugwah-Oguejiofor et al. [16] administered extract for 28 days and titled their study “subacute”. Sireeratawong et al. [4] did their study for 270 days and titled the study “chronic”. Also, Nsonwu-Anyanwu at al., [17], ran a study on “Chronic exposure to toluene and heavy metals and changes in indices of liver function, inflammation and oxidative DNA damage among automobile workers” which was restricted to participants within the vicinity of an automobile workshop or exposed to paint in their 

environment for a minimum of 1 year before the study. These are reflections of well-structured titles that followed the study duration protocol.

In light of the above, it is necessary for researchers to provide titles that are in consonance with the duration of exposure that such studies are meant for.

For the avoidance of non-conformity between the title of studies and duration of exposure, duration of exposure could be removed from the title such that testing duration is defined in the method section of such studies, but without mentioning any of the terms “subacute, subchronic, and chronic”. For example, Wattanathorn et al. [18] administered their extract for a period of 90 days in a study they titled “Toxicity Evaluation of Anacardium occidentale, the Potential Aphrodisiac Herb”. Similarly, a study titled “Toxicopathological Evaluation of Hydroethanol Extract of Dianthus basuticus in Wistar Rats” by Ashafa, and Kazeem [19] lasted for 28-days, yet subacute, subchronic and chronic toxicity did not appear in the title.

Conclusion

With respect to duration of exposure, there are flaws in the titles of some repeated toxicity articles published in high-ranking journals. This study highlighted the relevance of using the right terminology in repeated toxicity tests. In a nutshell, it is suggested that repeated toxicity studies lasting for 14 – 28 days should have the keyword “subacute” in their titles. Those lasting for 90 days should have the keyword “subchronic” in their titles, while those lasting for 6 months and above should have the keyword “chronic” in their titles. Also, articles without keywords; “subacute, subchronic, and chronic” in their titles, could state such keywords as subtitles in their method section and ensure that they comply with the exposure duration guidelines provided by the regulatory bodies. Whenever the duration of exposure does not match with subacute, subchronic, and chronic, the term “repeated toxicity evaluation or assessment can be used”. For instance, a study can be titled “42-day repeated dose toxicological evaluation”.

Acknowledgements: This goes to authors of references cited in this manuscript.

Conflict of interest: None to be declared.

Funding and support: None