Systemic toxicity tests of medical devices with repeated exposure (subacute, subchronic and chronic systemic toxicity). EN ISO 10993-11: 2018.

Test with the Certificate of Good Laboratory Practices (GLPs).

Acute toxicity tests determine adverse effects related to the administration of single doses (or limited exposures). However, the most common exposure of humans to many medical devices is repeated or continuous exposure. The effects of repeated or continuous exposure can manifest as a consequence of the accumulation of chemicals in the tissues or through other mechanisms, so long-term tests (subacute, subchronic and chronic toxicity tests) must be carried out in order to detect them.

Repeated exposure systemic toxicity tests provide general information on the physiological hazards that may occur after prolonged exposure according to the route of exposure and provide information on the toxic mode of action of a substance by the clinical route of exposure used. In addition, they provide detailed information on toxic effects, target organs, reversibility, and can be used as the basis for a safety assessment. The group sizes are designed to allow statistical evaluation of the recorded observations (see Table 1). Due to the variable duration of repeated exposure studies, test samples must ensure their stability.

According to EN ISO 10993-11, the subacute systemic toxicity test determines the side effects that occur after repeated or continuous exposure lasting from 24 hours to 28 days. Although the term is semantically incorrect, adverse effects occurring in the specified time interval can also be considered as a short-term repeated exposure systemic toxicity study. The selection of time ranges between 14 days and 28 days is included in most international regulatory guidelines. In subacute intravenous studies, the duration of treatment generally corresponds to a period greater than 24 hours, but less than 14 days.

Subchronic systemic toxicity tests determine adverse reactions that occur after repeated or continuous administration of a test sample for part of the animal´s life. The duration of subchronic toxicity studies is generally 90 days in rodents, but does not exceed 10% of the lifespan of other species. In subchronic intravenous studies, treatment times are generally between 14 days and 28 days, for rodents and other species, respectively.

In chronic systemic toxicity tests, adverse reactions that occur after repeated or continuous administration of a test sample for a large portion of the shelf life are determined. Chronic toxicity studies generally last between 6 months and 12 months.

Table 1.-Recommended minimum size of the groups (taken from EN ISO 10993-11: 2018)

Type of study

Rodents

Others species

Acutea

5

3

Subacute

10 (5 of each sex) a

6 (3 of each sex) a

Subchronic

20 (10 of each sex) a

8 (4 of each sex) a

Chronic

30 (15 of each sex) b, c

c

a It is acceptable to test single-sex animals. When the intended use of the device is for one sex only, it is appropriate to use animals of that sex in question.

b The recommendation for rodents relates to trials performed in a single dose level group. When additional groups of exaggerated doses are included, the recommended size can be reduced to 10 animals of each sex.

c It is recommended to consult a statistician to know the size of the group affected by a chronic study. The number of laboratory animals should be defined according to the minimum requirements to obtain meaningful results. There must be a sufficient number of animals at the end of the study to guarantee a correct statistical evaluation of the results.

In these studies, healthy young adult animals acclimated to laboratory conditions are used, which are randomly assigned to treatment groups. The most recommended animals are rodents (rat, mouse), and in case of using other animals, the choice must be justified from a scientific point of view. When scientifically justified, high-dose groups of satellite animals, with their corresponding controls, can be used for a predetermined period. This group, including its controls, can be used to examine the effects of treatment, including reversibility, persistence, or delayed toxicities. For subchronic toxicity tests, satellite animals must be kept alive for at least 28 days.

All animals are kept in environmental conditions of temperature and relative humidity and periodicity of lighting-darkness, feeding and drinking, as well as location by number and defined sex.

The dose to be used for toxicity testing of medical devices should be based on the results of the risk assessment, taking into account the dose of clinical exposure and the use of safety factors, if applicable. Except for the administration of the test substance, the animals in the control group should be handled in the same way as the test subjects.

Unlike conventional repeated exposure systemic toxicity studies, repeated dose studies of medical devices often do not produce a dose-response effect. Therefore, the occurrence of a toxic effect at the studied dose level is not mandatory.

Animals are administered one dose of the test sample daily, 7 days a week for the duration of the test. For longer repeated exposure studies, administration for 5 days a week is acceptable, in which case it should be documented and justified. The animals are weighed before the administration of the substance, and after once a week and at the end of the study. Throughout the study, animals should be observed with a frequency that depends on the type of exposure as described for each type of test. Observations take into account changes in skin and coat, eyes and mucous membranes, respiratory, circulatory, digestive, vegetative, and nervous systems, and somatomotor activity and behavior.

In addition, the animals are carried out some types of analytical tests of hematology, blood biochemistry (fluid and electrolyte balance, carbohydrate metabolism, liver and kidney function) and urine, at least at the end of the test, but also at intervals depending on the duration of the study.

All animals undergo a complete gross necropsy that includes an examination of the external surface of the body, all cranial, thoracic and abdominal openings and cavities and their contents. The adrenal glands, brain, epididymis, heart, kidneys, liver, ovaries, spleen, testes, thymus, and uterus, as appropriate, are weighed and preserved for later histopathological examination. The histopathological study is carried out both in the animals subjected to high doses, as well as in the control groups.

The final report includes all the indicated test findings, with information on their possible biological significance.

Histopathological analyzes and studies of the experimental animals are carried out by an external veterinary laboratory.