Table 3.

In vivo assays for evaluation of NP preparations.

Assay	Mechanism	Advantages/use	Limitations	References
Acute, subacute, subchronic, and chronic toxicity assays (Clinical chemistry tests, Hematological tests, Coagulation tests, Weight change assessment, Carcinogenicity tests, and Histopathology)	To evaluate systemic response by testing different routes of administration and durations of exposure.	Thorough assessment of biological changes as a result of substance administration.	Time-consuming, and expensive.	[171–175]
Reproductive toxicity tests (Mammalian germ cell cytogenetic assay, Heritable translocation assay, Mouse spot test, Micronucleus test, Chromosomal analysis)	To evaluate effects on fertility of the host anddevelopment of offspring by looking for changes at the genomic or embryonic level.	Accurate detection of mutations, deletions, and chromosomal aberrations.	Skill-demanding, time-consuming, and expensive.	[176–179]
Ocular- and skin-irritation tests (Eye irritation draize test, and Trans-epithelial water loss (TEWL) test)	Measurement of skin and eye irritancy by dripping the test substance on eye or skin of the host and looking for irritations such as inflammation, bleeding, ulceration, swelling, or permanent damage.	Accurate assessment of harmfulness of substances to the eye or skin.	Ethical concern over animal welfare.	[180–184]
Hypersensitivity tests (Skin prick test, Intradermal test, Patch test)	Treatment of skin with various concentration of test substance and observing for any immediate skin reactions, itching edema, erythema, urticaria, angioedema, signs of anaphylaxis; as well as testing for release of histamine, IgE, IgG, and Histopathological analysis.	Accurate detection of immediate contact reactions.	Difficult to attain sensitivity and specificity values, variations in symptoms.	[185–187]