**1. Introduction**

Nanoparticles are particles that have at least one dimension less than a 100nm [1]. Manufactured nanoscale particles find themselves in multitudes of applications such as industrial, food, cosmetic, medical, computing and so on. Distinctive properties such as increased surface area and enhanced reactivity largely fuel nanoingenuity and the net result is an exponential raise in the manufacture and use of nanoparticles. As such human exposure has also risen at an alarming rate. Deleterious effects of this exposure include pulmonary distress, hypertension, cardiovascular damage, irritation of the otolaryngological tissue, impairment of reproductive functions, neural injuries and blood disorders. Long term exposure can also lead to epigenetic alterations and cancer.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Benefits of nanoscale materials are numerous and the market for nanotechnology is enormous owing to the large turnout for profits. The only ethical way forward to manage the harmful effects of nanoparticles is in educated design with innovations, applications and in ethical disposal of nanowastes [2]. Understanding the mechanisms of toxicity attributed by nanoparticles is crucial to this endeavor. Some tools to assess dose and time dependent toxicity have already been developed. Toxicity can either be directly- cell death or the impairment of normal functions in the cell. There are different ways to test toxicity as there are different routes of exposure to the toxic substance.

in the extracellular matrix provides a local niche that provides the necessary growth factors, cytokines and integrin binding molecules that favors cell survival and growth. On the other hand, cells growing as a monolayer in a culture dish, also experience a growth arrest when they have crowded and have no more space to spread out. Cells stop proliferating once they fill out the culture dish. As such routine sub culturing or passaging is necessary, to not only maintain the proliferating pool of cells but also to keep them healthy. Tissue from most organs

*In Vitro* Toxicity Testing of Nanomaterials http://dx.doi.org/10.5772/intechopen.80818 99

Other than primary, there is also the suspension type cultures. These cells do not require a support matrix to grow. Examples include cells of the hematopoietic system. From an industrial perspective, suspension cultures are easier to maintain and implement in a large scale set up. Optimization and quantification of various culture parameters are quicker, and this helps efficient development of protocol for production. Like passaging in adherent cultures, suspension cultures need dilution for further growth and propagation. Cell population in a culture is finite and a function of the concentration of cells in the medium. Constant agitation is required to avoid flocculation of cells. Although do not require enzymatic and mechanical detachment as in adherent cultures. Adherent cultures best suit cytological studies while

Although primary cells retain genetic integrity of the source tissue, there is a limit to its life span and proliferative ability. It varies between donor tissues, requires optimization of culture conditions and is time consuming to grow. The other alternative to primary are continuous cell lines. They are primary cells transformed through subsequent culturing. Transformation can occur naturally or be induced through chemical and viral means. They greatly benefit from their ease of culturing methods. They are characterized for markers and often are available with well-established protocols of handling and propagation. They enable quicker enable quicker biochemical and cellular analysis of mammalian cells. A large number of experiments can be conducted and repeated to add accuracy to experimental evaluation. This is highly desirable for research and industrial applications. Antibody production, screening of toxic compounds and gene expression studies can all be achieved in a time and cost-efficient manner. Drawbacks however include disparity of investigations with *in vivo* systems. Thus, any use of continuous cell line as a model of study, needs to be followed by *in vivo* analysis and

All cultures face challenges of contamination through contact and lack of appropriate sterility techniques. Bacterial and fungal contaminations cloud the culture and shift the pH. pH imbalances also occur due to presence of incorrect salts, bicarbonate buffering and gaseous tension. pH changes may at times result in media precipitation, although this may also be the case for contamination with detergent phosphate used for cleaning the culture vessels and equipment. Contamination with magnesium and calcium ions can lead to cell clumping and lysis particularly in a suspension culture. Increased duration of enzyme treatment such as trypsin not only results in subsequent cell adherence issues but may adversely affect cell's

are candidates for adherent cultures.

suspension cultures enhance bulk protein production.

medical trials before consideration for implementation.

**3. Cell culture practices**

Conventionally 'acute toxicity testing' has been carried out on a model organism, with each candidate, being tested for a single dose and a single exposure time. Following the exposure, biochemical and histological changes observed from different tissue samples of the dead animal were documented for analysis. The determination of LD50; administration dose with 50% lethality thus required the sacrifice of many animals. LD50 has long been used the comparative standard in assessing the degree of toxicity. Several skin sensitivity tests and lymph analysis have also been performed to assess immunogenic potential of an exposing agent. There are other function-based toxicity tests to study effects on reproduction, mutagenic potential, neural management and embryonic development.

The advantage of cell-based toxicity assays is that large numbers of experiments can be conducted to screen the exponential dose-time combinations of exposure [3]. It is greatly time and cost effective as compared to *in vivo* testing. And ethical concerns of animal sacrifice and need for elaborate and regulated laboratories are avoided. No doubt, *in vitro* and *in vivo* results tend to vary depending on the case study, but the wider use of *in vitro* studies allows for only a fraction of the most promising outcomes to be further evaluated with live animal testing. Again, this alleviates many concerns associated with the using animal testing as the first line of investigation.
