It is a very interesting observation. Although small size AuNPs can show more toxicity than larger sized particles, the reason behind the more toxicity in less concentration of AuNPs is unknown. Can you just tell me did you dilute the AuNPs by any solvent to maintain the total volume of all doses equal?

Yes Sudip Sir, I did two sets of experiments where i diluted the samples once in PBS and second in distilled water to obtain desired concentration of AuNPs. I also kept a blank control where the eggs were inoculated with the diluent , but no mortality was seen in these blank controls. 

Did you perform CAM assay? High concentration of PBS may show little toxicity, but you also kept that as a control!! 

Yes Sir i did kept them as control and no mortality was seen.  I did not inoculate the test samples on CAM but in albumin for systemic exposure the test compound. 

Rohan

was the size  and shape of of gold nano particles Uniform?During dilution there are chances of partial segregation of bigger particles in the first diluent

because endocytotic uptake of smaller particles is faster and will become more toxic

Very important point raised by Dr. Madhuri, As PBS contains NaCl, during dilution may be your gold nanoparticles got aggregated due to Salt effect. Please check the size of your diluted AuNPs by DLS and TEM if possible.

Rohan (I his info also)ave mailed th

Limitation of binding sites: so at lower conc it blocks all site so no matter how much higher u go data will remain unaltered

Or

May be higher concentrations they cause oxidative stress that’s why

Moreover,

I think this should help

http://www.analysisonline.org/site/aoarticle_display.asp?sec_id=140002434&issue_id=5&news_id=140002554&pg=22

Understanding Risk

Toxicity Assessment

Toxicity assessment is a major component of risk assessment.*

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A toxicity assessment provides an estimate of how much of a substance causes what kind of harm.

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A toxicity assessment is a tool to investigate the potential for a substance to cause harm -- and how much causes what kind of harm.

All substances are toxic in quantity. Many therapeutic medications are acutely toxic, but beneficial when used at the appropriate level. Vitamin D, table salt, oxygen, and water are toxic in quantity. Thus, the mere presence of a substance does not automatically imply harm. This is why toxicity assessment is concerned with the type and degree of harm caused by differing amounts of a substance.

There is no one measure of toxicity. Effects may occur in the short term (acute effects) or after repeated exposures over a long time (chronic effects). They may affect only one part of the body or many, and they may vary greatly in severity. **

*Other components are exposure assessment (Chapter 2) and hazard identification and risk characterization (Chapter 1).

**The term toxicity refers to the inherent potential of a substance to cause systemic damage to living organisms. The term hazardous is very different. It refers to the potential of a substance to (1) cause any of several kinds of harm, through toxicity, flammability, explosiveness, corrosiveness etc., and (2) the ease with which people can come in contact with it. Hazardous is nota synonym for toxic.

Toxic Effects

Toxic effects are classified as two types -- either acute or chronic.

• Acute effects happen very rapidly after a single exposure has occurred (food poisoning, breathing fumes from a chlorine spill). Sweating, nausea, paralysis, and death are examples of acute effects.

• Chronic effects happen only after repeated long-term exposure (cigarette smoking, eating foods with low levels of contaminants, breathing polluted air). Cancer, organ damage, reproductive difficulties, and nervous system impairment are examples of chronic effects.

These chronic effects fall into two categories: carcinogenic effects and non-carcinogenic effects.

Examples of non-carcinogenic chronic effects:

• Organ damage: cirrhosis of the liver from long-term alcohol consumption; emphysema from long-term tobacco smoking.

• Reproductive difficulty: decreased fertility from the pesticide DBCP (dibromochloropropane).

• Nervous system impairment: mental retardation in people exposed to high levels of lead during early childhood.

Assessing Toxicity

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The dose-response concept is the basis for all toxicity assessments. It is used differently to evaluate acute effects and chronic effects.

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All quantitative toxicity assessments are based on the dose-response concept: as you increase the dose (exposure), the response (toxicity) also increases.

Scientists perform studies to determine exactly how high a dose causes what kind of a response, or effect. The smaller the dose needed to cause an effect, the more potent (toxic) the substance is.

For all compounds other than cancer-causing agents (carcinogens), it is assumed that there is a dose below which no effect occurs (a threshold). This is similar to a drug where too small of a dose has no beneficial effect.

For carcinogens, it is often assumed that even the smallest dose can cause an effect (no threshold).

Although the dose-response concept is used in all types of toxicity assessments, it is used somewhat differently for each of them to evaluate acute effects and chronic effects.

Acute toxic effects are estimated by LD50 studies or observation of accidental exposures.

Chronic toxic effects are estimated by dose-response studies on animals. Carcinogenic effects are estimated by a type of dose-response study called a carcinogenesis bioassay.

Assessing Acute Toxicity

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Acute toxicity is assessed using observations of accidental human exposures or by conducting LD50 tests on experimental animals, usually rodents.

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Most information about acute toxicity of chemicals to humans comes from accidental poisonings or exposures, such as drug overdoses or chemical spills. Physicians/researchers know or estimate the level of exposure and observe and document the effects.

Scientists also use animal tests called LD50 (L-D-fifty) studies to assess acute toxicity. These studies determine the amount of a substance that will kill half the test animals in 14 days. This amount is called the LD50--Lethal Dose for 50% of the animals.

LD50 is stated in milligrams per kilogram (mg/kg): milligram of chemical per kilogram of body weight.

* The lower the LD50--the lower the lethal dose-the more toxic the substance.

* The term LC50-Lethal Concentration is used to measure the toxicity of gases. The LC50 is stated in milligram of chemical per liter (or cubic meter) of air.

Example: A reported "rat oral LD50 of 50 mg/kg" means that half of the rats that ingested a dose of 50 milligrams of the substance per kilogram of body weight died within 14 days.

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LD50 values are unknown for humans, but animal LD50 values can be used to estimate lethal amounts for humans.

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LD50 values are unknown for humans, since LD50 experiments are not conducted on humans. Animal LD50 values can be used to estimate lethal amounts for humans. If a LD50 statement is applied to humans, it must in actuality be either:

• an animal LD50.

• the "average lethal dose" (ALD), calculated from the effects of accidental poisonings and exposures. Also called mean lethal dose (MLD).

• the "lethal amount" calculated from an animal LD50. This calculation is done by multiplying the LD50 by a number representing average human weight.

Example of lethal amount: If the LD50 is 50 mg/kg: The lethal amount for a child would be 50 mg/kg times 10 kg, which equals 500 mg (about 1/8 tsp.) The lethal amount for an adult would be 50 mg/kg times 70 kg, which is 3,500 mg (about 3/4 tsp.) *

* The 10 kg and 70 kg in the example above come from the weight of the "average" person-10 kg (22 lbs) for a child; 70 kg (154 lbs) for an adult.

Assessing Chronic Toxicity

Chronic toxicity can be divided into two categories:

• cancer (carcinogenic toxicity).

• all other effects (non-carcinogenic toxicity).

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Chronic toxicity is measured in two ways--depending on whether the concern is cancer or other chronic effects.

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Cancer is in a separate category because public concern about it is so great. People want to know if even one person in a million persons who are exposed to a substance will get cancer. To discover this, researchers conduct a specific type of dose-response study - a carcinogenesis bioassay.

Non-carcinogenic effects are usually assessed with a different type of dose-response study.

Non-Carcinogenic Assessment

Introduction

Scientists assess non-carcinogenic chronic toxicity by administering varying amounts of a substance (dose) to laboratory animals and noting the effects (responses), if any, at each dose. Essentially, the scientists look for the smallest dose that causes any detectable effect. This smallest dose is called the Lowest Observable Effect Level (LOEL). *

To conduct these dose-response studies, scientists:

• Administer different small doses of a substance to several groups of test animals every day over a lifetime.

• Periodically examine and finally autopsy the animals to determine if any effects have occurred. The effects may be:

o damage to an organ,

o behavioral modifications,

o change in the level of an essential body chemical.

• Determine the smallest dose at which an effect occurs--the Lowest Observable Effect Level (LOEL). **

• LOEL is measured in milligrams (mg) of substance per kilogram (kg) of body weight, or in parts per million (ppm) of substance in food.

* In addition to LOEL, the term LOAEL (Lowest Observable Adverse Effect Level) is sometimes used. The term LOAEL implies a judgment that the effect is adverse. A LOEL refers to any effect and may or may not be judged to be adverse.

** This dose may still be a high dose compared to environmental exposures.

Determining Safe Levels

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To protect the public, scientists also determine the highest dose at which no effects occur.

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When performing the experiments just described, scientists also determine the highest dose at which no effects occur to protect the public--the No Observable Effect Level (NOEL). *

The NOEL is considered the "safe level" for that chemical in the species studied.

The NOEL is not necessarily the "safe level" for humans, because:

• humans may be more/less sensitive to the substance than the animals studied.

• humans have more genetic, health, age, and other variabilities, which may affect individual human reactions. **

To account for these differences, public health officials divide the NOEL by a safety factor, usually 100, to arrive at a presumed "safe level" for humans. If the NOEL for a substance were 100 mg/kg, the "safe level" for humans would be considered 1 mg/kg.

This "safe level" is most likely lower than scientists' best estimate of the NOEL in humans. However, it is the number risk managers use to establish regulations, such as the maximum amount of a chemical allowed in drinking water, and to create guidelines such as fish consumption advisories.

* The term No Observable Adverse Effect Level (NOAEL) is sometimes used. The term NOAEL implies a judgment that the effect is adverse. A NOEL refers to any effect and may or may not be judged to be adverse.

** Lab animals are bred to be similar to one another, and an experiment will be conducted on animals of the same age and health.

Human Sensitivity and Variability

The "safe level" calculation for humans assumes that humans are more sensitive than animals, but humans are not more sensitive in all cases. Dividing the NOEL by a safety factor assumes that humans are more sensitive than animals. But humans are not always more sensitive. For some substances, they may be less sensitive, or less sensitive than some species.

This variation is usually due to the different degrees and rates of absorption, metabolism, and/or excretion of the substance by the different species.

Although it might be reasoned that the most humanlike animals-monkeys-would be the best test animals, they re-act to some substances more differently from humans than other animals. For example, dogs react to nitrobenzene similarly to humans, while monkeys do not.

Knowledge is still incomplete regarding the best test animals for different types of substances and whether humans can be expected to be more or less sensitive to any particular compound.

Examples of human sensitivity:

• More sensitivity: The drug Thalidomide caused no adverse effects in the animals studied, but caused severe birth defects in humans.

• Less sensitivity: Insecticides are often developed to be more toxic to insects than to humans. Since people are less sensitive to these chemicals, they can use them without injuring themselves.

• Genetic variability: People vary widely in their reactions to bee venom. Some show almost no reaction to a bee sting; others may die without immediate medical treatment.

Carcinogenesis Bioassay

Introduction

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Officially accepted methods of assessing carcinogens assume there is no safe level.

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Scientists assess carcinogenic toxicity very differently than they assess non-carcinogenic toxicity. This is in response to public fear about cancer.

People want to know if even one in a million individuals will get cancer from exposure to a suspected carcinogen. To find this out with any degree of confidence by traditional dose-response studies, scientists would have to use several million test animals. The impracticality of such experiments has led to the development of the carcino-genesis bioassay. *

With a carcinogenesis bioassay, scientists are not looking for the safe level of exposure (NOEL). Rather, harm is assumed, and they are looking for the incidence, or risk, of harm.

* The carcinogenesis bioassay is a method of testing substances for carcinogenic effects that utilizes high-dose studies on laboratory animals to look for even the rare case of cancer. It is not necessarily the best scientific approach to assess the carcinogenic effects of chemicals. Instead it is a way to respond to public concerns by generating carcinogenic risk values with large margins of safety.

Methodology

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To measure carcinogenic toxicity, scientists try to find even the rare case of cancer.

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Scientists assess carcinogenic toxicity by feeding large doses of the substance in question to animals in an effort to find even the rare case of cancer resulting from exposure to it.

• Test animals are administered different large doses of a substance daily over a lifetime (24-30 months in rats).

• At the end of the study, the animals are examined to see if cancer can be found.

• If cancer is found, scientists use available data and mathematical models to:

o estimate the cancer incidence at the lower doses more likely to occur in the environment.

o estimate the effect of the size and sensitivity differences between the test animals and human beings.

Example of a carcinogenesis bioassay: A carcinogenesis bioassay was performed for benzene on both rats and mice. Both sexes of each species got leukemia at the high doses administered.

Extrapolating the cancer incidence at high dose to low dose and from rodents to humans resulted in the risk estimate that a benzene dose of 1 mg/kg/day will result in 3 cancers per 100 people exposed daily for a lifetime to that dose. This dose is much higher than anyone would be exposed to in the environment under normal conditions.

Mathematical Models Vary

A mathematical model is a set of equations that mimic a real situation and predict what will happen under different circumstances.

In toxicity assessments, scientists do not know what will happen to humans exposed to the low doses found in the environment. So models are developed to apply information gained in animal studies to the human condition. Many educated assumptions must be made in developing these models. *

The choice of model will have a strong influence on the outcome of the toxicity assessment, because when scientists apply different models to identical data, they will get different results.

Non-Threshold vs. Threshold Models

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Public health officials generally rely on non-threshold models --models that make worst-case assumptions.

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Two fundamentally different types of mathematical models are applied to carcinogenic risk assessment. One is called a non-threshold model and is based on the assumption that even one molecule of a cancer-causing agent can lead to the disease. This type of model is also referred to as a "one-hit" model.

The second type of mathematical model is called a threshold model and is based on the premise that repeated exposures to a chemical are needed before a threshold of exposure is reached and cancer follows.

Scientists in regulatory agencies generally use non-threshold models for carcinogenesis bioassays. These assign to a substance a higher estimate of cancer potency than would threshold models. (However, the threshold model is currently used to assess risk of all non-carcinogenic chemicals.)

* These assumptions regard: similarities and differences between animal and human reactions; the effects of genetic, age, health, and other variations in humans; and whether only one molecule or many molecules of a carcinogen are sufficient to start a cancer process under appropriate conditions (see next page). Since not all carcinogens work the same way, a particular type of model may give a fairly realistic risk value for some, but a very unrealistic risk value for others.

Role of Toxicity Assessment

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The risk assessor estimates real world risk by combining information on toxicity and exposure.

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In the end, a toxicity assessment provides information on how much of a chemical causes what kind of harm.

If the toxicity assessment is based on an animal study, the degree of harm to humans must be extrapolated using mathematical models based on a variety of assumptions. Thus the toxicity assessment provides only an estimate of the harm to humans.

As more toxicity studies on a particular chemical are conducted-dose-response studies on different species of animals for example, or epidemiological and in vitro (test tube) studies-scientists become more confident in their characterization of the toxicity of the substance.

The risk assessor's job is to determine the real world risk to humans of a substance by combining information on toxicity and exposure. This job is made more complicated if data are collected from many different studies, but the results will be more likely to reflect the best estimates scientists can make

Ill check the change in size after dilution..thank you very much..Mam 

thank you very much Sudip Sir, for your help .

I found something similar with other nanoparticles. In higher concentrations the particles tend to aggregate. This phenomenon can inhibit the uptake, i.e. toxicity.