How is toxicity testing

Observations over time. For each interval of time, observations must be recorded for: Each of the treatment and control containers The numbers of organisms that are alive and normal The number not doing well The number dead Then apply a statistical procedure to estimate the median concentration of the toxin that maimed or killed half the organisms and write up the results. Quality control against bias Added to all this, the design of a toxicity test must include a number of features to insure there is no bias in the results.

Water quality must be monitored to ensure that temperatures and oxygen remain the same in all containers. Once the data is collected, the researcher must calculate the median lethal concentration, meaning the concentration of toxin that would kill half the test population.

Further, it is important not to rely only on one experiment. The whole thing should be repeated once or twice more to be convinced that the first effort was not a fluke.

Using toxicity data These are the steps scientists go through to determine if a substance is toxic and at what concentration levels. Back to top. Some of them fall in the category of regulatory policy; others involve scientific concerns.

The most commonly cited reason for noncompliance is lack of characterization of the test material. To improve the quality of testing and incorporate new scientific methods in its testing requirements, EPA is currently revising the 40 CFR Part data requirements for food and nonfood use pesticides.

The proposed revisions to these requirements can be found in Table Acute toxicity studies provide information on the potential for health hazards that may arise as result of short-term exposure.

Determination of acute oral, dermal, and inhalation toxicity is usually the initial step in evaluating the toxic characteristics of a pesticide. In each of these tests the animal is exposed to the test material only once on 1 day. Together with information derived from primary eye and primary dermal irritation studies also 1 dose on 1 day , which assess possible hazards resulting from pesticide contact with eyes and skin, these data provide a basis for precautionary labeling and may influence the classification of a pesticide for restricted use.

Acute toxicity data also provide information used to determine the need for child-resistant packaging, for protective clothing requirements for applicator, and for calculation of farm worker reentry intervals.

A minimum number of animals, usually adults, are used in these studies and only the end points of concern are monitored, i. One exception is the inclusion of microscopic examination of neural tissues in the newly required acute neurotoxicity study.

Guideline number EPA, , acute oral study in the rat, would be revised to include special visual system testing, which would be required for all organophosphate pesticide and other pesticides known to affect the visual system. Information on the pilot study and other problems associated with dose level selection. Information missing or requiring clarification of laboratory methods or results.

The additional acute study proposed in guideline number is acute neurotoxicity testing in the rat. This study would be required for all pesticide registrations food and nonfood and experimental use permits EUPs , and it would include assessments of function and activity as well as histopathological microscopic examination of selected neural tissue. EPA presently requires that this study be conducted by manufacturers wishing to reregister.

Subchronic exposures do not elicit effects that have a long latency period e. Subchronic tests also provide information necessary to select proper dose levels for chronic studies, especially for carcinogenicity studies for which an MTD must be selected. According to EPA , rats selected for these studies should be started on the test material shortly after weaning, ''ideally before the rats are 6 and, in any case, not more than 8 weeks old. Most subchronic toxicity studies monitor clinical or behavioral neurological signs of toxicity, body weight, food consumption, eye effects, certain plasma or serum and urine parameters, organ weights, and gross and microscopic pathology.

Clinical and behavioral signs of toxicity are observed and recorded daily. They can consist of activity, gait, excreta, hair coat, and feeding and drinking patterns. Body weight and food consumption data are routinely recorded throughout the study at intervals usually weekly determined by the length of the study.

Ophthalmoscopic examinations are conducted at the beginning of the study and, typically, just before it terminates. The laboratory parameters typically examined are summarized in Table The results of hematology testing indicate whether, for example, the chemical affects blood cell formation and survival, clotting factors, and platelets. Clinical chemistry and urinalysis results can indicate possible kidney, liver, pancreas, and cardiac function or toxicity as well as any electrolyte imbalance.

Urinalysis results can indicate adequacy of kidney, liver, and pancreas function. After necropsy, the weights of certain organs are also recorded. These organs generally include brain, gonads, liver, and kidneys, which are the four required according to EPA testing guidelines EPA, If toxicity is known to occur in another organ from previous testing, the weight of this organ should also be reported. For thyroid toxicity, for example, the weight of the thyroids should be recorded.

Changes from untreated control animals are generally an indication of potential toxicity in this organ. A complete necropsy is performed after sacrifice or death of the test animal. Generally all tissues are examined, and those saved for microscopic examination are aorta, jejunum, peripheral nerve, eyes, bone marrow, kidneys, cecum, liver, esophagus, colon, lung, ovaries, duodenum,.

Required when the product consists of, or under conditions of use will result in, an inhalable material e. Required unless repeated dermal exposure does not occur under conditions of use. Required for uncharged organophosphorus esters, thioesters, or anhydrides of organophosphoric, organophosphonic, or organophosphoramidic acids or of related phosphorothioic, phosphonothioic, or phosphorothioamidic acids, or other substances that may cause the neurotoxicity sometimes seen in this class.

Additional measurements such as cholinesterase determinations for certain pesticides e. The route of exposure should correspond to a primary route of human exposure. Required if intended use of the pesticide is expected to result in human exposure via the oral route. All day subchronic studies can be designed to simultaneously fulfill the requirements of the day neurotoxicity study.

Studies must include additional end points so as to provide an immunotoxicity screen in the rodent. An equivalent independent study may fulfill the requirements for an immunotoxicity screen. In most cases, where the theoretical maximum residue contribution TMRC exceeds 50 percent of the reference dose Rfd , a 1-year or longer interim report on a chronic 2-year feeding study is required to support a temporary tolerance.

This report is to be in addition to the day feeding studies in rodents and nonrodents. If the pesticide is found to leach into groundwater or may contaminate drinking water, a day drinking water study may be required unless data demonstrate that there are no significant differences in toxicity observed when the test material is administered in feed versus when the test material is administered in drinking water.

This study may be requested in addition to any day oral studies that may be required. Required if intended use of the pesticide is expected to result in human exposure via the dermal route and data from a subchronic day dermal toxicity study are not required.

For nonfood uses, a day dermal toxicity study is required, since intended use of the pesticide is expected to result in repeated dermal exposure of humans. For food uses, required if: a the active ingredient of the product is known or expected to be metabolized differently by the dermal route of exposure than by the oral route, and a metabolite of the active ingredient is the toxic moiety; b the active ingredient of the product is classified as toxicity category I or II on the basis of acute dermal toxicity data.

Required if the active ingredient is a gas at room temperature or if use of the product results in respirable droplets and use may result in repeated inhalation exposure at a concentration likely to be toxic, regardless of whether the major route of exposure is inhalation. Required for substances when statistically or biologically significant effects were seen in the acute study Guideline , or if other available data indicate that the substance can cause this type of delayed neurotoxicity.

Required if either of the following criteria is met: a use of the pesticide is likely to result in repeated human exposure over a significant portion of the human life span e. Studies designed to simultaneously fulfill the requirements of both the chronic feeding and carcinogenicity studies i.

Minimum acceptable study durations for chronic feeding and carcinogenicity studies are as follows: chronic rodent feeding study food use pesticide —24 months; chronic rodent feeding study nonfood pesticide —12 months in usually sufficient; chronic nonrodent i.

Required active ingredients or any of their metabolites, degradation products, or impurities are structurally related to a recognized carcinogen, cause mutagenic effects as demonstrated by in vitro or in vivo testing, or produce a morphologic effect in any organ e. The use requires a tolerance for the pesticide or exemption from the requirement to obtain a tolerance or requires the issuance of a food additive regulation.

Use of the pesticide product is likely to result in exposure of humans over a portion of the life span that is significant in terms of either the timing or duration of exposure e. Range-finding studies of at least 90 days duration in rats and mice are generally required to determine dose levels adequate to demonstrate an MTD in carcinogenicity studies. A subchronic day oral study conducted in accordance with Guideline may also be acceptable for this purpose.

Testing in two species is required for food uses. For products intended for nonfood uses, testing in two species is required if significant exposure of human females of child-bearing age may reasonably be expected.

For other nonfood uses, testing in at least one species is required. A study in one species is required to support a temporary tolerance. Testing in a second species is required if significant developmental toxicity is observed after testing in the first species. The test substance or vehicle is usually administered by oral intubation, unless the chemical or physical characteristics of the test substance or pattern of human exposure suggest a more appropriate route of administration.

Under certain conditions where a pesticide is determined to be a developmental toxicant e. Required to support products intended for food and nonfood uses if the use is likely to result in exposure of humans over a portion of the life span that is significant in terms of the frequency, magnitude, or duration of exposure e. Conditionally required to more fully assess any of the manifestations of developmental toxicity.

These studies permit assessment of potential functional deficits that cannot be evaluated in the classical developmental toxicity study Guideline Protocols for these studies are usually designed on a case-by-case basis.

For this type of postnatal study, a guideline is available. An initial battery of mutagenicity tests with possible confirmatory testing is minimally required. Also, results from other mutagenicity tests that may have been performed and as complete a reference list as possible shall be submitted. Subsequent testing may or may not be required based on the evidence available to EPA's Office of Pesticide Programs in accordance with the objective and considerations for mutagenicity testing.

Because of the rapid improvements in the field, applicants are encouraged to discuss test selection, protocol design, and results of preliminary testing with the agency. Choice of assays using either mouse lymphoma LY cells, thymidine kinase tK gene locus, maximizing assay conditions for small colony expression and detection; Chinese hamster ovary CHO or Chinese hamster lung fibroblast V79 cells, hypoxanthine-guanine phosphoribosyl transferase hgprt gene locus, accompanied by an appropriate in vitro test for clastogenicity; or CHO cells strain AS52, xanthine-guanine phosphoribosyl transferase xprt gene locus.

Choice of assays; initial consideration usually given to rodent bone marrow, using either metaphase analysis aberrations or micronucleus assay. Also may be required if significant adverse effects are observed in toxicology studies e.

May be required, on a case-by-case basis, to support registration of an end-use product if cats, dogs, cattle, pigs, sheep, horses, or other domesticated animals will be exposed to the pesticide product, including, but not limited to, exposure through direct application for pest control and consumption of treated feed.

Dermal penetration studies are required for compounds that have serious toxic effects, as identified in oral or inhalation studies, and for which a significant route of human exposure is dermal. Thus, this study is required when any of the following exposure studies are required: passive dosimetry—dermal exposure Guidelines , or , foliar dislodgeable residue dissipation Guideline , soil dislodgeable residue dissipation Guideline , and indoor surface residue dissipation, unless the toxicity studies including Guidelines , , , and that triggered the need for these exposure studies were conducted via the dermal route of dosing.

Registrants should work closely with the agency in developing an acceptable protocol for performing dermal penetration studies. Registrants should consult with the agency for development of protocols and methodology prior to initiation of studies. Testing of the end-use product dilution is required if it can be reasonably anticipated that the results of such testing may meet the criteria for restriction to use by certified applicators specified in 40 CFR The data described above are not required for all subchronic studies.

For the day dermal study, for example, only limited necropsy data are required. For the day oral study Guideline ; EPA, in the rodent and other test species, three changes are proposed:. A day study would be required for all food uses, except when acute dermal toxicity is observed. A day dermal study would ordinarily be required for all nonfood uses. Special tests for neurotoxicity or immunotoxicity could be added to these studies if these toxicity end points are not studied in other tests required for a particular pesticide.

A day inhalation study Guideline ; EPA, would be required more frequently whether or not the major route of exposure is inhalation, especially for a nonfood use pesticide that is a gas or whose use generates respirable droplets.

The requirement for a day inhalation study for a tobacco use pesticide would be deleted. Special tests for neurotoxicity or immunotoxicity could be added if those end points are not studied in other EPA-required toxicity studies. The conditionally required day neurotoxicity study in the hen or mammal Guideline ; EPA, would be replaced by two new studies:.

Testing would include assessments of function functional observation battery , motor activity, and histopathological examination of the nervous system. Information derived from chronic studies is used to assess potential hazards resulting from prolonged and repeated exposure to a pesticide over a large portion of the human life span. These studies usually last 12 to 24 months. Of particular importance are long-term carcinogenicity studies, the purpose of which is to observe the test animals for the development of neoplastic lesions after a lifetime of exposure at dose levels up to and including the MTD determined from subchronic testing.

The emphasis of the carcinogenicity study is the detection of tumors in animals. For these studies, both concurrent and historical control data are used to evaluate the relevance of tumors. Historical control data should.

Carcinogenicity studies should be 24 months long in rats and 18 months long in mice. The age of test animals in carcinogenicity rat and mouse studies and other chronic rat and dog studies is determined by the same criteria as for subchronic toxicity studies. The parameters to be examined in carcinogenicity studies are also generally the same as those discussed above for subchronic and chronic studies, except that clinical chemistry and urine parameters are not required and only limited hematology data are required.

Modifications to chronic feeding studies in two species rodent and nonrodent; Guideline ; EPA, may be required to include additional end points for neurotoxicity or immunotoxicity or special visual system toxicity for organophosphates if these were not tested in other studies.

Range-finding studies of at least 90 days duration in rats and mice will generally be required to determine dose levels that are adequate to test the carcinogenicity Guideline ; EPA, of a pesticide. Studies conducted to satisfy the requirement for Guideline EPA, will also be acceptable to satisfy this day study requirement. Developmental toxicity studies are designed to assess the potential of developmental effects in offspring resulting from the mother's exposure to the test substance during pregnancy.

For the second part of the study, the committee was asked to build on the work presented in this report and develop a long-range vision and strategic plan to advance the practices of toxicity testing and human health assessment of environmental contaminants. The second report is expected to be completed by fall To accomplish the task of preparing its first report, the committee held four meetings.

The first three, held from June to November , included public sessions. In Chapter 2 , the committee presents an overview of consensus-study protocols focusing primarily on EPA guidelines. The objective of that chapter is not to detail each type of testing protocol but rather to indicate the general types of whole-animal and in vitro toxicity testing now in use. Chapter 3 considers a variety of human-based studies ranging from clinical trials to epidemiologic studies.

Challenges that have often prevented the use of epidemiologic studies in regulatory risk assessment and possible advances and improvements in epidemiology are discussed. Chapter 4 examines applications of toxicity tests in testing strategies that are used to rank, screen, or characterize chemical toxicity.

Several examples of testing strategies are presented. The strategies identified are not meant to be exhaustive but to illustrate the array of toxicity tests that may be required under different circumstances. Chapter 5 provides an overview of risk-assessment guideline documents that deal with the use of toxicity data in human health risk assessment and concludes with observations regarding strengths and weaknesses of the current system for generating toxicologic data to assess environmental risks.

Chapter 7 discusses alternatives to animal testing and a few emerging technologies, such as -omics approaches and computational toxicology. It concludes with a discussion of validation to emphasize the importance of evaluating new toxicity-testing methods to ensure that the information obtained from them is at least as good as, if not better than, conventional mammalian models.

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