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Pesticides in the Diets of Infants and Children (1993)

Chapter: 4 METHODS FOR TOXICITY TESTING

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Suggested Citation:"4 METHODS FOR TOXICITY TESTING." National Research Council. 1993. Pesticides in the Diets of Infants and Children. Washington, DC: The National Academies Press. doi: 10.17226/2126.
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4
Methods for Toxicity Testing

THE PURPOSE OF THIS CHAPTER is to familiarize the reader with the testing that is currently conducted by a manufacturer prior to and during the process of submitting a petition to register a pesticide. Codified toxicologic evaluation of potential pesticides has been a requirement in the United States for approximately 50 years. The testing requirements and guidelines continue to evolve based on new science. This chapter identifies the current testing that is pertinent to the young animal and young human as well as aspects of testing that are needed to fill the data gaps to better  ensure the protection of infants and children. The current testing guidelines can be found in Pesticide Assessment Guidelines issued by the Environmental Protection Agency (EPA, 1991a,b).

Data, including those derived from toxicity testing, crop residue analyses, environmental fate testing, and ecotoxicology testing, are generated by the manufacturer of a pesticide to meet the mandatory requirements of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) for pesticide registration. Although these data are essential to the EPA's registration process, other data generated by EPA itself, as well as by other government institutions and academia, are considered in the registration decision-making process.

EPA has issued 194 registration standards on 350 chemicals used as active and inert ingredients in pesticide products. These standards are published by EPA and are intended to upgrade and update the data base on a previously registered pesticide or class of pesticide products. They call for additional studies in the areas of toxicity testing, crop residue analyses, environmental fate, and ecotoxicology testing. This testing must be conducted within an EPA-mandated time frame to allow for the continued

Suggested Citation:"4 METHODS FOR TOXICITY TESTING." National Research Council. 1993. Pesticides in the Diets of Infants and Children. Washington, DC: The National Academies Press. doi: 10.17226/2126.
×

TABLE 4-1 End Points for Various Toxicity Studies

Study

End Points

Developmental toxicity

Fetus: mortality, growth retardation, skeletal variations, gross external malformations, soft tissue/internal organ defects

Female parent: general toxicity

Reproductive Toxicity

Male parent: general toxicity, effects  on fertility, reproductive organ changes

Offspring: effects on viability, sex ratio, growth, behavior

Carcinogenicity

Tumor development and general toxicity

Neurotoxicity

Behavior, function, and motor activity deficits; microscopic nervous tissue  changes

Mutagenicity

Heritable lesions leading to altered phenotypes

 

SOURCE: EPA, 1984

registration of given product. The list of pesticides for which registration standards have been issued is referred to as List A and can be found in Appendix I of the Federal Register notice of February 22, 1989. Under the FIFRA Amendments of 1988, the data bases on the remaining registered pesticide products are being upgraded in five phases over a 9-year period.

The first sections of this chapter describe in detail the present toxicity testing procedures for pesticides in relation to their registered use patterns and EPA's proposed changes or additions to these procedures. The conclusions and recommendation of the committee for further changes and additions to the toxicity testing battery to allow for more adequate consideration of the special testing needs for infants and children are presented.

CURRENT METHODS: GENERAL CONSIDERATIONS

Toxicity studies are required to assess potential hazards to humans through the acute, subchronic, and chronic exposure of laboratory animals to pesticides. The more specific types of toxicity that are determined include carcinogenicity; developmental (including teratogenicity in offspring) and reproductive toxicity; mutagenicity; and neurotoxicity (Table 4-1). Detailed information on the metabolism or biotransformation of the pesticide is also obtained. Consideration is given to testing individual metabolites in animals, and in or on pesticide-treated plants to which humans could exposed through their diet. The extent of metabolite testing required depends on the level of potential toxicity and environmental persistence of the metabolite. With the exception of

Suggested Citation:"4 METHODS FOR TOXICITY TESTING." National Research Council. 1993. Pesticides in the Diets of Infants and Children. Washington, DC: The National Academies Press. doi: 10.17226/2126.
×

the acute toxicity tests, most tests are conducted to determine the nature of any toxicity that can be produced by repeatedly dosing animals over an extended period. The results enable toxicologists to estimate the safety of a material of humans (Loomis, 1978).

Weil (1972) published the following set of guidelines, which reflected a consensus among toxicologists. These should be considered before initiating a toxicity test:

  1. Use, wherever practical or possible, one or more species that biologically handle the material qualitatively and/or quantitatively as similarly as possible to man. For this, metabolism, absorption, excretion, storage and other physiological effects might be considered.

  2. Where practical, use several dose levels on the principle that all types of toxicologic and pharmacologic actions in man and animals are dose-related. The only exception to this should be the use of a single, maximum dosage level if the material is relatively nontoxic; this level should be a sufficiently large multiple of that which is attainable by the applicable hazard exposure route, and should not be physiologically impractical.

  3. Effects produced at higher dose levels (within the practical limits discussed in 2) are useful for delineating mechanism of action, but for any material effect, some dose level exists for man or animal below which this adverse effect will not appear. This biologically insignificant level can and should be set by use of a proper uncertainty factor and competent scientific judgment.…

  4. Statistical tests for significance are valid only on the experimental units (e.g., either litters or individuals) that have been mathematically randomized among the dosed and concurrent control groups….

  5. Effects obtained by one route of administration to test animals are not a priori applicable to effects by another route of administration to man. The routes chosen for administration  to test animals should, therefore, be the same as those to which man will be exposed. Thus, for example, food additives for man should be tested by admixture of the material in the diet of animals.

In general, Weil's guidelines are considered by EPA in its toxicity testing requirements and subsequent evaluation of results for pesticides. One exception to Weil's points is found in his guideline 3. EPA does not recognize the existence of a dose level at which a carcinogen will not exert its effect. For carcinogens, EPA generally accepts a risk of 10-6, as extrapolated from bioassays using the nonthreshold modification of the linearized multistage model of Armitage and Doll (1954), as adequate for the protection of humans.

The selection of animal species for toxicity tests depends on life span,

Suggested Citation:"4 METHODS FOR TOXICITY TESTING." National Research Council. 1993. Pesticides in the Diets of Infants and Children. Washington, DC: The National Academies Press. doi: 10.17226/2126.
×

behavior, availability, and overall costs. EPA recommends using rats for subchronic, chronic, carcinogenicity, and reproduction studies; mice for carcinogenicity studies; and dogs for subchronic and chronic studies. Rats are routinely used for acute oral and inhalation studies and rabbits for eye and skin irritation studies and acute dermal studies. One exception to this is the use of guinea pigs for dermal sensitization testing. The rat and rabbit are recommended for developmental toxicity (teratogenicity) testing. Justification must be provided for the use of species other than those outlined above.

The number of animals to be tested in each dose group depends on a number of factors, including the purpose of the experiment, the required sensitivity of the study, the reproductive capacity and the fertility of the species, economic aspects, and the availability of animals (IPCS, 1990). Table 4-2 lists the minimum number of animals required by EPA for some toxicity studies. For the most part, these numbers are consistent with those recommended by the International Program Chemical Safety (IPCS).

The selection of dose levels for subchronic studies should be based on the results of acute toxicity testing, on range-finding studies, and on pharmacokinetic (metabolism, including rate in various tissues) data. For subchronic studies, four dose groups of animals should be included: a control group; a low-dose group (a dose that produces no compound related toxicity); a mid-dose group (a dose that elicits some minimal signs of toxicity); and a high-dose group (a dose that results in toxic effects but not in an incidence of fatalities that would prevent a meaningful evaluation; for nonrodents, there should be no fatalities) (EPA, 1984). This same guidance is relevant to chronic toxicity and reproduction studies. For teratology studies, the highest dose tested should elicit some signs of maternal toxicity, but the toxicity should not obscure the results.

The one notable exception to this guidance pertains to carcinogenicity studies. The highest dose levels for these studies should be at a maximum tolerated dose (MTD), as determined in 90-day toxicity studies in the appropriate test species and from pharmacokinetic information on the material being tested. The Committee on Risk Assessment Methodology of the National Research Council (NRC) recently examined the criteria for the MTD and other doses used in carcinogenicity studies (NRC, 1993). The EPA has issued its own guidance for the selection of this dose level. Some of the factors to consider in selecting an MTD are: 10% decrement in body weight gain in 90-day study; observation of potential life-threatening lesions during microscopic examination of organs, e.g., liver necrosis; significant inhibition of cholinesterase activity in two biological compartments, such as brain and plasma; and significant signs of anemia or other biologically relevant effects on blood.

Suggested Citation:"4 METHODS FOR TOXICITY TESTING." National Research Council. 1993. Pesticides in the Diets of Infants and Children. Washington, DC: The National Academies Press. doi: 10.17226/2126.
×

TABLE 4-2  Animal Model Requirements in Toxicity Studies

 

Minimum No. of Animals Required

Study Type

Males

Females

Dosing

Age at Start of Study

Acute oral (rat), dermal, or inhalation (rat)

5

5

Single

Young adult

Eye and skin irritation (rabbit)

6a

 

Single

Young adult

Dermal sensitization (guinea pig)

b

 

Repeated

Young adult

21-Day dermal (rat, rabbit, or guinea pig)

5

5

Repeated

Rat, 200–300 g; rabbit, 2.0–3.0 kg; guinea pig, 350–450 g

90-Day oral (rat)

10

10

Repeated

6–8 weeks

90-Day inhalation (rat)

10

10

Repeated

Young adult

90-Day dermal (rat, rabbit, or guinea pig)

10

10

Repeated

Rat, 200–300 g; rabbit, 2.0–3.0 kg; guinea pig, 350–450 g

90-Day or chronic (1 ear) oral (dog)

4

4

Repeated

4–6 months

Reproduction (rat)c

20

20

Repeated

8 weeks

Teratology

Rat

 

20d

Repeated

Young adult

Rabbit

 

12d

Repeated

Young adult

Chronic toxicity (1 or 2 year) (rat)

20

20

Repeated

6–8 weeks

Oncogenicity (lifetime) (rat and mouse)

50e

50e

Repeated

6–8 weeks

a Either males or females may be used in this test.

b The number of animals used depends on the method used. Several different experimental methods are acceptable.

c EPA prefers that one male rat be housed with one female during mating.

d Number of pregnant females required.

e 50 rats and 50 mice of each sex.

SOURCE: EPA, 1984.

In general, EPA has set a cap on dosing of 1.0 g/kg/day for toxicity tests other than acute studies. This dose level is referred to as the limit dose and corresponds to approximately 20,000 ppm in the diet of rats, 7,000 ppm in the diet of mice, and 40,000 ppm in the diet of dogs.

The duration of exposure for toxicity testing of a pesticide depends on the expected duration of human exposure to the pesticide in practice. The typical length of various toxicity tests and the number of doses administered are shown in Table 4-2. Repeated dosing refers to dosing once per day for the designated number of days. When the material is

Suggested Citation:"4 METHODS FOR TOXICITY TESTING." National Research Council. 1993. Pesticides in the Diets of Infants and Children. Washington, DC: The National Academies Press. doi: 10.17226/2126.
×

given to the test animals in their diet, dosing is usually continuous for 7 days a week. If the material is administered by gavage (oral bolus dose), by dermal application, or by inhalation, doses are frequently given 5 days a week, which is acceptable to EPA because of practical considerations (EPA, 1984).

The type of statistical analysis performed on the toxicity data resulting from these studies depends on the type of data under consideration (see, for example, Gad and Weil, 1982, for review). Interpreting the meaning of statistical significance for any particular parameter depends on the dose level at which it was achieved, the biological significance of the finding, and the normal spontaneous occurrence of this finding in the strain and species being tested.

For regulatory purposes, the no-observed-effect level (NOEL) is defined as a dose level at which no effects attributable to the pesticide under test can be found. A no-observed-adverse-effect level (NOAEL) can also be determined for each study; however, EPA does not routinely use the NOAEL to regulate pesticide usage. To establish a NOAEL, the toxicologist must determine what is and what is not adverse effect, which can be defined differently by different scientists. For example, effects such as hair loss can be considered adverse by some and not by others. Plasma and red blood cell cholinesterase inhibition can be viewed as either an adverse effect or simply as a market of exposure to a pesticide.

EPA uses the NOEL to calculate the acceptable daily intake (ADI) of the pesticide under consideration. More recently, the EPA has replaced the ADI with the reference dose, or RfD. Chronic studies, such as reproduction studies and lasting 1 year or longer in the rat or dog are used for this purpose. EPA does not routinely use the NOEL determined from teratology (developmental toxicity) studies for calculating ADIs because the observed effect are not considered chronic; however, these NOELs can be used to support the calculated ADI. EPA does routinely use developmental toxicity NOELs for other types of risk assessments, such as calculating the risk from acute, daily dietary or occupational exposure or from exposure of homeowners to a developmental toxicant.

EPA's toxicity testing requirements for food and nonfood use pesticides have been published in 40 CFR Part 158. In general, for food use chemical with maximum human exposure, the following toxicity tests are required:

• acute oral toxicity

• acute dermal toxicity

• acute inhalation toxicity

• primary eye irritation

• primary dermal irritation

• chronic feeding toxicity

• dermal sensitization

• acute neurotoxicity

• 90-day toxicity

• 21-day dermal toxicity

• 90-day neurotoxicity study

• reproduction study

Suggested Citation:"4 METHODS FOR TOXICITY TESTING." National Research Council. 1993. Pesticides in the Diets of Infants and Children. Washington, DC: The National Academies Press. doi: 10.17226/2126.
×

• carcinogenicity

• developmental toxicity

• mutagenicity tests

• general metabolism study

More than 30% of the tests for pesticides submitted to EPA in the past have been rejected. Those rejected must be resubmitted until they are in conformance with EPA criteria before registrations can be granted. The criteria for rejection are summarized in Table 4-3. 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 158 data requirements for food and nonfood use pesticides. The proposed revisions to these requirements can be found in Table 4-4.

ACUTE TOXICITY STUDIES

General Description

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.e., mortality, observable skin or eye effects, dermal sensitization, and observable neurotoxic behavioral changes. One exception is the inclusion of microscopic examination of neural tissues in the newly required acute neurotoxicity study.

EPA's Proposed Changes

Guideline number 81-1 (EPA, 1984), 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.

Suggested Citation:"4 METHODS FOR TOXICITY TESTING." National Research Council. 1993. Pesticides in the Diets of Infants and Children. Washington, DC: The National Academies Press. doi: 10.17226/2126.
×

TABLE 4-3 Summary of EPA Rejection Factors

Guideline

Rejection Factor

Acute Oral Toxicity (81-1)

Lack of characterization of the test material

Inadequate dose levels to calculate LD50

Acute Dermal Toxicity (81-2)

Lack of characterization of the test material

Inadequate percentage of body surface area exposed

No quality assurance statement

Improper number of animals tested per dose group

Only one sex tested

Omitted source, age, weight, or strain of test animal

Acute and 90-Day Inhalation (81-3 and 82-4)

Less than 25% of particles were <1 µm; LC50 could not be calculated; highest concentration did not produce toxicity

Inadequate reporting of exposure methodology

Protocol errors

Lack of characterization of the test material

Compound preparation

Chamber concentration not measured

Primary Eye Irritation (81-5)

Lack of characterization of the test material

Primary Dermal Irritation (81-5)

Lack of characterization of the test material

No quality assurance statement and/or no Good Laboratory Practice (GLP) statement

Improper test material application/preparation

Omitted source, age, weight, or strain of test animal

Missing individual/summary animal data

Dermal Sensitization (81-6)

Control problems

Dosing level problems

Lack of characterization of the test material

Unacceptable protocol or other protocol problems

Individual animal scorers or data missing

Scoring method or other scoring problem

Reporting deficiencies or no quality assurance statement

90-Day Feeding—Rodent (82-1(a))

A NOEL was not established

Lack of characterization of the test material or incorrectly reported

Lack of clinical chemistry and/or lack of histopathology

90-Day Feeding—Nonrodents (82-1(b))

Reporting deficiencies

Lack of characterization of the test material

A NOEL was not established

An investigation parameter missing

Suggested Citation:"4 METHODS FOR TOXICITY TESTING." National Research Council. 1993. Pesticides in the Diets of Infants and Children. Washington, DC: The National Academies Press. doi: 10.17226/2126.
×

Guideline

Rejection Factor

90-Day Feeding—Nonrodents (cont.) (82-1(b))

Information on the pilot study and other problems associated with dose level selection

An investigational parameter missing

Information on the pilot study and other problems associated with dose level selection

21-Day Dermal Toxicity (82-2)

Lack of characterization of the test material

Raw data analyses incomplete or missing

A systemic NOEL was not established

Inadequate percentage of body surface area exposed in each dose group

Insufficient number of dose levels tested

90-Day Dermal Toxicity (82-3)

Lack of characterization of the test material

A systemic NOEL was not established

Incomplete/missing raw animal data analyses

Insufficient number of dose levels tested

Poorly controlled test environment

Chronic Feeding/

Carcinogenicity—Rats (82-3(a) and (83-2(a))

Missing histopathology information

Missing information in study reports

MTD was not achieved

Missing historical control data

Lack of characterization of the test material

Deficiencies in reporting the study data

Carcinogenicity—Mice (83-2(b))

Histopathology information missing

MTD was not achieved

Lack of historical control data

Information missing in study reports

Lack of characterization of the test material

Deficiencies in reporting of study data

Developmental Toxicity—Rodents (83-3(a))

Missing historical controls

Lack of characterization of the test material

Information missing or requiring clarification of the laboratories' methods

Information missing or requiring clarification of the laboratories' results

A NOEL was not established

Statistical problems

Did not use conventional assessments for skeletal or visceral examinations

Developmental Toxicity—Nonrodents (83-3(b))

Clarification of laboratory procedures of interpretation of the data

Individual maternal or fetal data missing

Missing historical controls

Lack of characterization of the test material

Excessive maternal toxicity

Suggested Citation:"4 METHODS FOR TOXICITY TESTING." National Research Council. 1993. Pesticides in the Diets of Infants and Children. Washington, DC: The National Academies Press. doi: 10.17226/2126.
×

Guideline

Rejection factor

Developmental Toxicity—Nonrodents (cont.) (83-3(b))

A NOEL was not established

Statistical problems

Reproduction (83-4)

Information missing from laboratory results

Lack of characterization of the test material

Information missing or requiring clarification of laboratory methods or results

Missing historical controls

A NOEL was not established due to effects at the lowest dose tested

Low fertility and/or inadequate number of animals were used per dose level

A NOEL was not established  in the absence of reproductive effects

Metabolism (85-1)

Inadequate or missing data on identification of metabolites

Improper methodology or dosing regimen

Inadequate number of animals were used in the dose groups

No individual animal data

Improper reporting

Inadequate or missing tissue residue analysis data

Testing at only one dose level

Only one sex of animal used

Lack of an intravenous dose group

No collection of 14 CO2

Dermal Penetration (85-2)

Incomplete/missing data evaluation

Improper test material preparation/application

Raw data missing and incomplete summary tables

No signed quality assurance statement

Missing purity or concentration of test material

 

SOURCE: P. Fenner-Crisp, EPA, personal communication, 1992

The additional acute study proposed in guideline number 81-4 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.

Suggested Citation:"4 METHODS FOR TOXICITY TESTING." National Research Council. 1993. Pesticides in the Diets of Infants and Children. Washington, DC: The National Academies Press. doi: 10.17226/2126.
×

SUBCHRONIC TOXICITY STUDIES

General Description

Subchronic exposures do not elicit effects that have a long latency period (e.g., carcinogenicity). However, they do provide information on health hazards that may result from repeated exposures to a pesticide over a period up to approximately 30% of the lifetime of a rodent. 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 (1984), 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." For dogs, dosing should begin when they are 4 to 6 months of age and "not later than 9 months of age."

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 4-5.

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, 1984). 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,

Suggested Citation:"4 METHODS FOR TOXICITY TESTING." National Research Council. 1993. Pesticides in the Diets of Infants and Children. Washington, DC: The National Academies Press. doi: 10.17226/2126.
×

TABLE 4-4 Toxicity Data Requirements Proposed by EPA for Food and Nonfood Uses of Pesticides

 

 

 

Data Requirements

Test Substance Data to Support

Kinds of Data Required

EPA Guideline Number

Comments (see Corresponding Numbers Beginning on Page 00)

Food Usesa

Nonfood Usesb

Manufacturing-Use Product

End-Use Product

Acute Testing

Acute oral toxicity—rat

81-1

1, 36, 37

[R]c

[R]

MP and TGAId

TGAI, EP,e and possibly EP dilution

Acute dermal toxicity—rabbit rat, or guinea pig

81-2

1, 2, 37

[R]

[R]

MP and TGAI

TGAI, EP, and possibly EP dilution

Acute inhalation toxicity—rat

81-3

3

[R]

[R]

MP and TGAI

EP and TGAI

Primary eye irritation—rabbit

81-4

2

[R]

[R]

MP

EP

Primary dermal irritation—rabbit

81-5

1, 2

[R]

[R]

MP

EP

Dermal sensitization—guinea pig

81-6

4

[R]

[R]

MP

EP

Delayed neurotoxicity (acute)—hen

81-7

5

[CR]f

[CR]

TGAI

TGAI

Acute neurotoxicity—rat

81-8

6

[R]

[R]

TGAI

TGAI

Subchronic Testing

90-day oral—two species, rodent and nonrodent

82-1

7, 8, 9, 10, 11, 36

[R]

CR

TGAI

TGAI

21-day dermal—rat, rabbit, or guinea pig

82-2

9, 12

R

None

TGAI

TGAI

90-day dermal—rat, rabbit, or guinea pig

82-3

8, 9, 13, 14

CR

R

TGAI

TGAI

90-day inhalation—rat

82-4

8, 9, 15

CR

CR

TGAI

TGAI

28-day delayed neurotoxicity—hen

82-6

16

CR

CR

TGAI

TGAI

90-day neurotoxicity—rat

82-7

6, 8

[R]

R

TGAI

TGAI

Suggested Citation:"4 METHODS FOR TOXICITY TESTING." National Research Council. 1993. Pesticides in the Diets of Infants and Children. Washington, DC: The National Academies Press. doi: 10.17226/2126.
×

Chronic Testing

Chronic feeding—two species: rodent and nonrodent

83-1

9, 10, 17, 18, 19, 36

[R]

CR

TGAI

TGAI

Carcinogenicity—two species: rat and mouse preferred

83-2

9, 19, 20, 21

R

CR

TGAI

TGAI

Developmental Toxicity and Reproduction

Developmental toxicity—two species: rat and rabbit preferred

83-3

22, 23, 24, 25

[R]

R

TGAI

TGAI

Reproduction—rat

83-4

26, 27

[R]

CR

TGAI

TGAI

Postnatal developmental toxicity—rat and/or rabbit

83-6

28, 29

CR

CR

TGAI

TGAI

Mutagenicity Testing

Salmonella typhimurium reverse mutation assay

84-2

30

[R]

R

TGAI

TGAI

Mammalian cells in culture

84-2

30, 31

[R]

R

TGAI

TGAI

In vivo cytogenetics

84-2

30, 32

[R]

R

TGAI

TGAI

General Metabolism—rat

85-1

33

R

CR

PAI or PAIRAg

PAI or PAIRA

Special Testing

Domestic animal safety

85-2

34

CR

CR

Choiceh

Choice

Dermal penetration

85-3

35

CR

CR

Choice

Choice

Visual system studies

85-4

36

[CR]

[CR]

TGAI

TGAI

a Food use includes terrestrial food and feed, aquatic food, greenhouse food, and indoor food.

b Nonfood use includes terrestrial nonfood, aquatic nonfood outdoor, aquatic nonfood industrial, aquatic nonfood residential, greenhouse nonfood, forestry, residential outdoor, indoor nonfood, indoor medical, and indoor residential.

c R = required; brackets [] indicate data requirements that apply when an experimental use permit is being sought.

d MP = manufacturing-use product; TGAI = technical grade of the active ingredient.

e EP = end-use product.

f CR = conditionally required.

g PAI = pure active ingredient; PAIRA = pure active ingredient, radiolabeled.

h Choice of several substances, depending on studies required.

Suggested Citation:"4 METHODS FOR TOXICITY TESTING." National Research Council. 1993. Pesticides in the Diets of Infants and Children. Washington, DC: The National Academies Press. doi: 10.17226/2126.
×

Notes for Table 4-4: Specific Conditions, Qualifications, or Exceptions to the Designated Test Procedures

1. Not required if test material is a gas or highly volatile.

2. Not required if test material is corrosive to skin or has pH <2 or >11.5; such a product will be classified as toxicity category 1 on the basis of potential eye and dermal irritation effects.

3. Required when the product consists of, or under conditions of use will result in, an inhalable material (e.g., gas, volatile substances, or aerosol/particulate).

4. Required unless repeated dermal exposure does not occur under conditions of use.

5. 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.

6. Additional measurements such as cholinesterase determinations for certain pesticides (e.g., organophosphates and carbamates) may also be required. The route of exposure should correspond to a primary route of human exposure.

7. Required if intended use of the pesticide is expected to result in human exposure via the oral route.

8. All 90-day subchronic studies can be designed to simultaneously fulfill the requirements of the 90-day neurotoxicity study.

9. 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.

10. 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 90-day feeding studies in rodents and nonrodents.

11. If the pesticide is found to leach into groundwater or may contaminate drinking water, a 90-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 90-day oral studies that may be required.

12. Required if intended use of the pesticide is expected to result in human exposure via the dermal route and data from a subchronic 90-day dermal toxicity study are not required.

13. For nonfood uses, a 90-day dermal toxicity study is required, since intended use of the pesticide is expected to result in repeated dermal exposure of humans.

Suggested Citation:"4 METHODS FOR TOXICITY TESTING." National Research Council. 1993. Pesticides in the Diets of Infants and Children. Washington, DC: The National Academies Press. doi: 10.17226/2126.
×

14. 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.

15. 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

16. Required for substances when statistically or biologically significant effects were seen in the acute study (Guideline 81-7), or if other available data indicate that the substance can cause this type of delayed neurotoxicity.

17. 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.g., products intended for use in and around residences, swimming pools, and enclosed working spaces or their immediate vicinity); (b) the use requires a tolerance for the pesticide or an exemption from the requirement to obtain a tolerance for the pesticide or an exemption from the requirement to obtain a tolerance, or requires issuance of a food additive regulation.

18. Based on acute and subchronic neurotoxicity studies, and/or on other available data, a functional observational battery, an assessment of motor activity, and perfusion neuropathology may be required.

19. Studies designed to simultaneously fulfill the requirements of both the chronic feeding and carcinogenicity studies (i.e., a combined study) may be conducted. 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.e. dog) feeding study—12 months; mouse carcinogenicity study-18 months; and rat carcinogenicity study—24 months.

20. 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.g., hyperplasia, metaplasia) in subchronic studies that may lead to neoplastic change. 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.g., pesticides used in treated fabrics for wearing apparel, diapers, or bedding; insect repellents applied directly to the skin; swimming pool additives; or constant-release indoor aerosol pesticides).

21. 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 90-day oral study conducted in accordance with Guideline 82-1 may also be acceptable for this purpose.

Suggested Citation:"4 METHODS FOR TOXICITY TESTING." National Research Council. 1993. Pesticides in the Diets of Infants and Children. Washington, DC: The National Academies Press. doi: 10.17226/2126.
×

22. 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.

23. Testing in a second species is required if significant developmental toxicity is observed after testing in the first species.

24. 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.

25. Under certain conditions where a pesticide is determined to be a developmental toxicant (e.g., after oral dosing), additional testing via other routes (e.g., dermal) may be required.

26. 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.g., pesticides used in treated fabrics for wearing apparel, diapers, or bedding; insect repellents applied directly to the skin; swimming pool additives; or constant-release indoor pesticides used in aerosol form). Also may be required for nonfood uses if adverse effects on organs of the reproductive system are observed in 90-day or other studies, and/or if developmental toxicity is demonstrated by available data (Guideline 83-3).

27. In most cases, where the TMRC exceeds 50% of the RfD, a first-generation (or longer) interim report on a multigeneration reproduction study is required to support a temporary tolerance.

28. 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 83-3). Protocols for these studies are usually designed on a case-by-case basis.

29. On the basis of acute and subchronic neurotoxicity studies, and/or on other available data, a developmental neurotoxicity study may be required. For this type of postnatal study, a guideline is available.

30. 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. Current protocols for tests in the initial battery and other mutagenicity tests are given in the EPA's Office of Pesticides and Toxic Substances Health Effects Testing Guidelines (40 CFR Part 798, Subpart F—Genetic Toxicity). 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.

Suggested Citation:"4 METHODS FOR TOXICITY TESTING." National Research Council. 1993. Pesticides in the Diets of Infants and Children. Washington, DC: The National Academies Press. doi: 10.17226/2126.
×

31. Choice of assays using either mouse lymphoma L5178Y 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.

32. Choice of assays; initial consideration usually given to rodent bone marrow, using either metaphase analysis (aberrations) or micronucleus assay.

33. Required for all food uses and when chronic and/or carcinogenicity studies are required. Also may be required if significant adverse effects are observed in toxicology studies (e.g., reproduction and developmental toxicity).

34. 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.

35. 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 133-3, 231 or 233), foliar dislodgeable residue dissipation (Guideline 132-1), soil dislodgeable residue dissipation (Guideline 132-1), and indoor surface residue dissipation, unless the toxicity studies (including Guidelines 82-6, 82-7 83-1, 83-2 83-3, 83-4 and 83-6) 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.

36. Special testing (acute, subchronic, and/or chronic) is required for organophosphates, and may be required for other cholinesterase inhibitors and other pesticides that have demonstrated a potential to adversely affect the visual system. Registrants should consult with the agency for development of protocols and methodology prior to initiation of studies.

37. 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 152.170(b) or the criteria for initiation of special review specified in 40 CFR 154.7(a)(1).

SOURCE: Code of Federal Regulations, Title 40, Parts 150 to 189, 11992.

Suggested Citation:"4 METHODS FOR TOXICITY TESTING." National Research Council. 1993. Pesticides in the Diets of Infants and Children. Washington, DC: The National Academies Press. doi: 10.17226/2126.
×

TABLE 4-5 Laboratory Parameters Measured in Various Data Categories

Data Type

Parameter Measured

Hematology

Erythrocyte count

Hemoglobin

Hematocrit

Leukocyte count

Differential count

Platelet count (or clotting parameter)

Clinical chemistry

Alkaline phosphatase

Alanine aminotransferase

Creatinine kinase

Lactic dehydrogenase

Glucose

Bilirubin

Cholesterol

Creatinine

Aspartate aminotransferase

Total protein

Albumin

Urea nitrogen

Inorganic phosphate

Calcium

Potassium

Sodium

Chloride

Urinalysis

Blood

Protein

Ketone bodies

Appearance

Glucose

Total bilirubin Urobilinogen

Sediment

Specific gravity

Volume

 

SOURCE: EPA, 1984.

lymph nodes, oviduct, brain, stomach, pancreas, skin, mammary gland, rectum, heart, spleen, spinal cord, testes, musculature, thyroid/parathyroid, pituitary, epididymis, salivary glands, ileum, adrenals, thymus, trachea, urinary bladder, accessory sex organs, and gallbladder.

The data described above are not required for all subchronic studies. For the 21-day dermal study, for example, only limited necropsy data are required.

EPA's Proposed Changes

For the 90-day oral study (Guideline 82-1; EPA, 1984) in the rodent and other test species, three changes are proposed:

  • the studies would be required for all pesticide uses that could result in oral exposure of humans and would not depend on frequency, magnitude, or duration of exposure;

  • this study could be modified to include additional end points for neurotoxicity and immunotoxicity; and

  • a separate drinking water study, in addition to the other dietary studies, could be required if the pesticide were found to leach into groundwater or contaminate drinking water.

Either a 21-day dermal (Guideline 82-2; EPA, 1984) or a 90-day dermal

Suggested Citation:"4 METHODS FOR TOXICITY TESTING." National Research Council. 1993. Pesticides in the Diets of Infants and Children. Washington, DC: The National Academies Press. doi: 10.17226/2126.
×

study (Guideline 82-3; EPA, 1984) would be required (not conditionally required as in the past) to support all registrations. A 21-day study would be required for all food uses, except when acute dermal toxicity is observed. A 90-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 90-day inhalation study (Guideline 82-4; EPA, 1984) 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 21-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 90-day neurotoxicity study in the hen or mammal (Guideline 82-5; EPA, 1984) would be replaced by two new studies:

  • a 28-day delayed neurotoxicity study in the hen (Guideline 82-6; EPA, 1984), which would be conducted under the same conditions as the 90-day study; and

  • a 90-day neurotoxicity study in the rat (Guideline 82-7; EPA, 1984) (previously required only conditionally) to support all registrations and food/feed use EUPs (this is now required only for organophosphate and carbamate pesticides).

Testing would include assessments of function (functional observation battery), motor activity, and histopathological examination of the nervous system.

CHRONIC TOXICITY STUDIES

General Description

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

Suggested Citation:"4 METHODS FOR TOXICITY TESTING." National Research Council. 1993. Pesticides in the Diets of Infants and Children. Washington, DC: The National Academies Press. doi: 10.17226/2126.
×

be derived from studies in the same species and strain and, preferably, in the same laboratory as used in the study under consideration. 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.

EPA's Proposed Changes

Modifications to chronic feeding studies in two species (rodent and nonrodent; Guideline 83-1; EPA, 1984) 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 83-2; EPA, 1984) of a pesticide. Studies conducted to satisfy the requirement for Guideline 82-1 (EPA, 1984) will also be acceptable to satisfy this 90-day study requirement.

DEVELOPMENTAL TOXICITY STUDIES

General Description

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. These effects include death of the developing organism, structural abnormalities, altered growth, and functional deficiencies. In addition to the classic teratology (now called developmental toxicity) study, a postnatal study is required by the EPA on a case-by-case basis. It is in this study that functional deficiencies are best studied.

The EPA prefers that the rat and the rabbit be used in these studies; however, hamster and mouse are also acceptable. Doses should be administered over the period of major organogenesis (major visceral and skeletal formation) in the fetus. The maternal animals only are dosed in this study and only for specified periods. When day 0 is the day that evidence of mating was observed, the rat  and mouse are dosed on days 6 through 15; the rabbit, days 6 through 18; and the hamster, days 6 through 14.

Suggested Citation:"4 METHODS FOR TOXICITY TESTING." National Research Council. 1993. Pesticides in the Diets of Infants and Children. Washington, DC: The National Academies Press. doi: 10.17226/2126.
×

Dosing is usually administered by gavage (oral bolus dose). The pregnant animal should be observed daily for signs of toxicity. Maternal body weight should be monitored at least every 2 to 3 days during gestation. At sacrifice, the maternal animals should be examined for any abnormalities or pathological changes that may have influenced the pregnancy. The uterus is then removed and examined. The number of corpora lutea and live and dead fetuses should be recorded. The sex of the fetuses should be determined. Each fetus is then weighed and examined externally and malformations recorded by litter along with weight and sex. A certain percentage (depending on the animal species used) of the fetuses are then prepared for visceral examination and the remainder for examination of skeletal anomalies. Although the litter is considered the most relevant unit for statistical analysis, data should also be presented and assessed for each fetus.

Historical control data are also useful for determining the biological importance of visceral or skeletal anomalies that are elevated to a statistically significant level by treatment. Again, only historical control data from studies on the same species and strain of animal should be used for comparison purposes.

EPA's Proposed Changes

At least one developmental toxicity (formerly teratogenicity) study (Guideline 83-3; EPA, 1984) would now be required for all nonfood uses. In the past it was required only if there was expected exposure of women of childbearing age. A second study could be required if concerns are raised from the results of the first study. For food use EUPs accompanied by a temporary tolerance request, a second study could also be required, depending on the results of the first study.

A postnatal development toxicity study (Guideline 83-6; EPA, 1984) is proposed as a conditional requirement. This study could be required to more fully assess the manifestations of developmental toxicity, especially potential deficits in function or developmental neurotoxicity.

The parameters that need to be studied in a postnatal study depend on the effects seen in the prenatal study. Guidelines are presently being developed by EPA.

REPRODUCTION STUDIES

General Description

Multigeneration reproduction studies are designed to provide information concerning the general effects of a test substance on overall reproductive

Suggested Citation:"4 METHODS FOR TOXICITY TESTING." National Research Council. 1993. Pesticides in the Diets of Infants and Children. Washington, DC: The National Academies Press. doi: 10.17226/2126.
×

capability. Such studies may also provide information about the effects of the test substance on neonatal morbidity and mortality and about the meaning of preliminary data for developmental toxicity. EPA requires that the study include a minimum of two generations and that one litter be produced each generation. Dosing of both parents should begin when they are 8 weeks old and continue for 8 weeks prior to mating. Dosing of parental males should continue at least until mating is completed. Dosing of parental females continues through a 3-week mating period and pregnancy and up to the time of weaning 3 weeks after delivery of the pups. Dosing of pups selected for mating to produce the second generation should begin at weaning and continue as discussed above. The dosing and breeding schedule is clarified in the timeline presented in Table 4-6.

Parental animals should be observed daily for signs of toxicity. This is especially important for females during pregnancy in order to detect signs of difficult or prolonged parturition. Weights of parental animals are recorded weekly. The duration of pregnancy should be determined from the time evidence of mating was first observed. Each litter should be examined for the number of dead and live pups and for gross abnormalities. Live pups should be individually weighed on days 0 (optional), 4, 7 (optional), 14, and 21 after birth. A complete gross necropsy should be performed on all parental animals, all pups found dead prior to day 21 (weaning), and all weanlings not selected as parental animals for a next generation. Pups culled on day 4 do not have to undergo gross necropsy. Histopathology is required for reproductive and target organs (those known from previous studies to be adversely affected by the test material) for all control and high-dose parental animals and should be conducted on weanling animals (except for those selected as parental animals in the next generation) as described for parental animals (EPA, 1988).

EPA's Proposed Changes

The addition of a fertility assessment of parental males is recommended by EPA if fertility or reproductive parameters are found to be affected by the test chemical. The parameters to be examined or reported in this assessment include weight of reproductive organs, spermatid count, total cauda epididymal sperm count, assessment of sperm morphology and motility, examination of epididymal fluid for debris and unexpected cell types, and additional histopathology of the testes. A reproduction study (Guideline 83-4; EPA, 1984) could also be required to support nonfood uses if adverse effects on the reproductive system or developmental toxicity are observed in other studies.

Suggested Citation:"4 METHODS FOR TOXICITY TESTING." National Research Council. 1993. Pesticides in the Diets of Infants and Children. Washington, DC: The National Academies Press. doi: 10.17226/2126.
×

TABLE 4-6 Approximate Dosing and Breeding Schedule for a Rat Two-Generation Reproduction Study

Weeks of Study

P1

F1

F2

0

Dosing of P1 males and females begins

 

 

8–14

P1 mating period

 

 

11–17

Dosing of P1 males may end at week 25; P1 females are killed

 

 

22–23

 

F1 mating; dosing of F1 males may end at week 40; F1 males killed

 

25–37

 

Remaining F1 females are killed

F2 born. Litter sizes randomly adjusted to 8 each

 

 

 

F2 offspring are killed

 

SOURCE: EPA, 1984.

Suggested Citation:"4 METHODS FOR TOXICITY TESTING." National Research Council. 1993. Pesticides in the Diets of Infants and Children. Washington, DC: The National Academies Press. doi: 10.17226/2126.
×

MUTAGENICITY STUDIES

General Description

A battery of mutagenicity tests is required to assess the potential of each test chemical to affect genetic material. The test selection criteria focus on the test's ability to detect, with appropriate assay methods, the capacity of the chemical to alter genetic material in cells. When mutagenic potential is demonstrated, these findings are considered in the assessment of potential heritable effects in humans, in the weight-of-the-evidence evaluation for carcinogenicity, and in the decision to require submission of a carcinogenicity study if otherwise conditionally required. Mutagenicity results per se are not used by themselves for risk assessment purposes, even when results suggest possible heritable genetic effects in humans.

EPA's Proposed Changes

EPA has already published changes to the 40 CFR Part 158 data requirements for mutagenicity (EPA, 1984).

As described in Pesticide Assessment Guidelines: Subdivision F (EPA, 1984), the original mutagenicity test battery consisted of three assays: one for gene mutations, one for structural chromosome aberrations, and one for other genotoxic effects. Other testing included DNA damage and repair. The revised guidelines would require an initial battery of tests consisting of:

  • Salmonella typhimurium reverse mutation assay;

  • mammalian cells in culture forward gene mutation assay allowing detection of point mutations, large deletions, and chromosome rearrangements; and

  • in vivo cytogenetics.

Results derived from these assays could trigger the requirement for further mutagenicity testing. The type of additional required testing would depend on the observed results from the initial battery and other toxicity testing results. For example, testing could involve cytogenetic testing in spermatozoa if other test results suggest that they are targets.

GENERAL METABOLISM STUDIES

General Description

Data from studies on the absorption, distribution, bioaccumulation, excretion, and metabolism of a pesticide may also allow more meaningful evaluation of test results and more appropriate risk assessment (as a result of more meaningful extrapolation from data on animals to humans). Such

Suggested Citation:"4 METHODS FOR TOXICITY TESTING." National Research Council. 1993. Pesticides in the Diets of Infants and Children. Washington, DC: The National Academies Press. doi: 10.17226/2126.
×

data may also aid in designing more relevant toxicology studies. Information on metabolites formed in laboratory animals is also used to determine whether further toxicity testing is required on plant metabolites. If a major metabolite forms in the plant but not in the test animal, separate toxicity testing on the plant metabolite could be necessary. The extent of testing required depends on the level of concern raised by the initial battery of toxicity tests (acute and subchronic studies, one teratology study, and a battery of mutagenicity tests).

As presently designed, the metabolism study consists of four separate parts: a single low, intravenous dose of radiolabeled test material (not required if the test material is insoluble in water or normal saline solution); a single low, oral dose of radiolabeled test material; 14 consecutive daily low, oral doses of unlabeled test material followed by a single low dose of radiolabeled material; and single high, oral dose of radiolabeled test material. Selection of the low dose is based on the NOEL. The high dose should elicit some signs of toxicity but not be so high that it results in mortality. The test species of choice is the rat.

Urine, feces, and expired air are collected for 7 days after administration of the radiolabeled material or until >90% of the radioactivity is recovered. Bone, brain, fat, testes, heart, kidney, liver, lung, blood, muscle, spleen, residual carcass, and tissues showing pathology in this or prior tests should be examined for radioactivity for all animals except those given the intravenous dose. This is done to determine if the test material or radiolabeled metabolite accumulates in any particular organ and to relate this information to the findings in toxicity studies.

In addition, quantities of radiolabeled material in feces, urine, and expired air must be monitored for all dose groups at appropriate intervals up to 7 days after dosing. Furthermore, urinary and fecal metabolites must be identified.

EPA's Proposed Changes

A metabolism study would also be required when significant adverse effects are observed in toxicology studies, including reproduction and developmental studies (Guideline 85-1; EPA, 1984). EPA is currently rewriting to guidelines for conducting metabolism studies and is including a tiered approach for study design and conduct.

NEUROTOXICITY STUDIES

General Description

Neurotoxicity studies are required to evaluate the potential of each pesticide to adversely affect the structure or function of the nervous

Suggested Citation:"4 METHODS FOR TOXICITY TESTING." National Research Council. 1993. Pesticides in the Diets of Infants and Children. Washington, DC: The National Academies Press. doi: 10.17226/2126.
×

system. The objectives of these studies are to detect and characterize the following:

  • effects on the incidence and severity of clinical signs, the alteration of motor activity, and histopathology in the nervous system following acute, subchronic, and chronic exposures;

  • the potential of cholinesterase inhibiting pesticides and related substances to cause a specific organophosphate-pesticide-type induced delayed neurotoxicity;

  • other neurotoxic effects based on screening studies on certain chemical classes; and

  • effects on organisms exposed prior to birth or weaning.

Results from these studies may be used for qualitative and quantitative risk assessment. The guidelines for these studies were published in March 1991 as addendum 10 to the EPA guidelines (EPA, 1991a).

EPA's Proposed Changes

The changes in the requirements for neurotoxicity testing were described above under ''Acute Toxicity" and "Subchronic Toxicity."

SPECIAL TESTING

EPA intends to develop better definitions of the conditions under which domestic animal safety (Guideline 85-2; EPA, 1984) testing and visual system studies (Guideline 85-4; EPA, 1984) would be required for all organophosphates and other pesticides shown to affect the visual system. These studies could be of acute, subchronic, or chronic duration, whichever is deemed appropriate for the pesticide under study. Since guidelines have not been formulated for these studies, they will be designed in conjunction with EPA scientists.

CONCLUSIONS AND RECOMMENDATIONS

Conclusions

Current and past studies conducted by registrants are designed primarily to assess pesticide toxicity in sexually mature animals. The protocols for these studies have evolved over several decades and have included some testing paradigms that allow extrapolation to infant and adolescent animals. These studies have produced some valuable information on toxicity and exposure. After reviewing EPA's current and proposed toxicity testing guidelines, however, the committee concluded that current studies do not directly address the following areas:

Suggested Citation:"4 METHODS FOR TOXICITY TESTING." National Research Council. 1993. Pesticides in the Diets of Infants and Children. Washington, DC: The National Academies Press. doi: 10.17226/2126.
×
  • toxicity of pesticides in neonates and adolescent animals;

  • metabolism of pesticides in neonates and adolescent animals; and

  • exposure during early developmental stages (after the second trimester through adolescence) and sequelae in later life.

Recommendations

  • Studies should be redesigned and expanded in scope to elucidate the differences in the metabolism and disposition of pesticides in the infant, adolescent, and young adult.

Current metabolism studies are designed to provide information about sex-related differences, metabolic pathways and excretion, bioaccumulation in tissues, and tissue distribution in adult rats. EPA uses the data to determine whether toxicity testing needs to be conducted on individual plant or animal metabolites in addition to the parent compound.

  • The metabolism of pesticides in newborn animals needs to be more thoroughly investigated.

Greater knowledge in this area would make it possible to develop computer programs for physiological pharmacokinetic modeling to forecast how information about metabolism in infant animals could be extrapolated to infant humans. The committee realizes that this is a very difficult area of investigation and application. Nevertheless, it urges that such investigations be pursued, since the resulting information could provide more realistic systemic exposure scenarios for risk assessment.

  • A study should be conducted to compare the toxicity of several representative classes of pesticides in both adult and immature animals.

Results of such a broad-range study designed to specifically address the infant and young adult animal should indicate whether comparative studies of this nature should routinely be required by EPA. This study should be designed to examine several critical end points in the developing animal, including neural (functional and behavioral), immune, and endocrine systems to cite a few examples. Because the battery of acute toxicity tests now required by EPA is generally performed in adult animals, very little information is available on acute toxicity in immature animals. Such data are important in determining dietary risk to infants and children for acutely toxic pesticides such as organophosphates and carbamates. The committee recognizes that some of these data can be obtained from multigeneration studies if specific observation requirements are added to the current studies.

  • Test animals should be exposed to the chemical of concern early in their lives so the risks of exposure of infants and children to the compound can be more adequately assessed.

Suggested Citation:"4 METHODS FOR TOXICITY TESTING." National Research Council. 1993. Pesticides in the Diets of Infants and Children. Washington, DC: The National Academies Press. doi: 10.17226/2126.
×

The committee recognizes the difficulty in dosing animals during lactation and is aware that testing requirements would have to be modified to accomplish these studies. EPA Guideline 83-5 for a chronic toxicity/carcinogenicity study states that exposure of rats to pesticides should begin at approximately 6 to 8 weeks of age, essentially when they are adolescents (EPA, 1984). Because the effects of the pesticide in the rat are not determined early in its lifetime, chronic toxicity/carcinogenicity studies in adolescent animals may not be representative of the responses of younger animals. Current reproduction studies (Guideline 83-4; EPA, 1984) partially address this period in the life of a rat, but the effects of early exposure are not addressed past 21 days of age for second-generation pups or past the death of the second-generation parents (first-generation pups used for mating to produce the second generation). The protocol does not indicate whether exposure early in life has any impact on the adults or whether continuous exposure from birth to young adulthood influences the severity of the toxicity over a lifetime. FDA has used the multigeneration studies to include the F2A or F3A generation of laboratory animals for direct and indirect food additives (Becci et al., 1982).

  • To obtain lifelong data on rodents for a given pesticide, the committee recommends that the testing guideline for a rat chronic toxicity/carcinogenicity study be modified to include in utero exposure during the last trimester, exposure through the mother's milk, and after weaning, oral exposure through diet.

This would mean that weanlings from the F1A or F2A generation would be selected from each dose group and tested throughout their lifetimes (see Table 4-6). In addition to this group, another smaller group of rats from the F1 generation would be killed at 6 months and 1 year and necropsied to examine the same parameters normally measured at the end of a lifetime feeding study.

The NTP tested three chemicals using a similar protocol and their standard protocol for an carcinogenicity study. One of the chemicals was ethylenethiourea, which is a breakdown product and metabolite of the ethylenebisdithiocarbamate fungicides and a thyroid toxicant (decreases T3 and T4). In utero exposure did not affect the occurrence of liver tumors in male and female mice, but did result in a sex-dependent increase in the number of malignant thyroid tumors in mice and rats (NTP, 1992).

  • Measurement of the serum thyroid hormones T3 and T4 and serum TSH should be routinely added to the EPA chronic/carcinogenicity study protocol or to the subchronic toxicity protocol for the rat so that adverse effects on thyroid function can be determined earlier.

When examining the parameters currently measured in the EPA chronic/ carcinogenicity study, the committee found that endocrine function

Suggested Citation:"4 METHODS FOR TOXICITY TESTING." National Research Council. 1993. Pesticides in the Diets of Infants and Children. Washington, DC: The National Academies Press. doi: 10.17226/2126.
×

was adequately covered for all but the thyroid. Although the thyroid is saved in these studies for microscopic examination and its weight is recorded, the committee believes that changes in the functioning capabilities of this organ can occur regardless of whether there are organ weight or histopathologic changes.

  • If abnormalities are found during histopathologic examination of the spleen, lymph nodes, thymus, and bone marrow, more detailed and specific studies should be conducted on a case-by-case basis relevant to the types of effects initially seen in immune system tests.

EPA has developed protocols for immunotoxicity testing for some pesticides that affect the immune system, and the agency is considering developing a generic testing protocol. The committee believes that because the human immune system is one of the most robust of systems in terms of resistance to pesticides or other chemical toxicity, initial evaluation using current histopathologic examination of spleen, lymph nodes, thymus, and bone marrow should be sufficient unless abnormalities are noted.

  • A modified reproductive/developmental toxicity study in the rat is suggested for registration of all food-use pesticides.

One set of dams in this study would be dosed continuously with the test material from day 6 of gestation through birth of the pups and until weaning of their offspring at 21 days of age. A developmental assessment would be performed on the pups as described in EPA's recently published developmental neurotoxicity testing guidelines (EPA, 1991a). In addition, a set of pups from each dam would undergo gross and histopathologic examination at day 60 post partum. The second set of dams would be dosed from day 6 of gestation to term; however, these animals would not be allowed to deliver but, rather, would be subjected to cesarean section as in a routine teratology study. The fetuses would be subjected to skeletal and visceral examination, as described for a teratology study (Guideline 83-3) designed to examine the prenatal development of pups. This study design allows a determination of the reversibility of postnatal significance of findings seen in fetuses at the time of cesarean section. EPA has indicated in its proposed changes to Part 158 that a similar study be required; however, the committee recommends that this study be made a requirement for registration of all food-use pesticides.

  • Because neurotoxicity is such an important consideration for the newborn, EPA should continue to revise its published guidelines on developmental and functional neurotoxicity testing as new information emerges from the actual conduct of preregistration studies and from ongoing research in rodent neurotoxicity.

Suggested Citation:"4 METHODS FOR TOXICITY TESTING." National Research Council. 1993. Pesticides in the Diets of Infants and Children. Washington, DC: The National Academies Press. doi: 10.17226/2126.
×

The committee supports EPA's proposed requirement for acute and subchronic neurotoxicity testing for pesticides and encourages the agency to make this a general requirement for all food-use pesticides—not just for organophosphate and carbamate pesticides. New approaches to neurotoxicity testing are described in the report Environmental Neurotoxicology (NRC, 1992).

  • EPA should develop a general guideline for visual system toxicity testing that can be modified and applied on a case-by-case basis.

The eye is exquisitely sensitive to changes in glucose metabolism, blood flow, and neuronal function, and several pesticides have been shown to be visual system toxicants (hexachlorophene, naphthalene, 2,4-DNP, and some organophosphates). In the past, scientists have examined the effects of chemicals that may irritate the eye by accidental contact. More recently, however, researchers have been examining the effects of chemicals on specific sections of the visual system, such as the optic nerve, iris, retina, and lens. The guideline proposed by the committee should be applied to species in which this type of testing appears to be appropriate, e.g., EPA has recently considered protocols for visual system testing in dogs.

Recent studies indicate that visual system damage may be associated with dietary exposure to some cholinesterase inhibiting compounds. Thus the committee supports EPA's proposed testing (the sensory evoked potential test) of such pesticides for visual system toxicity. However, it does not believe that a single protocol would suffice to cover all classes of compounds because different classes would affect different parts of the visual system.

REFERENCES

Armitage, P., and R. Doll. 1954. The age distribution of cancer and multi-stage theory of carcinogenesis. Br. J. Cancer 8:1–12.


Becci, P.J., K.A. Voss, F.G. Hess, M.A. Gallo, R.A. Parent, K.R. Stevens, and J.M. Taylor. 1982. Long-term carcinogenicity and toxicity study of zearalenone in the rat. J. Appl. Toxicol. 2(5):247–254.


EPA (U.S. Environmental Protection Agency). 1984. Pesticide Assessment Guidelines, Subdivision F: Hazard Evaluation—Human and Domestic Animals. Revised Ed. November 1984. PB-86-108958. Washington, D.C.: U.S. Environmental Protection Agency.

EPA (U.S. Environmental Protection Agency). 1988. FIFRA Accelerated Reregistration Phase 3 Technical Guidance. US EPA 540/09–0784. Washington, D.C.: U.S. Environmental Protection Agency.

EPA (U.S. Environmental Protection Agency). 1991a. Pesticide Assessment Guidelines, Subdivision F: Hazard Evaluation—Human and Domestic Animals, Carcinogenicity of Ethylene Thiourea [CAS No. 96-45-7] in F/344 Rats and B6C3F1 mice. Addendum 10—Neurotoxicity. Washington, D.C.: U.S. Environmental Protection Agency.

Suggested Citation:"4 METHODS FOR TOXICITY TESTING." National Research Council. 1993. Pesticides in the Diets of Infants and Children. Washington, DC: The National Academies Press. doi: 10.17226/2126.
×

EPA (U.S. Environmental Protection Agency). 1991b. Pesticide Assessment Guidelines, Subdivision F: Hazard Evaluation—Human and Domestic Animals Series 84. Addendum 9—Mutagenicity. PB-158394. 540/09-91-122. Washington, D.C.: U.S. Environmental Protection Agency.

Gad, S.C., and C.S. Weil. 1982. Statistic for toxicologists. Pp. 273–320 in Principles and Methods of Toxicology, A.W. Hayes, ed. New York: Raven Press.


IPCS (International Program on Chemical Safety). 1990. In Environmental Health Criteria 104: Principles for the Toxicological Assessment of Pesticide Residues in Food. Geneva, Switzerland: World Health Organization.


Loomis, T.A. 1978 Essentials of Toxicology. Philadelphia, Pa.: Lea & Febiger.


NRC (National Research Council). 1992 Environmental Neurotoxicology. Washington, D.C.: National Academy Press.

NRC (National Research Council). 1993. Issues in Risk Assessment. Washington, D.C.: National Academy Press.

NTP (National Toxicology Program). 1992. NTP Technical Report on the Perinatal Toxicology and Carcinogenesis Studies of Ethylene Thiourea (CAS No. 96-45-7) in F3441N Rats and B6C3F1 Mice (Feed Studies). NTP TR 388. Research Triangle Park, N.C.: National Toxicology Program.


Weil, C.S. 1972. Guidelines for experiments to predict the degree of safety of a material for man. Toxicol. Appl. Pharmacol. 21:194–199.

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Many of the pesticides applied to food crops in this country are present in foods and may pose risks to human health. Current regulations are intended to protect the health of the general population by controlling pesticide use. This book explores whether the present regulatory approaches adequately protect infants and children, who may differ from adults in susceptibility and in dietary exposures to pesticide residues.

The committee focuses on four major areas:

  • Susceptibility: Are children more susceptible or less susceptible than adults to the effects of dietary exposure to pesticides?
  • Exposure: What foods do infants and children eat, and which pesticides and how much of them are present in those foods? Is the current information on consumption and residues adequate to estimate exposure?
  • Toxicity: Are toxicity tests in laboratory animals adequate to predict toxicity in human infants and children? Do the extent and type of toxicity of some chemicals vary by species and by age?
  • Assessing risk: How is dietary exposure to pesticide residues associated with response? How can laboratory data on lifetime exposures of animals be used to derive meaningful estimates of risk to children? Does risk accumulate more rapidly during the early years of life?

This book will be of interest to policymakers, administrators of research in the public and private sectors, toxicologists, pediatricians and other health professionals, and the pesticide industry.

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