APPENDIX II-AG:  Synergy – Blood Brain Barrier – Kepner.


This article cites: 5 Abou-Donia, M. B., et al. 2001. Effects of daily dermal application of

DEET and permethrin, alone and in combination, on sensorimotor performance, blood-brain barrier, and blood-testis barrier in rats. Journal of Toxicology and Environmental Health 62:523-541.This article states: “The neurological indicators included: sensorimotor performance and permeability of the blood-brain barrier,5


This article is from:


Beyond Pesticides/National Coalition Against the Misuse of Pesticides

Vol. 23, No. 4, 2004 Pesticides and You Page 17

Synergy: The Big Unknowns of

Pesticide Exposure

Daily combinations of pesticides and pharmaceuticals untested

By John Kepner

Pesticide exposures in the real world are not isolated incidents.

Rather, they are a string of incidents marked by combinations of

exposures. As a result, scientists have argued for years that toxic

exposures to pesticides should be measured as they would normally

occur, in combination with one another. Yet, current federal

law does not require this type of testing for pesticides on the

market, except in very limited instances. This issue has been fueled

during the recent West Nile virus spray programs

by researchers at Duke University’s School of

Medicine, who found that exposure to a popular

insect repellent when combined with

exposure to a popular insecticide caused

a synergistic, or magnified, effect greater

than the individual chemical effects

added together. The debate has also

heated up around the question of potential

interactions between pesticides and

pharmaceuticals. A law requiring the testing

of drug-pesticide combinations was adopted

by Congress and then dropped by the Food and

Drug Administration (FDA) in the 1960’s.

How much do we really know about the

pesticides that are widely used in our communities,

schools, homes, offices, hospitals,

parks, on lawns and golf courses and in West

Nile virus and other mosquito spray programs?

Not as much as we should to be able to

make sound decisions that are protective of public

health and the environment. Sometimes limitations in protection

are a function of a regulatory failure to carry out the mandate of

a federal statute. But, in this case, the underlying statutes that

govern pesticide use, allowable residues (and exposure), and risk

assessment are wholly deficient. The laws simply do not require

testing that is ultimately essential in determining the safety of

pesticide use, as typically used every day. No amount of improved

enforcement of law or additional dollars for EPA will correct this

situation until the mandates in law change.

This piece by John Kepner tracks the current situation and history

on this critical issue of public health and safety. It leaves us

with a greater sense of the importance of efforts to eliminate on a

daily basis exposure to pesticides and opt for alternative pest management

approaches that do not rely on pesticides. The burden

must shift to those who want to use pesticides to show that basic

questions of health protection have been answered. Pointing to a

pesticide label or citing an EPA pesticide product registration is no

assurance of safety. —JF


In the summer of 2001, the mosquito-borne West Nile virus

hit Maryland. As the media fueled a local panic, the

Maryland Departments of Health and Agriculture worked

together to monitor, treat, contain and eradicate the disease.

Permanone, a synthetic pyrethroid-based insecticide

containing the active ingredient permethrin, emerged as

the pesticide of choice for combating the adult

mosquitoes that could be carrying the virus. Aside

from spraying Permanone from foggers mounted

on the backs of trucks, the state also instructed

its residents to empty standing water on

their property to reduce mosquito

breeding grounds, and encouraged

residents to use mosquito repellants

containing the active ingredient N, Ndiethyl-

m-toluamide (DEET).

Both DEET and permethrin are registered

as pesticides by the Environmental

Protection Agency (EPA) and

have been, or are in the process of being,

individually tested for adverse health effects.

Based on these results, EPA has determined that

the risks posed by these pesticides do not outweigh

the benefits, namely killing and repelling mosquitoes.

However, many of the residents of Maryland will not be exposed

to these pesticides individually. Real world pesticide exposures

rarely occur as individual, isolated incidents. Many residents

could have applied DEET to their bodies as recommended

by the state when the mosquito trucks fog their neighborhoods.

Or because permethrin has a half-life of 30 to 38 days, they

could be exposed to the combination anytime they are wearing

DEET for weeks to come. Although not all pesticide combinations

show increased toxicity, recent studies out of Duke

University’s School of Medicine suggest that the residents of

Maryland should be concerned about the potentially damaging

synergistic effects of this particular pesticide combination. These

studies will be discussed in greater detail below.

What is synergy?

The concept of interaction is fundamental to understanding

the processes by which chemical mixtures act. If the effect is

simply additive, the sum of the effects is the same as if we were

exposed to each chemical individually. Synergy occurs when

Beyond Pesticides/National Coalition Against the Misuse of Pesticides

Page 18 Pesticides and You Vol. 23, No. 4, 2004

the effect of a mixture of chemicals is greater than the sum of

the individual effects.1 (If the effect of a mixture is less than the

sum of the individual effects, it is called antagonism).

For example, a population exposed to neither “Pesticide

A” nor “Pesticide B” experiences a background level

of a certain health effect at 5%. In a population exposed

only to “Pesticide A,” the effect is seen at 10%

(5% + the 5% background). In a population exposed

only to “Pesticide B,” the effect is seen at 20% (15%

+ the 5% background). If the two pesticides are simply

additive, and not synergistic, we would expect

the effect to be observed at 25% (5% + 15% + the

5% background). If the observed effect is greater

than 25%, the combination is synergistic.

Prior to 1957, the combined effects of exposure

to a group of pesticides was assumed to be additive.

However, a study2 published that year documented

for the first time a case of pesticide synergy. The authors

postulated that the combined effects of exposure

to the organophosphate insecticides ethylpnitrophenyl

benzenethiophosphate (EPN) and

malathion would be additive. Instead, there was a 10-

fold synergistic effect in rats and a 50-fold synergistic

effect in dogs for the acute toxicity of EPN and

malathion administered simultaneously.

Regulatory history

Faced with potential interactions between pesticides and pharmaceuticals,

the Food, Drug and Cosmetic Act was amended

with the following in 1962: “Pesticide chemicals that cause

related pharmacological effects will be regarded, in the absence

of evidence to the contrary, as having an additive deleterious

action. For example, many pesticide chemicals within

each of the following groups have related pharmacological

effects: chlorinated organic pesticides, arsenic-containing

chemicals, metallic dithiocarbamates, cholinesterase-inhibiting

pesticides.” While this language assumed only additive

and not synergistic effects, it still considered, for the first time,

the adverse impact of cumulative chemical exposures. However,

in 1967, FDA abandoned the regulation on the grounds

that the “requirement has failed to serve any useful purpose.”

During its first 85 years, federal pesticide law did not require

testing for adverse health effects of pesticide combinations.

In 1996, EPA was required for the first time to consider

cumulative pesticide exposures in limited circumstances

under the Food Quality Protection Act (FQPA). FQPA, which

amends the Federal Insecticide, Fungicide and Rodenticide Act

(FIFRA), recognizes that real-world pesticide exposures do

not occur as single discrete exposures to a specific pesticide,

but rather in combination to several pesticides at once. Considering

dietary exposure alone, U.S. Department of Agriculture

(USDA) data shows that apples surveyed from across

the U.S. contained 22 different pesticide residues, and peaches

surveyed contained 40 different pesticide residues. Many of

these residues remain even after thorough washing and preparation

of food.

To address the issue of multiple pesticide exposures, FQPA

directs EPA to consider combinations of pesticides that have a

common mechanism of toxicity when setting tolerances. This

means that only if EPA determines that two chemicals have

the same toxic mechanism in the body will the agency

aggregate the exposure value in its risk assessment calculation.

The first result of this mandate was released

in June 2002 when EPA published its Revised Organophosphate

Cumulative Risk Assessment,14 in which

the agency examined the combined hazard of exposure

to all organophosphate pesticides. Although

the report was seen as incomplete by the environmental

community and criticized by FIFRA’s Scientific

Advisory Panel, the intent of the report is an

important first step in evaluating the combined effects

of several pesticides. Unfortunately, the current

Guidance on Cumulative Risk requires that only chemicals

sharing both a common toxic effect and a common

mechanism of toxicity be considered in determining

pesticide tolerances. In the real world, a liver

cannot tell the difference between two cancer-causing

chemicals because of the biochemical route each chemical

takes to cause that cancer. In other words, if a number

of pesticides and other substances cause liver cancer via a

number of different pathways, the end result is the same,

a diseased liver. EPA should not use common mechanisms of

toxicity as a filter to decrease the number of chemicals it considers.

This leaves the majority of potential pesticide interactions

untested and potentially dangerous.

Medical studies: proof of

pesticide synergy

While the first study showing pesticide synergy was published

in 1957, the topic has not been studied at the level necessary

to adequately inform officials making decisions regarding

human health. Despite the lack of depth, many studies demonstrating

synergy between pesticides and other commonly

used chemicals have been documented in medical literature.

In the late 1960’s and early 1970’s, researchers Samuel Epstein,

MD, at the time with the Children’s Cancer Research Foundation

in Boston, MA and Keiji Fujii, MD, of the National

Institute of Hygienic Sciences in Tokyo, Japan published a

series of papers3,4 on the synergistic effects of carcinogens and

co-carcinogens found in a variety of common pesticide products.

“Co-carcinogens” is a term used to describe non-carcinogenic

chemicals that increase the rate of cancer when used

in combination with carcinogens. These papers highlighted

carcinogenicity between two chemicals used in combination,

even when the individual dosages were applied at sub-carcinogenic

levels. One study produced the effect even when the

chemicals were applied as far as 200 days apart.

Much of the latest research on the synergistic effects of pesticides

used in combination has come out of the Duke University

Medical Center in Durham, NC. In 2001, researchers in

the Department of Pharmacology and Cancer Biology published

Beyond Pesticides/National Coalition Against the Misuse of Pesticides

Vol. 23, No. 4, 2004 Pesticides and You Page 19

a series of papers in the Journal of Toxicology and Environmental

Health and Experimental Neurology looking closely at the synergistic

health effects of DEET, the active ingredient in most

insect repellents, and permethrin, a pesticide commonly used

in community mosquito spray programs, as well

as many household bug killers.

To determine the effect of subchronic dermal

application of these chemicals on the brain,

the researchers evaluated neurological indicators

after daily dermal doses of DEET,

permethrin or a combination of the two pesticides

for varying periods of time, from 24 hours

to 60 days. The neurological indicators included:

sensorimotor performance and permeability of

the blood-brain barrier,5 increased urinary excretion

of 6B-hydroxycortisol (a marker chemical

poisoning),6 release of brain mitochonrial cytochrome-

c (a result of cell death)7, and diffuse

neuronal cell (cells specialized to conduct nerve

impulses) death and cytoskeletal (structural components of the

cell) abnormalities.8 In the first study, DEET alone caused a

decrease in the permeability of the blood-brain barrier and impairment

of sensorimotor performance, and permethrin alone

showed no effect. In combination, the effect on the blood brain

barrier and sensorimotor performance was amplified, a “0+1=2”

example of pesticide synergy. This “0+1=2” pattern was also

seen in the study examining increased urinary excretion of 6Bhydroxycortisol.

When the researchers looked

at the release of cytochrome c as an indicator

of brain cell death, no effect was seen when

the pesticides were used individually. However

in combination, a significant increase in

the release of cytochrome c was seen 24 hours

after dosing, a “0+0=1” example of pesticide

synergy. In the study examining neuronal cell

death, damage was seen in all treatment

groups, but was accelerated in rats treated with

both DEET and permethrin.

The purpose of the Duke studies was to

determine a possible link between pesticides

and other chemicals used during the Persian

Gulf War and “Gulf War Syndrome,” neurological

disease characterized by headache, loss of memory,

fatigue, muscle and joint pain, and ataxia, which causes an

inability to coordinate muscular movements. The first work

in this area by this team of researchers, published in 1996,

studied the combination of DEET and permethrin with pyridostigmine

bromide, a drug taken prophylactically to counteract

toxic gas warfare agents.9 The study found that test animals

exposed to the three chemicals in combination experienced

neurological deficits similar to the symptoms of the

Gulf War veterans. However, when the chemicals were administered

alone, even at doses three times the level soldiers

received, no effects were observed, a “0+0+0=1” effect. The

researchers theorized that many of the symptoms might be

seen without the pyrido-stigmine bromide and continued to

study the interactions of DEET and permethrin.

Neurology experts give three possible reasons for the synergistic

effects seen in the above experiments. First, the stress endured

by animals when exposed to a combination of chemicals

undermines the protective role of the blood brain barrier, allowing

the level of toxics to cross into the brain to be 100 times

higher. Second, tissue that has been exposed becomes more sensitive

and receptive to other toxic substances. Third, certain

chemicals bind to enzymes that detoxify the body, making the

enzymes unavailable to protect the body from other intruding

chemicals. Dr. Goran Jamal, a neurologist at the West London

Regional Neuro-Science Center of the Imperial College of Medicine,

makes the following comparison, “It’s like releasing 200

criminals in London and taking away the police officers that are

usually on duty. There is bound to be some damage.”


Synergistic effects between multiple pesticides and/or other

chemicals represent one of the greatest gaps in EPA’s ability to

protect the public from the adverse health effects associated

with pesticide use and exposure. The U.S. government recognizes

that pesticide exposures occur in combinations and not

as unique events, yet has rules and regulations to test only a

Pesticide-Drug Synergy

In the summer of 1985, 30 year-old Thomas Latimer

was leading a good life in the suburbs of Dallas, TX. He

was a vigorous, athletic man with a promising engineering

career. On one particular Saturday afternoon,

Mr. Latimer spent the day mowing the lawn, picking

up the clippings and edging the walkways. After about

an hour, he began to feel dizziness, nausea, tightness

in his chest and a pounding headache. Ten days later,

he felt even worse and went to see his doctor.

Over the next six years, Mr. Latimer found himself

unable to exercise and suffering from brain seizures.

He visited 20 different doctors and underwent numerous

tests to determine the source of his medical problems.

His symptoms were consistent with organophosphate

poisoning, most likely from the insecticide

diazinon that had been applied to his lawn. But because

his symptoms were so severe and the amount of

pesticide he was exposed to was so low, the doctors

continued to look for a complicating factor. After further

research, a toxicologist, three neurologists and two

neuro-ophthalmologists all concluded independently

that the popular ulcer drug Tagamet that Mr. Latimer

was taking had suppressed his liver, making him more

susceptible to pesticide poisoning.

Alfredo A Sudan, a professor of neurology and ophthalmology

at the University of Southern California, who

conducted extensive tests evaluating an eye disorder that

Mr. Latimer developed, estimates that taking a medication

like Tagamet “can make a person 100 to 1,000 times

more sensitive to organophosphate poisoning.”10

Beyond Pesticides/National Coalition Against the Misuse of Pesticides

Page 20 Pesticides and You Vol. 23, No. 4, 2004

limited number of possible interactions. Given that there are

over 875 active ingredients currently registered for use, it

would be impossible to test all possible combinations, but we

must start somewhere. One approach would be to prioritize

pesticides most likely to act in combination. The following

recommendations would serve as a basis for beginning to look

at this very important aspect of pesticide safety:

Test for interactions between pesticides commonly

used in combination in both agricultural

and non-agricultural settings. This would include

testing of groups of pesticides that are commonly used on

the same crops, like atrazine and chlorpyrifos, the most

common herbicide and insecticide applied to corn.11 Another

example would be DEET, used as an insect repellent

and permethrin, used as a mosquito fog.

Test for interactions between agricultural pesticides

and the most persistent food contaminants.

FDA data shows chlordane, DDE (a breakdown product

of DDT), DDT, dieldrin, dioxin, endrin, heptachlor,

hexachlorobenzene, and toxaphene are frequent contaminants

of the typical U.S. diet.12

Test for interactions between the pesticides that

most commonly contaminate drinking water. Like

all pesticide use patterns, water contamination will vary

greatly around the country, so it is imperative that these

combinations are tested for synergistic effects. The Wisconsin

State Laboratory of Hygiene has found 14 different

pesticides contaminating state water supplies.13

Test pesticides that are most likely to drift and

cause non-target exposure. Based on formulations

and methods of application, pesticides often drift far from

their point of application. A July 2000 survey of air samples


For a fully cited version of this article, see

1 Working Group on Synergy in Complex Mixtures, Harvard School of

Public Health. 1986. Synergy: positive interaction among chemicals in

mixtures. Journal of Pesticide Reform, Summer.

2 Frawley, J.P., et al. 1957. Marked potentiation in mammalian toxicity

from simultaneous administration of two anticholinesterase compounds.

J. Pharmacol. Exper. Therap. 121:96-106.

3 Epstien, Samuel S., et al. 1967. Synergistic toxicity and carcinogenicity

of freons and piperonyl butoxide. Nature, 214:526-528.

4 Epstein, Smauel S. and Keiji Fujii. 1970. Synergism in Carcinogenesis

with particular reference to synergistic effects of piperonyl butoxide and

related insecticidal synergists. Chemical Tumor Problems. Ed. Nakahara,

W. Tokyo: Japanese Society for the Promotion of Science, 1970.

5 Abou-Donia, M. B., et al. 2001. Effects of daily dermal application of

DEET and permethrin, alone and in combination, on sensorimotor performance,

blood-brain barrier, and blood-testis barrier in rats. Journal of

Toxicology and Environmental Health 62:523-541.

6 Abu-Qare, Aqel W. and Mohamed B. Abou-Donia. 2001. DEET (N,N-Diethyl-

m-Toluamide) alone and in combination with permethrin increased

urinary excretion of 6B-hydroxycortisol in rats, a marker of hepatic cyp3a

induction. Journal of Toxicology and Environmental Health 64:373-384.

7 Abu-Qare, Aqel W. and Mohamed B. Abou-Donia. 2001. Combined exposure

to DEET (N,N-Diethyl-m-Toluamide) and permethrin-induced

release of rat brain mitochondrial cytochrome c. Journal of Toxicology

and Environmental Health 63:243-252.

8 Abdel-Rahman, Ali, et al. 2001. Subchronic Dermal Application of N,NDiethyl

m-Toluamide (DEET) and Permethrin to Adult Rats, Alone or

in Combination, Causes Diffuse Neuronal Cell Death and Cytoskeletal

Abnormalities in the Cerebral Cortex and the Hippocampus, and Purkinje

Neuron Loss in the Cerebellum. Experimental Neurology 172:153-171.

9 Abou-Donia, M.B., et. al. 1996. Neurotoxicity resulting from coexposure

to pyridostigmine bromide, DEET, and permethrin: Implications of Gulf

War chemical exposures. J. Toxicol. Environ. Health 48:35-56.

10 Allen, Frank Edward. 1991. One Man’s Suffering Spurs Doctors to Probe

Pesticide-Drug Link. The Wall Street Journal. October 14.

11 U.S. Department of Agriculture. 2002. Agricultural Chemical Usage 2001

Field Crops Summary. <


12 Schafer, K. S. and S. E. Kegley. 2002. Persistent toxic chemicals in the

U.S. food supply. J. Epidemiol. Community Health 56:813–817.

13 Wisconsin State Laboratory of Hygiene. 2002. Pesticides in drinking

water. <>

14 Environmental Working Group. 2001. Every breath you take: airborne

pesticides in the San Joaquin Valley. <


15 Cecchine, Gary, et. al. 2000. Review of the Scientific Literature as it Pertains

to Gulf War Illnesses, Volume 8: Pesticides. RAND.

in Fresno, CA, on four separate sampling dates, detected

carbaryl, chlorpyrifos and trifluralin.14

Test interactions between the most common pharmaceuticals

and the most common pesticides. According

to the National Defense Research Institute, DEET

has been reported to accelerate the dermal absorption of

pharmaceuticals and possibly other pesticides.15

Recognizing the unlikely reality of testing even the most common

pesticide combinations, another approach would be to

reduce pesticide risk by limiting exposure. When weighing

the benefits of a pesticide against the risks to public health,

we must err on the side of safety. In registering pesticides,

EPA should assume interactions between chemicals will occur.

Limiting exposure, and therefore limiting synergistic

health effects, could be accomplished through decreased pesticide

use and tighter restrictions to minimize pesticide drift

and runoff. For example, ban drift-prone application technologies,

like cropdusting and ultra-low volume foggers; establish

buffer zones around populated areas; require notification

to nearby residents before a pesticide application, so

appropriate precautions may be taken; and encourage lower

exposure formulations such as containerized baits. By taking

the appropriate steps, we could minimize harmful synergistic

health effects.

Overall, this deficiency in data and the difficulty associated

with its collection calls for a national policy of pesticide

use reduction and national adoption of the Precautionary Principle

that seeks to avoid pesticide use in favor of alternatives.

John Kepner, project director at Beyond Pesticides, has been with

the organization since 1999. He graduated with a B.S. in biology

from Pennsylvania State University minor in Human Environmental

Relations in 1998.