UNITED STATES ENVIRONMENTAL PROTECTION
AGENCY WASHINGTON, D.C. 20460
APPENDIX D: Memo
Requesting Phototoxicity Study Protocol for Light-Dependent Peroxidizing
SUBJECT: Request for Phototoxicity Study Protocol for Light-Dependent
DATE: March 7, 2001
TO: Elizabeth Leovey, Acting Director Environmental Fate and
Effects Division Office of Pesticide Programs
FROM: Norman B. Birchfield, Ph.D. Thomas M. Steeger, Ph.D. Brian
Montague Aquatic Biology Tech Team
The light-dependent peroxidizing herbicides (LDPHs) are a growing
class of weed control chemicals (see partial listing attached).
They act in plants by inhibiting the enzyme protoporphyrinogen
oxidase (protox), which is the last common enzyme in the heme
and chlorophyll biosynthetic pathways.1 Protox exists in both
plants and animals and the enzyme from both sources has been shown
to be highly sensitive to many LDPHs.2.
LDPH protox inhibition in plants results in a rapid accumulation
of protoporphyrin IX, a phototoxic heme and chlorophyll precursor.
In the presence of light, protoporphyrin IX is a powerful generator
of singlet oxygen which in plants causes lipid membrane peroxidation
leading to a rapid loss of turgidity and foliar burns. LDPH exposure
in mammals has been shown to result in excretion of porphyrins
in urine (porphynuria) and feces, increased liver weight, elevated
blood porphyrin levels, developmental abnormalities, and cancer.
Humans with a hereditary protox disorder (variegate porphyria)
which results in lowered protox activity exhibit many symptoms
similar to LDPH exposure in addition to photosensitivity. However,
photosensitivity is not a commonly reported symptom of LDPH exposure
An LDPH-induced occurrence of phototoxicity in rats 3 and increased
cytotoxicity to human skin cells grown in culture in the presence
of light and an LDPH 4 have been reported but many other LDPH
toxicity studies make no mention of phototoxicity in animals.
The scarcity of phototoxicity data in animals could result from
physiological or biochemical distinctions from plants. For instance,
animals exposed to LDPHs may not normally accumulate protoporphyrin
IX in their epidermis. However, phototoxicity may not be reported
in many LDPH toxicity tests because of relatively low light conditions
in laboratories and/or protection afforded by the animals' fur
or feathers. Animals without fur or feathers existing in sunny
environments would be expected to be at highest risk for potential
The Aquatic Biology Tech Team (ABTT) recommends that phototoxicity
studies be conducted on herbicides with this mode of action to
determine if animals exposed to LDPHs and intense light (similar
to sunlight) show increased toxicity relative to controls exposed
to LDPHs and low intensity light. The results of these studies
will help to determine if animals that are exposed to sunlight
in LDPH use areas are at higher risk than guideline toxicity studies
The ABTT is requesting that a LDPH phototoxicity protocol be
submitted for review and agreement by EFED and the registrant
prior to study initiation. Protocols for standard toxicity tests
have also been published.5 In nature, fish and other aquatic organisms
are expected to be exposed to LDPHs through run-off and spray
drift. Aquatic organisms inhabiting small, shallow water bodies,
exposed to high levels of solar radiation would be expected to
be at greatest risk for potential phototoxic effects. Therefore,
the ABTT is requesting a small fish species be used in a phototoxicity
assay to assess the potential of light to increase LDPH toxicity.
The ABTT requests that the study adequately address the following
issues and suggests the paper, "Photoenhanced Toxicity of a Carbamate
Insecticide to Early Life Stage Anuran Amphibians",5 and other
studies in the peer-reviewed scientific literature serve as sources
of additional guidance:
The fathead minnow may be an appropriate test species because
of existing toxicity protocols which may be adapted for this study.
A subchronic exposure duration would be adequate for proof of
principle. A single exposure may not allow adequate time for porphyrin
accumulation, however, a life-cycle is not necessary to identify
a phototoxic effect.
A range finding study should be conducted under defined low light
conditions to identify an LC50 value and lower dose levels expected
to be similar to controls. Doses used in the phototoxicity study
should not be expected to result in significant mortality in low
light controls. Dissolved concentrations of the test chemical
should be confirmed by an appropriate analytical method.
Behavioral observations should be made in addition to measurements
of mortality, growth, weight, morphology, and appearance. Ideally,
measurements of protoporphyrin and heme concentrations in the
blood and protox activity in the liver of each test organisms
should be made.
Artificial light may be preferred to natural light that will vary
in different regions and seasons as well as with weather. If artificial
light is used, the light should resemble full, natural sunlight
as closely as possible, particularly around 400 nm. The most important
wavelength for porphyrin induced phototoxicity in ~400 nm. No
matter what the light source, the duration and intensity of UV
and visible light should be reported at all wavelengths (200-800
nm). At this point EFED does not have a specific recommendation
for an artificial light source.
Dark, light, and positive controls
As this study is intended to identify potential effects of light
on LDPH toxicity, an appropriate study protocol should include
a dark, or low light, control group. Another group not exposed
to chemicals but exposed to full light should be included (a full
light control). In addition to the dark and light controls, a
positive control group using protoporphyrin IX may be useful.
Exposure chambers and light filters
Light intensity should be measured inside test chambers if glass
or any other material is placed between the light source and the
test animals. Any filters should be cured under the study light
for 72-hours prior to study initiation to ensure consistent transmittance.
1 Matringe, M., J.-M. Camadro, P. Labbe,
and R. Scalla. 1989. Protoporphyrinogen oxidase as a molecular
target for diphenyl ether herbicides. Biochem. J. 260: 231-235.
2 Birchfield, N.B., and J.E. Casida. 1997.
Protoporphyrinogen oxidase of mouse and maize: Target site selectivity
and thiol effects on peroxidizing herbicide action. Pesticide
Biochemistry and Physiology 57, 36-43.
3 Halling, B.P., D.A. Yuhas, V.F. Fingar,
and J.W. Winkleman. 1994. "Protoporphyrinogen oxidase inhibitors
for tumor therapy" in Porphyric Pesticides: Chemistry, Toxicology,
and Pharmaceutical Applications, (S.O. Duke and C.A. Rebeiz, Eds.)
pp. 280-290, American Chemical Society Symposium Series 559, Am.
Chem. Soc., Washington, D.C., 1994.
4 Birchfield, N.B. Protoporphyrinogen Oxidase
as a Herbicide Target: Characterization of the [ 3 H]Desmethylflumipropyn
Binding Site. Dissertation. University of California, Berkeley.
5 American Society for Testing and Materials.
1994. Standard guide for conducting the frog embryo teratogenesis
assay-Xenopus. E 1439-91. In Annual Book of ASTM Standards, Vol
11.5, pp. 825-835. Philadelphia, PA.
11, 2001 - US EPA. Revised
Environmental Fate and Effects Division Preliminary Risk Assessment
for the Oxyfluorfen Reregistration Eligibility
Also of interest:
January 2000 Pharmacology
and Toxicology Guidance for Industry Photosafety Testing.
U.S. Department of Health and Human Services; Food and Drug Administration;
Center for Drug Evaluation and Research (CDER)