Document ID: EPA-HQ-OPP-2009-0302-0002
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2009-06-10T04:00Z

Notice of Filing of Pesticide Petition 9E7548 by Interregional Research
Project Number 4 (IR-4)

EPA Registration Division contact: Barbara Madden

	EPA has received a pesticide petition (PP9E7548) from Interregional
Research Project Number 4 (IR-4), 500 College Road East, Suite 201W,
Princeton, NJ 08540 proposing, pursuant to section 408(d) of the Federal
Food, Drug, and Cosmetic Act (FFDCA), 21 U.S.C. 346a(d), to amend 40 CFR
part 180 by establishing a tolerance for residues of diazinon in or on
the raw agricultural commodity mushrooms at 0.75 parts per million
(ppm).  EPA has determined that the petition contains data or
information regarding the elements set forth in section 408 (d)(2) of 
FDDCA; however, EPA has not fully evaluated the sufficiency of the
submitted data at this time or whether the data supports granting of the
petition. Additional data may be needed before EPA rules on the
petition.

A. Residue Chemistry

	1. Plant metabolism.

The qualitative nature of the residue in plants is adequately understood
for the nature of this tolerance.  Plant metabolism has been studied in
apple, potato, sweet corn, lettuce and green bean.  EPA concluded that
the residues of concern in plants and animals were diazinon, diazoxon
and hydroxydiazinon.  For enforcement purposes only diazinon was
included in the tolerance expression but diazinon and its metabolites
were included in the dietary risk assessment.  Storage stability of
diazinon and hydroxydiazinon indicated frozen stability of residues on
raw agricultural commodities (RAC’s) for up to 26 months.  Diazoxon
was not stable (<3 months).

	2. Analytical method

Adequate analytical methodology is available for data collection and
enforcing tolerances of diazinon.  The enforcement method (AG-550 and
its modifications) is a GC/FPD method that can be used for determination
of residues of diazinon, diazoxon and hydroxyl diazinon in plant and
animal matrices.  There is also a confirmatory method that uses GC/MS.

The FDA PESTDATA database dated 1/94 (PAM Vol. 1, Appendix I) indicates
diazinon is completely recovered using FDA Multi-residue Protocols D and
E (PAM, Vol. 1 Sections 232.4 and 311.1/212.2).  Diazoxon and hydroxyl
diazinon are also completely recovered using protocol D. 

	3. Magnitude of residues. 

Field trial data from IR-4 studies (MRID No. 00140118 and 42322401) are
available for mushrooms treated with diazinon using one pre-spawn
application (2.5 lb a.i./50 gal), one soil drench application (0.585 lb
a.i./50 gal) and two post-spawn applications (2.5 and 2.5 lb
a.a./50gal).  Residues in 13 samples of mushrooms ranged from 0.07 to
0.17 ppm.  EPA has determined that the residue database for the use of
diazinon on mushrooms is complete.

The RDF for partially-blended mushrooms for acute dietary exposure is
comprised of 13 detected residues (0.095, 0.070, 0.097, 0.12, 0.12,
0.12, 0.14, 0.14, 0.13, 0.14, 0.17, 0.12, 0.11 ppm) based on data from
the two IR-4 studies.

For diazinon, the PDP data include samples analyzed for diazinon in
2001, 2002, and 2003, with a combined total of 1464 samples for the 3
years.  Of these, 348 samples had residues, with a range of 0.003 - 0.19
ppm.

B. Toxicological Profile

	1. Acute toxicity.  The acute oral LD50 for diazinon was 1250
milligrams/ kilograms body weight (mg/kg bw) (combined male and female
rats).  The rat acute dermal LD50 for diazinon was greater than 2020
mg/kg bw.  The rat acute inhalation LC50 was >2.33 mg/L (Four hour
exposure with a MMAD of 2.046 µm).  Diazinon was minimally irritating
to the eyes and moderately irritating dermally.  Diazinon was not a
dermal sensitizer.  

In a special study with human a group of 56 human volunteers were
induced and later challenged  with diazinon technical l (1% emulsified
in water with Tween 80) or diazinon 2E diluted in water. Nine inductions
were made over 2-3 days. Challenge was made 14 days after last
induction.  Six out of 56 patients showed a fairly persistent
sensitization, positive results were also obtained from second and third
challenges. Diazinon was considered a positive dermal sensitizer in
humans.

In an acute delayed neurotoxicity study, hens were dosed between 10 and
100mg/kg.  At 10mg/kg and above transient symptoms including reduced
activity and impaired gait were observed.  Three hens died at 100mg/kg. 
There was no evidence of delayed neuropathology and it was concluded
diazinon does not induce delayed type neuropathology in hens.

In an acute neurotoxicity study both behavioral effects and inhibition
of ChE/AChE were assessed.  The LOEL for behavioral effects based on
meiosis and hyperactivity was 250 mg/kg. The NOEL was 100mg/kg and was
considered a threshold dose.  Brain AChE was significantly inhibited at
250 and 500mg/kg in female and male rats respectively.  RBC AChE was
significantly inhibited at 25 and 100 mg/kg in female and male rats
respectively.  Plasma ChE was inhibited at 2.5 and 10 mg/kg in female
and male rats respectively.  The NOEL was 0.25 mg/kg based on plasma ChE
inhibition in female rats at 25 mg/kg.

In an acute neurotoxicity screening study the LOEL for neurotoxicity was
150 mg/kg based on ataxia gait. The NOEL for neurotoxicity was 2.5mg/kg.
 RBC AChE was inhibited at 150mg/kg at the time of peak effect (9 hours)
and plasma ChE was inhibited at the lowest dose, 2.5 mg/kg.

In a further acute study the NOEL for brain AChE inhibition was
established at 2.5 mg/kg based on significant inhibition of AChE in four
regions of the brain and spinal cord at 150mg/kg.

	2. Genotoxicty. 

The potential for genetic toxicity of diazinon was evaluated in several
in vitro and in vivo assays and all acceptable mutagenicity studies
showed a negative mutagenic response. Diazinon was devoid of mutagenic
activity in the Ames (bacterial reverse mutation tests) and an in vitro
mammalian gene mutation test.  Diazinon was negative in an in vivo mouse
micronucleus assay.  A study on unscheduled DNA synthesis in rat
hepatocyte cultures was negative for all doses. Diazinon was negative in
both in vitro and in vivo sister chromatid exchange assays.  It was
concluded diazinon was not mutagenic or genotoxic.

	3. Reproductive and developmental toxicity.

In a multi generation reproduction study parental groups showed
decreased weight gain at 6.69 /7.63 mg/kg/day (♂/♀). The NOEL was
therefore 0.67mg/kg/day.  In the pups at 6.69/7.63 mg/kg there was
mortality and decreased weight gain during lactation, this was the LOEL.
 The NOEL was 0.67mg/kg/day.

Groups of rats were dosed by oral gavage on day 6-15 of gestation and
sacrificed on day 20 of gestation.  The maternal toxicity LOEL was
100mg/kg/day based on a decrease of body weight gain.  The NOEL for
maternal toxicity was 20 mg/kg/day.  In the pups there were no
significant findings at 100 mg/kg/day and the NOAEL was concluded to be
>100mg/kg/day.

In a developmental toxicity study in rabbits, diazinon was administered
by oral gavage at four dose levels.  Systemic toxicity, including death
(9 animals) tremors and convulsions was observed at 100mg/kg/day.  The
NOAEL for maternal toxicity was 25 mg/kg/day.  No compound related
effects on the fetuses were evident therefore the NOAEL for development
toxicity was >100mg/kg/day.

Overall effects on offspring were only seen at a dose that caused
parental toxicity and there was no evidence of abnormalities in the
development of the nervous system.

	4. Subchronic toxicity. 

Rats were dosed with diazinon for 42 days with doses in the range 0.02
to 29 mg/kg/day.

No systemic effects were reported and the NOEL was >29 mg/kg/day.  Brain
AChE was inhibited at 8.60/9.27 and 25.8/29 mg/kg/day ((♂/♀).  RBC
AChE was inhibited at 1.68/1.82 mg/kg/day and plasma ChE significantly
inhibited in and in males and females at 0.17/0.19 mg/kg/day.  The LOEL
was 0.19 mg/kg/day based on the plasma ChE inhibition in females.  The
NOEL was 0.05 mg/kg/day.

In a six week dietary study to rats systemic toxicity was observed in
the 165/168 mg/kg/day (♂/♀) dose group with decreased bodyweight
gain, food consumption and soft faeces. The NOEL for systemic toxicity
was 9.4 mg/kg/day.  At 8.4/9.4 mg/kg/day brain AChE was significantly
inhibited only in females but RBC AChE was significantly inhibited in
both sexes at these dose levels.  Plasma ChE was only significantly
inhibited in female rats at 0.2 mg/kg/day giving the LOEL.  The NOEL was
0.05 mg/kg/day. 

The subchronic toxicity of diazinon has been studied in a 13 week
feeding study in rats

The NOEL for systemic effects was 19 mg/kg /day.  From measurements of
brain, red blood cell (RBC) and plasma cholinesterase (ChE) the NOEL was
0.04 mg/kg/day based on significant inhibition of plasma and RBC ChE in
female rats at 0.4 mg/kg/day.

In a 4-week pilot feeding study in dogs the LOEL for systemic toxicity
was 14.68 mg/kg/day based on body weight effects.  The NOEL was 0.80
mg/kg/day.  From ChE measurements, significant inhibition of brain and
RBC AChE was only observed at the highest doses administered 14.68/15.99
mg/kg/day (♂/♀).  Plasma ChE was inhibited in females but not males
at the lowest dose tested. The LOEL was <0.023mg/kg/day and NOEL not
established.

In a 90 day oral gavage study in dogs the systemic LOEL was 5.6
mg/kg/day based on decreased bodyweight.  The NOEL was 0.021 mg/kg/day. 
Significant inhibition of brain and RBC AChE was only observed at
5.9/5.6 mg/kg/day (♂/♀).  Plasma ChE was significantly inhibited in
males but not in females at 0.020 mg/kg/bw. The LOEL was 0.020mg/kg/day
and NOEL 0.0037 mg/kg/day based on plasma ChE inhibition.

In a 21 day dermal toxicity study in rabbits the systemic toxicity
animals were dosed at 1, 5, and 100 mg/kg bw/day, due to deaths in male
rabbits at 100 mg/kg this was reduced to 50 mg/kg.  The NOEL for
systemic toxicity was 50mg/kg/day.  RBC AChE was significantly inhibited
at 50 mg/kg/day.  A NOAEL of 1 mg/kg was concluded  based on inhibition
of  brain AChE and plasma AChE in females at 5 mg/kg for use in short,
intermediate and long-term dermal exposure risk assessments.  .

In a 21 day rat inhalation study no systemic symptoms were reported at
doses up to 100µg/L, thus this dose was the NOEL.  Brain AChE was
significantly inhibited at 1µg/L in males and females.  RBC AChE was
inhibited at 1µg/L in females and 0.1µg/L in males.  Plasma ChE was
significantly inhibited at 0.1µg/L in both sexes. A definitive  NOAEL
was not determined.  However, the LOAEL was <0.1µg/L (equivalent to
0.026 mg/kg bw/day) based on plasma ChE inhibition in both sexes and RBC
AChE inhibition in male rats. This LOAEL was selected as the endpoint
for inhalation exposure risk assessments.  

In a 90 day subchronic neurotoxicity study the LOEL for systemic and
neurotoxic effects was 180 mg/kg based on weight gain and nervous system
perturbation.  The NOEL was 18 mg/kg.  The LOEL for plasma ChE and RBC
AChE inhibition was 1.8 mg/kg and the NOEL 0.018 mg/kg.

In a special study designed to assess the time course for and regional
brain inhibition of ChE/AChE rats were dosed via the diet and groups
sacrificed after 1, 2 and 4 weeks. At 2.4 mg/kg plasma and RBC AChE were
inhibited.  At 23 mg/kg brain AChE was inhibited with maximum inhibition
at 2 weeks and no marked sensitivity of any one brain region was noted. 
The NOELs for brain and plasma inhibition were 2.4 and 0.02 mg/kg
respectively.

	5. Chronic toxicity. 

at the highest dose tested 12 mg/kg/day, therefore the NOEL is ≥
12mg/kg/day.  Significant inhibition of Brain and RBC AChE was observed
at 5/6 mg/kg/day (♂/♀).  Plasma ChE was inhibited at 0.06/0.07
mg/kg/day but only at termination.  The NOEL was therefore 0.005
mg/kg/day

In a chronic feeding study in dogs dosed with diazinon for 52 weeks The
LOEL for systemic toxicity was 4.5 mg/kg/day based on decreased body
weight.  The NOEL was 0.02mg/kg/day.  Brain AChE was not significantly
inhibited in male dogs at the highest dose tested but was inhibited in
females at 4.5 mg/kg/day.  RBC AChE was significantly inhibited at
4.7/4.5 mg/kg/day in both sexes.  Plasma ChE was significantly inhibited
at 0.02 mg/kg/day in female dogs only giving a LOEL.  The NOEL was
0.0037 mg/kg/day.

In a carcinogenicity study rats were dosed at about 20 and 40 mg/kg/day
for 103 weeks.  No systemic toxicity was reported and there was no
evidence of compound related tumors.

In a carcinogenicity study in mice diazinon was administered in the diet
for 103 weeks.  There were no significant systemic effects or increased
tumor incidence at the highest dose tested, 29 mg/kg/day.

Diazinon was classified as non likely human carcinogen. 

	6. Animal metabolism. 

In all species tested diazinon is rapidly and almost completely absorbed
and the metabolites formed predominately eliminated via the kidneys in
urine.  

The main biotransformation pathways of diazinon in mammals can be
summarised as follows: 

Cleavage of the ester bond of diazinon and diazoxon (G 24576) leading to
the pyrimidinol G 27550 and either diethylphosphorothioate or diethyl
phosphate respectively.  This cleavage is mediated by multi-function
oxidase / hydrolase activity.

Transformation by oxidative desulfuration of the P-S moiety to the P-O
derivative to form diazoxon and other oxons.

Oxidation of the isopropyl functional group leading to the corresponding
tertiary and primary alcohols e.g. formation of GS 31144
(2-hydroxypyrimidinol) and CL-XIX-29 (1-hydroxypyrimidinol).

Oxidation of the methyl functional group on the pyrimidine ring to the
corresponding alcohol e.g. formation of isohydroxy diazinon.

Dehydration of the hydroxyisopropyl group to give isopropenyl compounds.

Glutathione mediated cleavage of the ester bond leading to a glutathione
conjugate of G 27550.

Conjugation of the hydropyrimidinols with glucuronic acid/sulphate. 

Hydrolytic and oxidative cleavage of the phosphorus ester bond, leading
directly or via diazoxon to the pyrimidinol metabolite, G 27550, is the
most significant and important route of diazinon detoxification.
Metabolites maintaining the phosphorus ester e.g diazoxon or
hydroxydiazinon are of very transient nature and are only ever observed
in minor quantities. The tertiary alcohol GS 31144 formed from either G
27550 or hydroxydiazinon is the terminal phase 1 metabolite. No cleavage
of the pyrimidinyl ring to form carbon dioxide has been reported. The
excretion of a water-soluble fraction, which has not been fully
characterised, suggests the GS 31144 metabolite is also excreted as a
conjugate. There is evidence that the diethylphosphorothioate and
diethylphosphate metabolites resulting from cleavage of diazinon or the
oxons respectively are further metabolised to inorganic acids and carbon
dioxide. No O-dethylation of diazinon has been reported to occur in
mammals.

	7. Metabolite toxicology. 

The toxicity of any metabolites has been evaluated in the toxicity
studies for the parent compound.  However the acute inhalation and
subchronic toxicity of the major metabolite –oxypyrimidine (G27750)
have been studied in rat.  The 4-hour inhalation LC50 was >5.22 mg/L in
male and female rats.  In a 5 week dietary study the NOEL was concluded
to be >20 mg/kg/day.

	8. Endocrine disruption. 

In the available toxicity studies on diazinon, there was no estrogen or
androgen, mediated toxicity. When additional appropriate screening
and/or testing protocols being considered under the Agency’s EDSP have
been developed, diazinon will be subjected to further screening and/or
testing to better characterize effects related to endocrine disruption. 

C. Aggregate Exposure

	1. Dietary exposure. 

Dietary Exposure.  The aggregate exposures result from direct food and
indirect food, uses for diazinon.   Indirect exposure to diazinon
residues results from the use as an insecticide in mushroom premises.  
The chronic population adjusted dose (cPAD) was 0.0002 mg/kg/day, from a
weight of evidence NOAEL of 0.02 mg/kg/day.  This was concluded based on
plasma inhibition of ChE in a number of subchronic and chronic studies
in rats and dogs.  An uncertainty factor of 100 was applied to the NOAEL
to account for intra and inter species variation.  An additional FQPA
safety factor applied to the reference doses for protection of infants
and children was not required for diazinon.

For acute dietary exposure and risk assessment, the dose EPA selected
was the NOAEL of 0.25 mg/kg based on plasma ChE inhibition at the LOAEL
of 2.5 mg/kg in an acute neurotoxicity study.  An uncertainty factor of
100 was applied to the NOAEL to account for intra and inter species
variation.  The resultant acute RfD (PAD) was 0.0025 mg/kg/day.

Food. 

Food. The EPA’s chronic dietary assessments (HED April 2000) included
all of the uses for diazinon at that time. This included sufficient
trials data on the magnitude of residues on mushrooms according to
maximum use patterns at that time.  No significant contribution was seen
for the use on mushrooms.  For the most highly exposed subgroup (non
Hispanic/non white/non-black) the mushrooms contributed 2.9% of the
cPAD.  For children 1-6 years old, mushrooms contributed 2.5% of the
cPAD. 

The estimated chronic risk from all sources in (Revised HED assessment,
2000), including mushrooms, ranged from 10% of the cPAD for the US
population to 16% of the cPAD for the most highly exposed subgroup,
Hispanic/non-white/non-black and 10% for children 1-6.  Risk estimates
for all subgroups analyzed were less than 100% of the cPAD and therefore
below EPA’s level of concern.

The estimated acute risks at the 99.9th percentile of exposure for all
uses, including mushrooms, ranged from 35% of the aPAD for the US
population to 64% of the aPAD for the most highly exposed subgroup
Hispanic/non-white/non-black, and 60% for children 1-6 years old. Risk
estimates for all subgroups analyzed were less than 100% of the aPAD and
therefore below EPA’s level of concern.

	ii. Drinking water. 

The EPA has determined a drinking water level of concern (DWLOC) for
diazinon which is the maximum concentration in drinking water which,
when considered together with dietary exposure, does not exceed a level
of concern. 

It was concluded the acute and chronic risk from exposure to diazinon
residues in drinking water from ground and surface water sources water
did not exceed the EPA’s level of concern even though some screening
model estimates exceeded the chronic and acute DWLOC.

 

	2. Non-dietary exposure.

All residential indoor uses, including pet collars and residential
outdoor uses were phased out between 2000 and 2004.  No diazinon
products with residential uses are now sold in the US.

An occupational risk assessment was conducted for mixers, loaders,
applicators and other handlers using the toxicology endpoints EPA used
in the November 30, 2000 ORE Chapter of the diazinon RED. 
Specifically, the inhalation LOAEL from a 21 day inhalation toxicity
study is 0.026 mg/kg/day and the dermal NOAEL from a 21 day dermal
toxicity study is 1 mg/kg/day.  The risk assessment utilized the water
soluble bag formulation, an application rate of 2 lb active
ingredient/50 gallons and a spray volume of 50 gallons per day.  The
personal protective equipment included chemical resistant clothing (rain
suits or Tyvek coveralls) over long-sleeved shirt and long pants,
chemical resistant gloves, and a respirator with an organic-vapor
removing cartridge.  Exposure to diazinon from the individual routes
(dermal and inhalation) indicated acceptable risks.  The short-term
combined inhalation and dermal ARI exceeds 1, indicating acceptable
risks.  The intermediate-term combined MOE is 310, therefore satisfying
EPA’s criteria for an MOE of at least 300.  

D. Cumulative Effects

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ive effects of a particular pesticide’s residues and “other
substances that have a common mechanism of toxicity” when considering
whether to establish, modify, or revoke a tolerance.  Diazinon is an
organophosphate (OP) and has a common mechanism of toxicity with other
organophosphate plant protection products.

EPA has made a cumulative risk assessment associated with exposures to
31 OP’s and concluded that (1) the pesticides covered by the IREDs
(including diazinon) that were pending the results of the OP cumulative
assessment are eligible for re-registration. (2) the pesticide
tolerances covered by the IRED’s ( including mushrooms and diazinon at
0.75 ppm) and TRED’s that were pending the results of the OP
cumulative assessment meet the safety standard under Section 408(b)(2)
of the FFDCA.

Thus, although the use of diazinon in mushrooms was cancelled, the
dietary risk form mushrooms was included in the cumulative risk
assessment.

E. Safety Determination

	1. U.S. population. 

As described above (Section C1) both acute and chronic exposure to
diazinon through food and drinking water sources was below the EPA’s
level of concern. and met the standards of FQPA (IRED, 2006).   These
risk estimates contained many uses, that have since been cancelled or
for which risk mitigation measures have been introduced.

The HED (2000) concluded the acute and chronic aggregate risk to
diazinon from food and drinking water did not exceed the level of
concern for any population sub-group even though some screening model
estimates exceeded the chronic and acute DWLOC.

	2. Infants and children. 

EPA has concluded that no Special FQPA Safety Factor is necessary to
protect the safety of infants and children.  EPA reduced the FQPA Safety
Factor to one for diazinon based on acceptable developmental toxicity
and reproductive studies for diazinon showing no evidence of increased
toxicity to offspring.  Adequate actual data, surrogate data and
modeling outputs were all available to satisfactorily assess dietary and
residential exposure and to provide screening level drinking water
exposure assessment.

F. International Tolerances

Although CODEX MRLs for diazinon have been established for many
commodities, a CODEX MRL for mushrooms has not been established.

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