Document ID: EPA-HQ-OPP-2004-0292-0028
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2007-09-12T04:00Z

EPA Primary Reviewer:  Ghazi Dannan, Ph.D. 				Signature: 					___

	Date: 							

EPA Secondary Reviewer:  Stanley Gross, Ph.D. 	Signature: 					___

	Date: 							

Registration Action Branch 3, Health Effects Division (7509P)

	

EPA Work Assignment Manager:  P.V. Shah, Ph.D.	Signature: 						

Registration Action Branch 1, Health Effects Division (7509P)	Date: 				
		

	Template version 02/06

DATA EVALUATION RECORD

STUDY TYPE:	Subchronic Inhalation Toxicity - [rat];
OPPTS 870.3465 [82-4]; OECD 413.

PC CODE:  099100	DP BARCODE:  D322409 

TXR#:  0053805

TEST MATERIAL (PURITY):  BAS 500F technical (98.7% a.i.)

SYNONYMS:	Pyraclostrobin; methyl
[2-[[[1-(4-chlorophenyl)-1H-pyrazol-3-yl]methyl] phenyl]methoxycarbamate

CITATION:	Gamer, A.O., K. Deckardt, S. Burkhardt, et al. (2005) BAS 500F
– Subacute inhlation study in Wistar rats – 20 aerosol exposures
during 4 weeks.  Experimental Toxicology and Ecology, BASF
Aktiengesellschaft, Ludwigshafen/Rhein, Germany.  Laboratory Project
Id.# 4010494/96073, BASF doc. No. 2005/1013950, August 9, 2005.  MRID
46638801.  Unpublished.

SPONSOR:	BASF Corporation, Agricultural Products, P.O. Box 13528,
Research Triangle Park, NC

EXECUTIVE SUMMARY:  In a subchronic inhalation toxicity study (MRID
46638801), Pyraclostrobin (BAS 500F) technical (98.7% a.i., Batch #
LJ27882/199/b) was dissolved in acetone and administered as an aerosol
to 10 Wistar rats/sex/concentration by nose-head only exposure at
concentrations of 0 (air), 0 (vehicle control), 0.001, 0.030, or 0.300
mg/L for 6 hours per day, 5 days/week for 28 days (i.e., 20 exposure
days).

There were no treatment-related effects on any parameters examined
during the FOB or on locomotor activity, ophthalmoscopy, clinical
chemistry, or organ weights except for decreased thymus weight and
relative weight (27-29%) in females and increased relative spleen weight
(33%) in males of the 0.300 mg/L group.

At 0.300 mg/L, several clinical signs of toxicity were observed among
males and females during daily or weekly examinations including urine
odor, slight visually increased respiration, and piloerection in
addition to abdominal position and moderate labored respiration observed
in one male on Day 21.  Additionally, at this concentration, four males
and three females died prior to scheduled termination.  Prior to death,
these animals exhibited visually increased respiration, urine odor, and
piloerection.

At 0.300 mg/L, decreases in body weights of 4-7% (not significant [NS]
except on Day 21) and decreases in cumulative body weight gains of
43-141% (p<0.05) were noted in the males throughout the study.  Food
efficiency was decreased (p<0.05) at this concentration on Day 7 

(-2.6% treated vs 4.0% controls) and on Day 21 (6.5% treated vs 9.9%
controls).  Additionally on Day 21, food efficiency was decreased in the
0.030 mg/L males (5.8% treated vs 9.9% controls).  In both sexes at this
concentration, an initial decrease of 11-13% (p<0.05) in food
consumption was observed on Day 7.    

At 0.300 mg/L, absolute and relative (to body) thymus weights were
decreased (27-29%, p<0.01) in males while relative spleen weight was
increased in females (33%, p<0.01).  Minor changes were also seen in
weights of liver, kidneys, and lungs but were not considered
treatment-related. 

At 0.300 mg/L, leaukocytes and neutrophils were increased in both sexes.
 As discussed below, the increased white blood cells (specifically
neutrophils) may be an inflammatory response to the treatment-related
effects on the respiratory tissues.

At 0.030 and 0.300 mg/L, diffuse mucosal hyperplasia of the duodenum was
observed in 5-7 males (vs 0 controls) and in 5-10 females (vs 1
control).  This finding increased with dose in both incidence and
severity.  The duodenum was also a target organ in the 90-day dietary
feeding studies in the rat, mouse, and dog (MRIDs 45118321, 45118320 and
45118323).

Other microscopic changes were effects on the respiratory system at
0.030 and 0.300 mg/L, including minimal to slight alveolar histiocytosis
in the females (5 treated vs 1 control) and minimal to moderate
olfactory atrophy/necrosis in nasal cavities II through IV in the males
(2-10) and females (3-9) compared to 0 controls.

Additional necropsy and microscopic findings in the respiratory system
in both sexes were noted at 0.300 mg/L including lung discoloration,
lung congestion, minimal to moderate hyperplasia of the respiratory
epithelium in nasal cavities I through IV, reactive inflammation in
nasal cavity I, slight olfactory regeneration/repair in nasal cavity IV,
minimal to slight respiratory hyperplasia in level III of the larynx
(males only), and minimal to slight squamous cell metaplasia in nasal
cavity III.

The LOAEL is 0.030 mg/L based on findings of hyperplasia in the
duodenum, alveolar histiocytosis in the lungs, and olfactory
atrophy/necrosis in the nasal tissues.  The NOAEL is 0.001 mg/L.

At the request of the Agency, this study was conducted for 28 days,
instead of the 90 days required by Guideline OPPTS 870.3465.  Aside from
the different study duration, this study was conducted in accordance
with Guideline OPPTS 870.3465.

This 28-day study is classified as acceptable/guideline and satisfies
the guideline requirement (OPPTS 870.3465; OECD 413) for a subchronic
inhalation study in the rat.

COMPLIANCE - Signed and dated Data Confidentiality, GLP Compliance, and
Quality Assurance statements were provided.  A Flagging statement was
not provided.

I.	MATERIALS AND METHODS:

A.	MATERIALS:

1.	Test material:	BAS 500F

	

Description:	Red-brown, clear solid melt

	

Lot/batch #:	

LJ27882/199/b

	

Purity:	98.7% a.i.

	

Compound stability: 	

It was stated that the stability of the test article under the storage
conditions was confirmed by reanalysis; however, only one purity value
was reported.  Stability of the test substance in the vehicle was not
reported.

	

CAS # of TGAI: 	

175013-18-0

	

Structure:	

2.	Vehicle and/or positive control:  Acetone

3.	Test animals	

	

Species:	

Rat

	

Strain:	

Wistar [Crl:WI(Han)]

	

Age/weight at study initiation:	

Approximately 9 weeks old; 225.4-267.5 g males, 158.9-193.9 g females

	

Source:	

Charles River Deutschland GmbH (Sulzfeld, Germany)

	

Housing:	

Individually in type MD III Makrolon wire cages, except during the motor
activity measurements which were conducted in polycarbonate cages with
wire covers.

	

Diet:	

Milled mouse/rat laboratory diet “GLP” (Provimi Kliba SA,
Kaiseraugst, Basel Switzerland), ad libitum, except during exposure and
motor activity measurements, and overnight (16-20 hours) prior to blood
collection and termination

	

Water: 	Tap water, ad libitum, except during exposure and motor activity
measurements

	

Environmental  conditions:	

Temperature:

Humidity:

Air changes:

Photoperiod:	

20-24(C

30-70%

Not reported

12 hrs dark/ 12 hrs light

	

Acclimation period:	Approximately 2 weeks

B.	STUDY DESIGN:

1.	In life dates:  Start:  January 24, 2005 		End:  February 25, 2005

2.	Animal assignment:  Animals were randomly assigned, stratified by
body weight, to the test groups noted in Table 1.  Individual body
weights at randomization were within ± 20% of the mean body weight for
each sex.

TABLE 1:  Study design a

Test group	Target conc. 

(mg/L) b	Analytical conc. 

(mg/L) b	MMAD

(m	

GSD	Rats/sex

Control (air)	0	0	---	---	10

Control (vehicle)	0	0	---	---	10

Low (LCT)	0.001	0.00117 ± 0.0003	1.6 ± 0.3	4.0 ± 0.2	10

Mid (MCT)	0.030	0.0304 ± 0.0012	1.9 ± 0.1	2.6 ± 0.1	10

High (HCT)	0.300	0.299 ± 0.015	1.9 ± 0.1	2.5 ± 0.2	10

a	Data were obtained from pages 22 and 176 of the study report.

b	It was stated that the nominal concentrations could be calculated from
the pump rates and air flows during exposure.  However, these values
were not determined because in aerosol studies they are not comparable
and are of no significance to the analytically measured concentrations. 
Thus, target concentrations are presented.

3.	Concentration selection rationale:  It was stated that the
concentrations were requested by the Sponsor, with the 0.300 mg/L
concentration expected to cause toxic effects and the 0.001 mg/L
concentration expected to be the NOAEL.  However, no further
concentration-selection rationale was provided.

4.	Test material administration:  The test substance was administered in
the vehicle as a liquid aerosol via nose-only inhalation for 6 hours per
day, 5 days per week for 4 consecutive weeks (20 total exposures).

5.	Generation of the test atmosphere / chamber description:  Diagrams of
the test atmosphere generation system and exposure chamber were included
as Figures 1a and 1b on page 71 of the study report.  These figures are
included in the Appendix of this DER.  The test substance was diluted at
a ratio of 1 part test substance to 9 parts acetone.  For each
concentration, test atmospheres were generated as aerosols by supplying
the test substance in air (Group 1) or in the vehicle (Groups 2-5) to a
two-compartment atomizer (pressure of 0.8-1.2 bars) at a constant rate
by means of a metering pump/infusion pump.  The aerosol was generated
with filtered compressed (approximately 6 bar) air inside the inhalation
system at a rate of 1 m3/h.  In the vehicle control group (Group 2),
acetone was sprayed at a pump rate equivalent to that of the high
concentration group.  Supply air was filtered via an activated charcoal
filter, conditioned to approximately 50 ± 20% relative humidity and 22
± 2ºC, and supplied at a rate of 5.0-6.0 m3/h.  Actual measurements of
the environmental conditions in the exposure chambers resulted in:  mean
relative humidity ranging between 35.6 and 50.3%; mean temperature of
21.2-22.3ºC, and approximately 67 air changes per hour.  Although
oxygen concentration was not measured, it was stated that the air change
rate within the inhalation system was judged to be sufficient to prevent
oxygen depletion and that the concentrations of the test substance were
not expected to affect the oxygen partial pressure.  Time to equilibrium
was not reported.

The head-nose exposure system consisted of a cylindrical inhalation
chamber made of stainless steel sheeting and cone-shaped outlets and
inlets.  During exposure, the rats were restrained in glass exposure
tubes with their snouts projecting into the inhalation chamber.  In
order for the animals to acclimate to the exposure system, the rats were
treated with supply air under conditions comparable to exposure on the 2
days prior to initiation of the first exposure.  Rats were exposed under
positive pressure, with an exhaust air flow rate of 5.4 m3/h.

Test atmosphere concentrations – The concentrations of the inhalation
atmospheres were determined by gravimetric analyses and reported as
daily means based on two measured samples per concentration per
exposure.  Results are presented in Table 1 above.  The constancy of the
concentration of the test atmosphere in each inhalation chamber was
continuously monitored using scattered light photometers.  Acetone
concentration was measured once per exposure in the control and high
concentration groups using gas chromatography.  It was stated that the
stability of the test article under the storage conditions was confirmed
by reanalysis; however, only one purity value was reported.  Stability
of the test substance in the vehicle was not reported.

Particle size determination – Aerosol particle size was determined for
each exposure group using cascade impactors.  Mass median aerodynamic
diameter (MMAD) and geometric standard deviation (GSD) results are
presented in Table 1 above.  In addition to these parameters, the
percent of particles less than 3 µm was measured and ranged from
65.4-71.9%.

6.	Statistics:  The following statistical analyses were performed:

Parameter	Statistical test

Body weight

Body weight gain

Food consumption

Food efficiency	All test groups were compared with the vehicle control
group using a two-sided Dunnett’s test.  The vehicle control group was
compared with the air control group using a two-sided Welch t-test

Feces

Rearing

Fore- and hind-limb grip strength

Landing foot splay

Motor activity

Hematology, except reticulocytes and differential blood count

Clinical chemistry

Organ weights and terminal body weight	A non-parametric analysis of
variance was conducted using a two-sided Kruskal-Wallis test to
determine differences among any of the groups.  If a difference
(p≤0.05) was detected, then groups exposed to the test material were
compared with the vehicle control group using a two-sided Wilcoxon test.
 The vehicle control group was compared with the air control group using
a two-sided Wilcoxon test

Significance was denoted at p≤0.05 and p≤0.01.  The statistical
methods were considered appropriate.

C.	METHODS

1.	Observations

	1a.	Cageside observations:  All animals were observed for mortality and
moribundity twice daily (in the morning and late afternoon) during the
week and once daily (in the morning) on weekends and holidays.

	1b.	Clinical examinations:  The clinical condition of each animal was
recorded at least three times on exposure days (before, during, and
after exposure) and once during pre-flow and on days on which the
animals were not exposed.  During exposure, only a limited examination
was possible due to the animals’ location in the exposure tubes. 
Detailed physical examinations were performed prior to exposure and
weekly thereafter.  The following CHECKED (X) parameters were examined
during handling and in a standard arena:

X	Abnormal behavior during handling	X	Abnormal movements

X	Fur	X	Impairment of gait

X	Skin	X	Lacrimation

X	Posture	X	Palpebral closure

X	Salivation	X	Exophthalmia

X	Respiration	X	Feces (appearance/consistency)

X	Activity/arousal level	X	Urine

X	Tremors	X	Pupil size

X	Convulsions

	1c.	Neurological evaluations:  A functional observational battery (FOB)
and an assessment of motor activity were conducted on all rats on Day
22.  Animals were not exposed on the day that the FOB and motor activity
tests were conducted.  At least 30 minutes prior to testing, the cages
were placed in racks in a randomized order, and food and water were
withheld.  The FOB started with passive observations without disturbing
the animals in their home cage, followed by removal from the home cage,
and open field observations in a standard arena.  Thereafter,
sensorimotor tests and reflex tests were conducted.  It was stated that
a trained technician conducted the FOB, and this technician had
performed positive control studies as part of the training.  Scoring
criteria were provided on pages 424-434 of the study report.  The
following CHECKED (X) parameters were evaluated during the FOB:

	HOME CAGE OBSERVATIONS

HANDLING OBSERVATIONS

OPEN FIELD OBSERVATIONS

X	Posture	X	Reactivity

Mobility

	Biting	X	Lacrimation / chromodacryorrhea	X	Rearing

X	Convulsions	X	Salivation	X	Arousal/ general activity level

X	Tremors

Piloerection	X	Convulsions

X	Abnormal Movements	X	Fur appearance	X	Tremors

X	Palpebral closure	X	Palpebral closure	X	Abnormal movements

	Feces consistency	X	Respiratory rate	X	Urination / defecation

X	Impairment of gait	X	Red/crusty deposits

Grooming

	SENSORY OBSERVATIONS	X	Mucous membranes /eye /skin color	X	Gait
abnormalities / posture

X	Approach response	X	Eye prominence

Gait score

X	Touch response

Muscle tone	X	Bizarre / stereotypic behavior

X	Startle response	X	Pupil size

Backing

X	Pain response	X	Behavior during handling

Time to first step

X	Pupil response

	Eye blink response

PHYSIOLOGICAL OBSERVATIONS

NEUROMUSCULAR OBSERVATIONS

	Forelimb extension

Body weight

Hind limb extensor strength

	Hind limb extension

Body temperature	X	Fore limb grip strength

X	Righting reflex

	X	Hind limb grip strength

	Olfactory orientation

	X	Landing foot splay

X	Visual placing response

OTHER OBSERVATIONS

Rotarod performance

X	Pinna reflex	X	Vocalization

Motor activity was measured in the dark using a Multi-Varimex-System
(Columbus Instruments International Corp., Ohio) with four infrared
beams per cage.  During the measurements, the animals were placed (in
randomized order) into polycarbonate cages with absorbent material, and
food and water were withheld.  The numbers of beam interruptions were
counted over 12 intervals, with each interval lasting 5 minutes.

2.	Body weight:  Animals were weighed on Day –7, at the start of the
pre-flow acclimation, at the start of the exposure period, once weekly
thereafter, and one day prior to scheduled termination.  Cumulative body
weight gain was reported weekly throughout the study.

3.	Food consumption and food efficiency:  Food consumption was
determined for each animal on Day –5 and weekly thereafter throughout
the study, and mean daily diet consumption was calculated for each
weekly interval as g/animal/day.  Food efficiency was calculated from
the individual values for each rat by dividing the differences in body
weights over an interval by the food consumption during that period and
multiplying by 100.

4.	Ophthalmoscopic examination:  Ocular examinations were conducted
prior to the initiation of exposure (Day –1 or Day –3) on all
animals and on Day 22 for rats in the 0 mg/L (air and vehicle controls)
and 0.300 mg/L groups.

5.	Hematology and clinical chemistry:  At scheduled termination, all
surviving animals were fasted overnight (but were allowed ad libitum
water), anesthetized with isoflurane, and blood was collected from the
retro-orbital venous plexus for hematology and clinical chemistry
analysis.  The CHECKED (X) parameters were examined.

	a.	Hematology

X	Hematocrit (HCT)*	X	Leukocyte differential count*

X	Hemoglobin (HGB)*	X	Mean corpuscular HGB (MCH)*

X	Leukocyte count (WBC)*	X	Mean corpuscular HGB concentration (MCHC)*

X	Erythrocyte count (RBC)*	X	Mean corpuscular volume (MCV)*

X	Platelet count*	X	Reticulocyte count

	Blood clotting measurements*

	(Activated partial thromboplastin time)

	(Clotting time)

X	(Prothrombin time)

* Recommended for subchronic inhalation studies based on Guideline
870.3465

	b.	Clinical chemistry

	ELECTROLYTES

OTHER

X	Calcium	X	Albumin*

X	Chloride	X	Creatinine*

X	Magnesium	X	Urea nitrogen*

X	Phosphorus	X	Total Cholesterol*

X	Potassium*	X	Globulins

X	Sodium*	X	Glucose*

	ENZYMES (more than 2 hepatic enzymes eg., *)	X	Total bilirubin

X	Alkaline phosphatase*	X	Total serum protein (TP)*

	Cholinesterase	X	Triglycerides

	Creatine phosphokinase

Serum protein electrophoresis

	Lactic acid dehydrogenase (LDH)

Albumin/globulin (A/G) ratio

X	Alanine aminotransferase (ALT/also SGPT)*

X	Aspartate aminotransferase (AST/also SGOT)*

	Sorbitol dehydrogenase*

X	Gamma glutamyl transferase (GGT)*

	Glutamate dehydrogenase

* Recommended for subchronic inhalation studies based on Guideline
870.3465

6.	Urinalysis:  Urinalysis was not conducted and is not required under
Guideline 870.3465.

7.	Sacrifice and pathology:  Following blood collection, all animals
were euthanized by exsanguination from the abdominal aorta and vena cava
while under Narcoren anesthesia and were subjected to a gross necropsy. 
The CHECKED (X) tissues were collected from all animals and were fixed
in neutral-buffered 4% formaldehyde.  Additionally from the liver,
samples of the right and left lateral lobes were preserved in Carnoy’s
solution.  These tissues were processed routinely, stained with
hematoxylin and eosin, and examined microscopically in the control (air
and vehicle) and high concentration groups.  Additionally in the
intermediate concentration groups, the nasal cavities, lungs, duodenum,
and any gross lesions were examined in both sexes, and the larynx was
examined in the males.  The (XX) organs, in addition, were weighed.

	DIGESTIVE SYSTEM

CARDIOVASC./HEMAT.

NEUROLOGIC

	Tongue	X	Aorta, thoracic*	XX	Brain*+

X	Salivary glands*	XX	Heart*+	X	Peripheral nerve (sciatic)*

X	Esophagus*	X	Bone marrow*	X	Spinal cord (3 levels)*

X	Stomach*	X	Lymph nodes*	X	Pituitary*

X	Duodenum*	XX	Spleen*+	X	Eyes (optic nerve )*

X	Jejunum*	XX	Thymus*+

GLANDULAR

X	Ileum*

	XX	Adrenal gland*+

X	Cecum*

UROGENITAL	X	Lacrimal gland

X	Colon*	XX	Kidneys*+	X	Parathyroid*

X	Rectum*	X	Urinary bladder*	XX	Thyroid*

XX	Liver*+	XX	Testes*+

OTHER

	Gall bladder* (not rat)	XX	Epididymides*+	X	Bone (sternum and/or femur)

	Bile duct* (rat)	X	Prostate*	X	Skeletal muscle

X	Pancreas*	X	Seminal vesicles*	X	Skin

	RESPIRATORY	XX	Ovaries*+	X	All gross lesions and masses*

X	Trachea*	XX	Uterus*+

XX	Lung*	X	Mammary gland (females)*

X	Nasal cavities*	X	Oviducts

X	Pharynx*	X	Vagina

X	Larynx*	X	Coagulation glands

* Recommended for subchronic rodent studies based on Guideline 870.3465

+  Organ weights required

II.	RESULTS:  Unless otherwise stated, the acetone control group was
comparable to the air control group, indicating no effect of the
vehicle.  Any mention of comparison of treated groups to controls refers
to the vehicle control.

A.	OBSERVATIONS

1.	Clinical signs of toxicity:  Selected daily clinical observations are
presented in Tables 2a and 2b.  At 0.300 mg/L, abdominal position and
moderate labored respiration were observed in one male on Day 21. 
Additionally at this concentration, the following clinical signs of
toxicity were observed [# affected (day of mean onset)]:  (i) urine odor
in 4 males (Day 29) and in 7 females (Day 24); (ii) slight visually
increased respiration in 10 males (Day 9) and in 10 females (Day 8); and
(iii) piloerection in 3 females (Day 20).  No other clinical
observations could be attributed to treatment.  During the detailed
clinical observations, similar findings were observed [# affected (day
of mean onset)], including: (i) urine odor in one male (Day 28) and in 3
females (Day 26); (ii) slight visually increased respiration in 8 males
(Day 12) and in 8 females (Day 14); and (iii) piloerection in 3 females
(Day 21).  No other clinical observations could be attributed to
treatment.

TABLE 2a. Clinical signs of toxicity in male rats during 28 days of
treatment with BAS 500 F via inhalation a

Observation/parameter	

Analytical concentration (mg/L)

	0	0.001	0.030	0.300

Abdominal position b

Number of animals	0	0	0	1

Number of observations	0	0	0	1

Mean onset (days)	0	0	0	21

Labored respiration, moderate b

Number of animals	0	0	0	1

Number of observations	0	0	0	1

Mean onset (days)	0	0	0	21

Urine odor

Number of animals	0	0	0	4

Number of observations	0	0	0	10

Mean onset (days)	0	0	0	29

Visually increased respiration, slight

Number of animals	0	0	0	10

Number of observations	0	0	0	319

Mean onset (days)	0	0	0	9

a	Data obtained from Tables IA and IIA on pages 76-77 and 188 in the
study report.

b	Observed in the same animal (#47).

TABLE 2b. Clinical signs of toxicity in female rats during 28 days of
treatment with BAS 500 F via inhalation a

Observation/parameter	

Analytical concentration (mg/L)

	0	0.001	0.030	0.300

Urine odor

Number of animals	0	0	0	7

Number of observations	0	0	0	41

Mean onset (days)	0	0	0	24

Visually increased respiration, slight

Number of animals	0	0	0	10

Number of observations	0	0	0	336

Mean onset (days)	0	0	0	8

Piloerection

Number of animals	0	0	0	3

Number of observations	0	0	0	23

Mean onset (days)	0	0	0	20

a	Data obtained from Table IA page 78 in the study report.

2.	Mortality:  At 0.300 mg/L, four males died prior to scheduled
termination (one each on Days 10, 12, 21, and 22), and three female rats
died (one each on Days 7, 11, and 24).  These animals exhibited visually
increased respiration, urine odor, and piloerection prior to death.  All
other rats survived until scheduled termination.

(p≤0.05) in the 0.030mg/L males compared to vehicle controls during
Interval 4.  However, this increase was considered unrelated to
treatment because it was transient, did not occur at the highest
concentration, and locomotor activity in all other 5-minute intervals
and in the overall (12-interval) session was comparable to controls for
both sexes and all concentrations.  Habituation was unaffected by
treatment.

BODY WEIGHT AND WEIGHT GAIN:  In the 0.300 mg/L male group, minor
decreases in body weights (↓4-7%) were noted throughout the study, and
attained significance on Day 21 (Table 3).  Cumulative body weight gains
were decreased (↓43-141%; p≤0.05) in these rats throughout the
study.  Body weights and body weight gains in the other treated males
and in all of the treatment groups in the females were comparable to
controls throughout the study.

TABLE 3. Mean ± SD body weights and cumulative body weight gains (g)
during 28 days of treatment a

Day	

241.0 ± 17.3 (↓4)

14	259.3 ± 13.8	264.9 ± 14.5	255.2 ± 10.0	246.4 ± 16.3 (↓5)

21	272.7 ± 14.8	276.2 ± 15.0	263.0 ± 11.3	254.8 ± 18.1* (↓7)

28	280.4 ± 15.0	285.0 ± 16.7	273.0 ± 12.0	265.6 ± 18.3 (↓5)

0-7	5.4 ± 5.3	4.0 ± 7.6	2.0 ± 4.7	-2.2 ± 7.7* (↓141)

0-14	14.7 ± 7.2	15.1 ± 11.8	11.0 ± 4.3	4.6 ± 6.3* (↓69)

0-21	28.0 ± 7.7	26.4 ± 13.7	18.8 ± 5.8	13.0 ± 8.0** (↓54)

0-28	35.7 ± 9.1	35.2 ± 17.7	28.8 ± 7.2	20.4 ± 7.9* (↓43)

Females

0	174.5 ± 10.2	176.2 ± 8.1	172.9 ± 7.0	172.7 ± 10.7

28	191.2 ± 8.3	187.5 ± 9.3	187.0 ± 8.8	189.0 ± 11.2

0-28	16.7 ± 5.8	11.3 ± 4.4	14.2 ± 5.1	13.5 ± 9.0

a	Data obtained from pages 81-82 and 85-86 in the study report; n =
6-10.  Percent difference from controls, calculated by the reviewers, is
included in parentheses.

↓11-13%; p≤0.05) in food consumption was observed in both sexes on
Day 7.  There were no other treatment-related differences in food
consumption in either sex.

2.	Food efficiency:  In the males, food efficiency was decreased
(p≤0.05) at 0.300 mg/L on Day 7 (-2.6% treated vs 4.0% controls) and
on Day 21 (6.5% treated vs 9.9% controls).  Additionally on Day 21, food
efficiency was decreased in the 0.030 mg/L males (5.8% treated vs 9.9%
controls).  Food efficiency was comparable to controls in the females
throughout the study.

D.	OPHTHALMOSCOPIC EXAMINATION:  There were no treatment-related ocular
lesions.

E.	BLOOD ANALYSES

1.	Hematology:  At 0.300 mg/L, the numbers of leukocytes were increased
by 36% and 46% (both not significant [NS]) in the males and females,
respectively (Table 4).  This increase may be accounted for by increased
lymphocyte numbers in females (↑39%) and increased number and percent
of neutrophils (↑38-215%) in both sexes.  In this group, reticulocytes
were also increased by 68% and 37% (NS) in males and females,
respectively (not shown in Table 4).   A minor decrease (↓4%;
p≤0.05) in mean corpuscular hemoglobin concentration was noted in the
0.300 mg/L males.  There were no other treatment-related findings in
hematology.  

In the vehicle control group, minor differences (p≤0.05) compared to
the air control group were noted in the females, including decreased
erythrocytes (↓4%), increased mean corpuscular volume and mean
corpuscular hemoglobin (↑4% each), and decreased platelets (↓8%). 
All other findings in the treated groups were comparable to their
respective controls.

TABLE 4. Selected mean (± SD) hematology findings after 28 days of
treatment with BAS 500F via inhalation a

Parameter	

↑ 18)	7.63 ± 3.60 (↑36)

Neutrophils (109/L)	0.92 ± 0.29	1.00 ± 0.50	1.11 ± 0.78 (↑ 20.7)
2.90 ± 3.70 (↑215)

Neutrophils (%)	16.3 ± 2.0	17.3 ± 5.4	16.1 ± 6.2	31.0 ± 20.0 (↑90)

Females

Leukocytes (109/L)	3.57 ± 0.99	3.72 ± 1.08	3.80 ± 1.20	5.24 ± 1.35
(↑46)

Neutrophils (109/L)	0.56 ± 0.21	0.47 ± 0.11	0.56 ± 0.27	1.13 ± 0.49
(↑102)

Neutrophils (%)	15.7 ± 3.5	13.0 ± 2.9	15.0 ± 4.8	21.6 ± 5.9 (↑38)

a	Data obtained from pages 125-130 in the study report.  Percent
difference from controls, calculated by the reviewers, is included in
parentheses.  n = 10, except in the 0.300 mg/L group, where n = 6 males
and n = 7 females.

2.	Clinical chemistry:  With the exception of a slight increase of 32 %
(p≤0.05) in alkaline phosphatase in the 0.300 mg/L female group, there
were no changes in clinical chemistry parameters that may be attributed
to exposure to Pyraclostrobin.  Minor differences (p≤0.05) were noted
in the vehicle control group compared to the air control group,
including:  (i) decreased alanine aminotransferase (↓20%), aspartate
aminotransferase (↓18%), and potassium (↓6%) in the females; (ii)
increased total bilirubin in the males (↑18%); and (iii) increased
total protein in the males (↑2%).

F.	URINALYSIS:  Not conducted.

G.	SACRIFICE AND PATHOLOGY

1.	Organ weight:  At 0.300 mg/L, absolute and relative (to body) thymus
weights were decreased by 27-29% (p≤0.01) in the females, and relative
spleen weight was increased by 33% (p≤0.01) in the males (Table 5). 
Even though there were little or no corroborating microscopic findings
in these organs, the weight changes may be related to treatment.  In the
90-day oral feeding study (MRID 45118321), rats of both sexes had a
dose-dependent increase in spleen weights in addition to histopathology
changes including sinus distension, extramedullary hematopoiesis, and
histiocytosis.  

TABLE 5. Selected mean (± SD) organ weight findings after 28 days of
treatment with BAS 500F via inhalation a

Parameter	

Analytical concentration (mg/L)

	0 	0.001	0.030	0.300

Males

Number of Animals	10	10	10	6

Terminal body weight (g)	249.9	253.5	243.7	234.8

Thymus: Absolute weight (mg)

               % Relative weight	224.8 ± 56.6

0.09 ± 0.021	257.2 ± 61.0

0.101 ± 0.021	219.3 ± 37.0

0.09 ± 0.016	223.5 ± 82.5

0.094 ± 0.033

Spleen: Absolute weight (mg)

             % Relative weight	452 ± 73

0.18 ± 0.025	497 ± 74

0.196 ± 0.025	463 ± 56

↑ 25)

0.24 ± 0.049** (↑ 33)

Females

Number of Animals	10	10	10	7

Terminal body weight (g)	167.2	165.4	165.3	165.2

Thymus: Absolute weight (mg)

               % Relative weight	260.8 ± 54.3

0.155 ± 0.028	234.1 ± 44.8

0.141 ± 0.025	213.5 ± 54.0

0.128 ± 0.028	186.1 ± 33.5** (↓ 29)

0.113 ± 0.022** (↓ 27)

Spleen: Absolute weight (mg)

             % Relative weight	389 ± 75

0.232 ±0.041	367 ± 45

0.222 ± 0.023	382 ± 43

0.231 ± 0.028	429 ± 34

0.260 ± 0.015

a	Data obtained from pages 161-168 in the study report.  Percent
difference from controls, calculated by the reviewers, is included in
parentheses.

mg/L females, increased (p≤0.05) relative weights of the kidney
(↑6%) and liver (↑12%) were observed; however, these increases were
minor and were not corroborated by findings in clinical chemistry or
histopathology.  In the male vehicle controls, absolute and relative (to
body) lung weights were decreased by 7-8% (p≤0.05), and relative liver
weight was decreased 4% compared to the air control group; however,
these decreases were minor and were not considered to be a toxic effect
of the vehicle.  There were no other significant differences from
controls in absolute or relative organ weights in either sex.

2.	Gross pathology:  Lung discoloration was observed in 2/10 males and
3/10 females compared to 0/10 animals in each control group.  There were
no other macroscopic findings which could be attributed to treatment.

3.	Microscopic pathology:  Treatment-related microscopic findings are
included below in Tables 6a (males) and 6b (females).  At 0.030 and
0.300 mg/L, diffuse mucosal hyperplasia of the duodenum was observed in
5-7 males (vs 0 controls) and in 5-10 females (vs 1 control).  This
finding increased with dose in both incidence and severity.  In 90-day
or 28-day oral administration studies in rats, there was duodenal
thickening and hyperplasia (MRIDs 45118321/45118322).  The duodenum was
also a target organ in the 90-day dietary feeding studies in the mouse
and dog (MRIDs 45118320 and 45118323).  In addition, spleen
extramedullary hematopoiesis (multifocal, grade 1 or 2) was found in one
animal per sex of the 0.300 mg/L group; all other groups had none.

The following remaining microscopic findings were observed in the
respiratory system (number affected per group out of 10 vs 0 controls,
unless otherwise noted):  (i) minimal to slight alveolar histiocytosis
in the 0.030 and 0.300 mg/L females (5 vs 1); (ii) lung congestion in
the 0.300 mg/L males and females (3); (iii) minimal to moderate
hyperplasia of the respiratory epithelium in nasal cavities I through IV
at 0.300 mg/L in the males (2-10 vs 0-1 controls) and females (7-9);
(iv) reactive inflammation in nasal cavity I in the 0.300 males (2) and
females (4); (v) minimal to moderate olfactory atrophy/necrosis in nasal
cavities II through IV in the 0.030 and 0.300 mg/L males (2-10) and
females (3-9); (vi) slight olfactory regeneration/repair in nasal cavity
IV in the 0.300 mg/L males (9) and females (8); (vii) minimal to slight
respiratory hyperplasia in level III of the larynx in the 0.300 mg/L
males (3); and (viii) minimal to slight squamous cell metaplasia in
nasal cavity III in the 0.300 mg/L females (4).  No other microscopic
findings could be attributed to treatment.

TABLE 6a. Selected microscopic findings in male rats after 28 days of
treatment with BAS 500F via inhalation a

Microscopic finding	Analytical concentration (mg/L)

	0  (air)	0 (acetone)	0.001	0.030	0.300

Duodenum

	Diffuse mucosal hyperplasia - 	Total	0	0	0	5	7

>6 – 8 mm2	0	0	0	5	4

>8 – 10 mm2	0	0	0	0	3

Lungs

	Congestion	0	0	0	0	3

Nasal cavity I

	Respiratory hyperplasia - 		Total	0	2	0	0	9

Minimal	0	2	0	0	2

Slight	0	0	0	0	7

Reactive inflammation	0	0	0	0	2

Nasal cavity II

	Olfactory atrophy/necrosis- 	Total	0	0	0	2	10

Minimal	0	0	0	2	2

Slight	0	0	0	0	1

Moderate	0	0	0	0	7

Respiratory hyperplasia - 		Total	0	0	0	0	2

Slight	0	0	0	0	1

Moderate	0	0	0	0	1

Nasal cavity III

	Olfactory atrophy/necrosis - 	Total	0	0	0	5	10

Minimal	0	0	0	5	0

Slight	0	0	0	0	6

Moderate	0	0	0	0	4

Squamous cell metaplasia - 	Total	0	0	0	0	1

Minimal	0	0	0	0	1

Respiratory hyperplasia - 		Total	0	0	0	0	10

Minimal	0	0	0	0	2

Slight	0	0	0	0	8

Nasal cavity IV

	Olfactory atrophy/necrosis -	Total	0	0	0	9	10

Minimal	0	0	0	2	1

Slight	0	0	0	6	5

Moderate	0	0	0	1	4

Olfactory regeneration/repair - 	Total	0	0	0	0	9

Slight	0	0	0	0	9

Respiratory hyperplasia -	 	Total	1	0	0	0	8

Minimal	0	0	0	0	4

Slight	1	0	0	0	4

Larynx , level III

	Respiratory hyperplasia - 		Total	0	0	0	0	3

Minimal	0	0	0	0	2

Slight	0	0	0	0	1

a	Data obtained from pages 62, 69, and 170-175 in the study report; n =
10 except for the duodenum findings in the 0.300 mg/L group where n=8
due to advanced autolysis in two animals.

TABLE 6b. Selected microscopic findings in female rats after 28 days of
treatment with BAS 500F via inhalation a

Microscopic finding	Analytical concentration (mg/L)

	0  (air)	0 (acetone)	0.001	0.030	0.300

Duodenum

	Diffuse mucosal hyperplasia - 	Total	1	1	0	5	10

>6 – 8 mm2	0	1	0	5	1

>8 – 10 mm2	1	0	0	0	5

>10 – 12 mm2	0	0	0	0	3

>12 mm2	0	0	0	0	1

Lungs

	Alveolar histiocytosis - 		Total	1	1	2	5	5

Minimal	0	0	1	5	5

Slight	1	1	1	0	0

Congestion	0	0	0	0	3

Nasal cavity I

	Respiratory hyperplasia  -		Total	0	2	0	0	9

Minimal	0	1	0	0	1

Slight	0	1	0	0	7

Moderate	0	0	0	0	1

Reactive inflammation	0	0	0	0	4

Nasal cavity II

	Olfactory atrophy/necrosis - 	Total	0	0	0	3	9

Minimal	0	0	0	3	1

Slight	0	0	0	0	6

Moderate	0	0	0	0	2

Respiratory hyperplasia - 		Total	0	0	0	0	7

Minimal	0	0	0	0	2

Slight	0	0	0	0	5

Nasal cavity III

	Olfactory atrophy/necrosis - 	Total	0	0	0	4	9

Minimal	0	0	0	2	0

Slight	0	0	0	1	5

Moderate	0	0	0	1	4

Squamous cell metaplasia - 	Total	0	0	0	0	4

Minimal	0	0	0	0	1

Slight	0	0	0	0	3

Respiratory hyperplasia - 		Total	0	0	0	1	7

Minimal	0	0	0	0	3

Slight	0	0	0	1	4

Nasal cavity IV

	Olfactory atrophy/necrosis - 	Total	0	0	0	8	9

Minimal	0	0	0	2	0

Slight	0	0	0	2	5

Moderate	0	0	0	4	4

Olfactory regeneration/repair - 	Total	0	0	0	1	8

Minimal	0	0	0	1	1

Slight	0	0	0	0	7

Respiratory hyperplasia - 		Total	0	0	0	1	7

Minimal	0	0	0	0	5

Slight	0	0	0	1	2

a	Data obtained from pages 62, 69, and 170-175 in the study report; n =
10.

III.	DISCUSSION AND CONCLUSIONS

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.the respiratory tract at 0.300 mg/L, including hyperplasia of the
respiratory epithelium in the nasal and laryngeal tissues and damage to
the lungs (as indicated by perivascular inflammatory cell infiltration
and alveolar histiocytosis).  Aside from effects of the respiratory
system, treatment-related findings at 0.300 mg/L included increased
mortality and clinical signs of toxicity and decreased body weights,
body weight gains, food consumption, and food efficiency.

B.  REVIEWER COMMENTS:  There were no treatment-related effects on any
parameters examined during the FOB or on locomotor activity,
ophthalmoscopy, or clinical chemistry. Additionally, the few differences
noted between the air and vehicle controls (e.g., red blood cell and
some clinical chemistry indices) were minor and not considered
biologically important.

	

decreased (p≤0.05) at this concentration on Day 7 and 21. 
Additionally on Day 21, food efficiency was decreased in the 0.030 mg/L
males.  In both sexes at this concentration, an initial decrease in food
consumption was observed on Day 7.

	At 0.300 mg/L, leaukocytes and neutrophils were increased in both
sexes.  As discussed below, the increased white blood cells
(specifically neutrophils) may be an inflammatory response to the
treatment-related effects on the respiratory tissues.

	At 0.300 mg/L, the thymus weight and relative weight were decreased
(27-29%, p<0.01) in females while the relative spleen weight was
increased (33%, p<0.01) in males.  There were no effects on weights of
other organs.

At 0.300 mg/L, abdominal position and moderate labored respiration were
observed in one male on Day 21.  Additionally at this concentration, the
following clinical signs of toxicity were observed [# affected (day of
mean onset)]:  (i) urine odor in 4 males (Day 29) and in 7 females (Day
24) during daily clinical observations and in one male (Day 28) and 3
females (Day 26) during weekly detailed examinations; (ii) slight
visually increased respiration in 10 males (Day 9) and in 10 females
(Day 8) during daily clinical observations and in 8 males (Day 12) and 8
females (Day 14) during weekly detailed examinations (iii) piloerection
in 3 females (Day 20) during daily clinical observations and in 3
females (Day 21) during weekly detailed examinations.  Additionally at
this concentration, four males died prior to scheduled termination (one
each on Days 10, 12, 21, and 22), and three female rats died (one each
on Days 7, 11, and 24).  Prior to death, these animals exhibited
visually increased respiration, urine odor, and piloerection.

At 0.030 and 0.300 mg/L, diffuse mucosal hyperplasia of the duodenum was
observed in 5-7 males (vs 0 controls) and in 5-10 females (vs 1
control).  This finding increased with dose in both incidence and
severity.  Duodenal thickening and hyperplasia were also reported in
90-day or 28-day oral administration studies in rats (MRIDs
45118321/45118322).  The duodenum was also a target organ in the 90-day
dietary feeding studies in the mouse and dog (MRIDs 45118320 and
45118323).

Respiratory effects were observed at 0.030 and 0.300 mg/L, including
minimal to slight alveolar histiocytosis in the females (5 treated vs 1
control) and minimal to moderate olfactory atrophy/necrosis in nasal
cavities II through IV in the males (2-10) and females (3-9) compared to
0 controls.

Additional effects on the respiratory system at 0.300 mg/L were
observed.  Lung discoloration was noted at necropsy in 2/10 males and
3/10 females compared to 0/10 animals in each control group.  The
following findings were observed microscopically (number affected per
group out of 10 vs 0 controls, unless otherwise noted):  (i) lung
congestion in males and females (3); (ii) minimal to moderate
hyperplasia of the respiratory epithelium in nasal cavities I through IV
in the males (2-10 vs 0-1 controls) and females (7-9); (iii) reactive
inflammation in nasal cavity I in the males (2) and females (4); (iv)
slight olfactory regeneration/repair in nasal cavity IV in the males (9)
and females (8); (vii) minimal to slight respiratory hyperplasia in
level III of the larynx in the males (3); and (viii) minimal to slight
squamous cell metaplasia in nasal cavity III in the females (4). 
Leukocyte numbers were increased at this concentration in both sexes
(36-46%).  Also, the number and percent of neutrophils were increased by
38-215% in both sexes.  The increased white blood cells (specifically
neutrophils) may be an inflammatory response to the treatment-related
effects on the respiratory tissues.

The LOAEL is 0.030 mg/L based on findings of hyperplasia in the
duodenum, alveolar histiocytosis in the lungs, and olfactory
atrophy/necrosis in the nasal tissues.  The NOAEL is 0.001 mg/L.

At the request of the Agency, this study was conducted for 28 days,
instead of the 90 days required by Guideline OPPTS 870.3465.  Aside from
the different study duration, this study was conducted in accordance
with Guideline OPPTS 870.3465.

This 28-day study is classified as acceptable/guideline and satisfies
the guideline requirement (OPPTS 870.3465; OECD 413) for a subchronic
inhalation study in the rat.

STUDY DEFICIENCIES:  It was stated that the stability of the test
article under the storage conditions was confirmed by reanalysis;
however, only one purity value was reported.  Furthermore, stability of
the test substance in the vehicle was not reported.  However, the
constancy of the concentration of the test atmosphere in each inhalation
chamber was continuously monitored.  This deficiency does not alter the
conclusions of this DER.

APPENDIX

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BAS 500F (PYRACLOSTROBIN)/099100	OPPTS 870.3465/ DACO 4.3.6/ OECD 413