---

---

870.3700a	Developmental Toxicity (rodent)

870.3700b	Developmental Toxicity (nonrodent)

870.3800	Reproduction

yes

yes

yes

yes

yes

yes

870.4100a	Chronic Toxicity (rodent)

870.4100b	Chronic Toxicity (nonrodent)

870.4200a	Oncogenicity (rat)

870.4200b	Oncogenicity (mouse)

870.4300	Chronic/Oncogenicity

yes

yes

yes

yes

yes

yes

yes

yes

yes

yes

870.5100	Mutagenicity—Gene Mutation - bacterial

870.5300	Mutagenicity—Gene Mutation - mammalian

870.5375	Mutagenicity—Structural Chromosomal Aberrations

870.5395	Mutagenicity—Other Genotoxic Effects

yes

yes

yes

yes

yes

yes

yes

yes

870.6100a	Acute Delayed Neurotox. (hen)

870.6100b	90-Day Neurotoxicity (hen)

870.6200a	Acute Neurotox. Screening Battery (rat)

870.6200b	Chronic Neurotox. Screening Battery (rat)

870.6300	Develop. Neuro

no

no

yes

yes

yes

---

---

yes

yes

yes

870.7485	General Metabolism

870.7600	Dermal Penetration

yes

no

yes

---

A.2  Toxicity Profiles

Table 1. Acute Toxicity Data on Fluazinam Technical

Guideline No./ Study Type	Test Substance	MRID No.	Results	Toxicity
Category

870.1100

Acute oral toxicity

rats	Technical grade fluazinam (lot #109; 95.3%)	42248603

M: LD50 = 4500 mg/kg

F: LD50 = 4100 mg/kg	III

	Technical grade fluazinam (lot #8412-20; 95.3%)	42248602	M: LD50 >5000
mg/kg

F: LD50 >5000 mg/kg

IV

	Technical grade fluazinam (lot #1/87; 97.9%)	42248604	M: LD50 >5000
mg/kg

F: LD50 >5000 mg/kg

IV

870.1200

Acute dermal toxicity

rats

Technical grade fluazinam (lot #8303-2; 98.5%)

42248605

M: LD50 >2000 mg/kg

F: LD50 >2000 mg/kg

III

870.1300

Acute inhalation toxicity

rats

Technical grade fluazinam (lot #109; 95.3%)

42270601

M: LC50 = 0.463 mg/L

F: LD50 = 0.476 mg/L

II

870.2400

Acute eye irritation

rabbits

Technical grade fluazinam (lot # SNPE B-1216, No. 1006; 97.9%)

42248606

Extremely irritating.  Corneal opacity did NOT reverse in 21 days.

I

870.2500 Acute dermal irritation

rabbits

Technical grade fluazinam (lot # SNPE B-1216, No. 1006; 97.9%)

42248607

Slightly irritating

IV

870.2600 Dermal sensitization

guinea pigs

Technical grade fluazinam (lot # 1030/91; 96.7%)

42274401

Positive

NA

Ultra-purified fluazinam (lot #Y910401; 100%)

42248608

Negative

NA

Table 2. Acute Toxicity Data on Fluazinam (formulation)

Guideline No./ Study Type	Test Substance	MRID No.	Results	Toxicity
Category

870.1100

Acute oral toxicity

rats

Omega 500F

(40% fluazinam)

42974907

M: LD50 >5000 mg/kg

F: LD50 >5000 mg/kg

IV

870.1200

Acute dermal toxicity

rabbits

Omega 500F

(40% fluazinam)

42974908

M: LD50 >2000 mg/kg

F: LD50 >2000 mg/kg

III

870.1300

Acute inhalation toxicity

rats

Fluazinam 50% WP (1)

(51.3% fluazinam)

42311001

M: LC50 = 3.0 mg/L

F: LD50 = 3.4 mg/L

IV

870.2400

Acute eye irritation

rabbits

Omega 500F

(40% fluazinam)

42974910

Slightly irritating

III

870.2500 Acute dermal irritation

rabbits

Omega 500F

(40% fluazinam)

42974911

Moderately irritating

II

870.2600 Dermal sensitization

guinea pigs

Omega 500F

(40% fluazinam)

42974912

Positive

NA

(1)   Study satisfies requirement for testing on Omega 500F.

Table 3. Subchronic, Chronic and Other Toxicity Table

Guideline No.

 Study Type

MRID No./ (year) Classification /Doses

Results

870.3100

90-Day oral toxicity rats

42248610 (1984); 44807214 (1998)

Acceptable/guideline

M : 0, 0.15, 0.77, 3.8, 38 mg/kg/day;

F: 0, 0.17, 0.86, 4.3, 44 mg/kg/day

NOAEL: Males: 3.8 mg/kg/day; Females: 4.3 mg/kg/day

LOAEL Males = 38 mg/kg/day; Females = 44 mg/kg/day based on increased
liver weights and liver histopathology in males, and increased lung and
uterus weights in females.

870.3150

90-Day oral toxicity

dogs

42248611 (1991); 44807215 (1998)

Acceptable/guideline

M & F: 0, 1, 10, 100 mg/kg/day

NOAEL = 10 mg/kg/day

LOAEL = 100 mg/kg/day based on retinal effects, increased relative liver
weight, liver histopathology and possible increased serum alkaline
phosphatase in females and possible marginal vacuolation of the cerebral
white matter (equivocal)

870.3200

21-Day dermal toxicity

rats

42270602 (1985)

Acceptable/guideline

M & F: 0, 10, 100, 1000 mg/kg/day

Systemic NOAEL = 10 mg/kg/day

 LOAEL = 100 mg/kg/day based on increased AST and cholesterol levels in
clinical chemistry determinations (males)

Dermal NOAEL = not identified

LOAEL = 10 mg/kg/day based on erythema, acanthosis, and dermatitis

870.3250

90-Day dermal toxicity

NA

NA

870.3465

90-Day inhalation toxicity

NA

NA

870.3700a

Prenatal developmental toxicity

rats

42248613 (1985)

Acceptable/guideline

F: 0,10, 50, 250 mg/kg/day

Maternal NOAEL = 50 mg/kg/day

LOAEL = 250 mg/kg/day based on decreased body weight gain and food
consumption and increased water consumption and urogenital staining

Developmental NOAEL = 50 mg/kg/day

LOAEL = 250 mg/kg/day based on decreased fetal body weights and
placental weights, increased facial/cleft palates, diaphragmatic hernia,
and delayed ossification in several bone types, greenish amniotic fluid
and possible increased late resorptions and postimplantation loss

870.3700b

Prenatal developmental toxicity

rabbits

42248616 (1988)

Acceptable/guideline

F: 0, 2, 4, 7, 12 mg/kg/day

Maternal NOAEL = 4 mg/kg/day

LOAEL = 7 mg/kg/day based on decreased food consumption and increased
liver histopathology.

Developmental NOAEL = 7 mg/kg/day

LOAEL = 12 mg/kg/day based on an increase in total litter resorptions
and possible fetal skeletal abnormalities

870.3700b

Prenatal developmental toxicity

rabbits

42248615 (1985); 42248614 (1984); 42248617 (1984)

Unacceptable/guideline

F: 0, 0.3, 1, 3 mg/kg/day

Maternal NOAEL = 3 mg/kg/day

LOAEL = not identified (>3 mg/kg/day)

Developmental NOAEL = 3 mg/kg/day

LOAEL = not identified (>3 mg/kg/day)

870.3800

Reproduction and fertility effects

rats

42248619 (1987); 42208406 (1985); 42248618 (1986)

Acceptable/guideline

F0 males: 0, 1.5, 7.3, 36.6 mg/kg/day

F0 females: 0, 1.7, 8.4, 42.1 mg/kg/day

F1 males: 0, 1.9, 9.7, 47.3 mg/kg/day

F1 females: 0, 2.2, 10.6, 53.6 mg/kg/day

Parental/Systemic NOAEL = 1.9 mg/kg/day

LOAEL = 9.7 mg/kg/day based on liver pathology in F1 males

Reproductive NOAEL = 10.6 mg/kg/day

LOAEL = 53.6 mg/kg/day based on decreased number of implantation sites
and decreased litter sizes to day 4 post-partum for F1 females (F2
litters).

Offspring NOAEL = 8.4 mg/kg/day

LOAEL = 42.1 mg/kg/day based on reduced F1 and F2 pup body weight gains
during lactation.

870.4100a

Chronic toxicity

rats

44839901 (1993)

Acceptable/guideline

M: 0, 1.0, 1.9, 3.9 mg/kg/day

F: 0, 1.2, 2.4, 4.9 mg/kg/day

NOAEL = Males: 1.9 mg/kg/day; Females: 4.9 mg/kg/day

LOAEL = Males: 3.9 mg/kg/day; Females: not identified (>4.9 mg/kg/day)
based on increased testicular atrophy in males and no effects in females

870.4100b

Chronic toxicity

dogs	42270603 (1987); 44807219 (1998)

Acceptable/guideline

M & F: 0, 1, 10, 50 mg/kg/day

NOAEL = 1 mg/kg/day

LOAEL = 10 mg/kg/day based on gastric lymphoid hyperplasia in both sexes
and nasal dryness in females

870.4300

Combined chronic toxicity/carcino-genicity

rats

42248620 (1988); 44807223 (1999); 45150201 (2000)

Acceptable/guideline

M: 0, 0.04, 0.38, 3.8, 40 mg/kg/day

F: 0, 0.05, 0.47, 4.9, 53 mg/kg/day

NOAEL = Males: 0.38 mg/kg/day; Females: 0.47 mg/kg/day

LOAEL = Males: 3.8 mg/kg/day; Females: 4.9 mg/kg/day based on liver
toxicity in both sexes, pancreatic exocrine atrophy in females and
testicular atrophy in males.

Some evidence of carcinogenicity (thyroid gland follicular cell tumors)
in male rats, but not in females.

870.4200b

Carcinogenicity

mice

42208405 (1988); 4807220 (1996)

Acceptable/guideline

M: 0, 0.12, 1.1, 10.7, 107 mg/kg/day

F: 0, 0.11, 1.2, 11.7, 117 mg/kg/day

NOAEL =  Males:1.1 mg/kg/day; Females: 1.2 mg/kg/day

LOAEL = Males: 10.7 mg/kg/day; Females: 11.7 mg/kg/day based on
increased incidences of brown macrophages in the liver of both sexes,
eosinophilic vacuolated hepatocytes in males, and increased liver weight
in females

Clear evidence of carcinogenicity (hepatocellular tumors) in male mice,
but not in females

870.4200b

Carcinogenicity

mice

44807222 (1996); 44807221 (1998); 45201301 (2000)

Acceptable/guideline

M: 0, 126, 377, 964 mg/kg/day

F: 0, 162, 453, 1185 mg/kg/day

NOAEL =  Males:<126 mg/kg/day, Females: <162 mg/kg/day

LOAEL = Males: 126 mg/kg/day; Females: 162 mg/kg/day based on increased
liver weights and liver and brain histopathology in both sexes

Equivocal/some evidence of carcinogenicity (hepatocellular tumors) in
male mice, but not in females

870.5100

Bacterial reverse mutation assay (Ames test)

42270605 (1988)

Acceptable/Guideline

Up to 2 μg/plate for S. typhimurium strains and up to 250 μg/plate for
E. coli (-S9).  Up to 100 μg/plate for S. typhimurium strains and up to
500 μg/plate for E. coli (+ S9)

Negative with and without S9 up to cytotoxic concentrations.

870.5100

Bacterial reverse mutation assay (Ames test)

42270604 (1989)

Acceptable/Guideline

Up to 1μg/plate for S. typhimurium strains and up to 250 μg/plate for
E. coli (-S9) and  up to 100 μg/plate for S. typhimurium strains and up
to 500 μg/plate for E. coli (+S9)

Negative with and without S9 up to cytotoxic concentrations.

870.5300

In vitro mammalian gene mutation assay

45261801 (2000)

Acceptable/guideline

Up to 9 μg/ml (+S9), up to 0.3  μg/ml (-S9)

Negative with S9 activation up to 9 μg/ml.  Negative without S9
activation up to 0.3 μg/ml.

Compound tested to cytotoxic concentrations.

870.5300

In vitro mammalian gene mutation assay

45156902 (1986)

Acceptable/guideline

Up to 5 μg/ml (+/-S9)

Negative with and without S9 activation up to 5 μg/ml.

Compound tested to cytotoxic concentrations.

870.5375

In vitro mammalian chromosome aberration (CHL cells)

42270606 (1988)

Acceptable/guideline

Up to 9.5 μg/ml (+S9) up to 4 μg/ml (-S9)

Negative with and without S9 up to cytotoxic concentrations.  Cells
harvested at 24 and 48 hours in nonactivated studies and at 24 hours in
activated studies.

870.5395

Mammalian erythrocyte micronucleus test

44807224 (1999)

Acceptable/guideline

500, 1000, 2000 mg/kg (oral gavage)

Negative at 24 hour sacrifice (500, 1000, 2000 mg/kg).

Negative at 24, 48, and 72 hour sacrifices (2000 mg/kg).

870.5550

UDS in primary rat hepatocytes

45156901 (1984)

Unacceptable/guideline

0.05 to 6.25 μg/ml

Negative; however there were several serious study deficiencies:
treatment time shorter than recommended, no data supporting the claim of
cytotoxicity, data variability for major endpoints.

870.5550

Differential killing/growth inhibition in B. subtilis

42270607 (1988)

Unacceptable/guideline

0.003 to 0.3 μg/disk (-S9)

0.3 to 30 μg/disk (+S9)

Negative, however only one replicate plate/dose was used.

870.6200a

Acute neurotoxicity screening battery

rats

44807210 (1995)

Acceptable/guideline

M & F: 0, 50, 1000, 2000 mg/kg

Systemic NOAEL = 50 mg/kg

LOAEL = 1000 mg/kg based on soft stools and decreased motor activity on
day of dosing.

Neurotoxicity NOAEL = 2000 mg/kg

LOAEL = not identified (>2000 mg/kg)

870.6200b

Subchronic neurotoxicity screening battery

rats

44807217 (1998); 44807218 (1998)

Acceptable/guideline

M :0, 21, 69, 74, 149, 233 mg/kg/day;

F: 0, 23, 81, 89, 175, 280 mg/kg/day

Neurotoxicity NOAEL = Males: 233 mg/kg/day; Females: 280 mg/kg/day

LOAEL = not identified (Males: >233 mg/kg/day; Females: > 280 mg/kg/day)

870.6300

Developmental neurotoxicity

46534401, 47018301 and 47037001.

Acceptable/Non-Guideline

0, 2, 10 or 50 mg/kg/day

Maternal:

NOAEL not established.  No toxicity at highest dose tested.

Developmental:

NOAEL = 2 mg/kg/day.

LOAEL = 10 mg/kg/day based on decreased pup weight and gain and delayed
balano-preputial separation.

870.7485

Metabolism and pharmacokinetics

rats

44807233 (1995); 43521004 thru 43521008, 43553001 (1993-1995)

Acceptable/guideline

0.5, 50 mg/kg

Only 33-40% of the administered dose was absorbed.  Most of the
administered dose was recovered in the feces (>89%).   Excretion via the
urine was minor (<4%).  Total biliary radioactivity, however,
represented 25-34% of the administered dose, indicating considerable
enterohepatic circulation.

A.3 Executive Summaries TC \l2 "A.3  Executive Summaries

A.3.1	Subchronic Toxicity

870.3100		90-Day Oral Toxicity – Rat

Executive Summary: In a subchronic oral toxicity study (MRID 42248610,
44807214), technical grade fluazinam (98.5% a.i.) was administered in
the diet to 10 CD (remote Sprague-Dawley strain) rats/sex/dose level at
dose levels of 0, 2, 10, 50, or 500 ppm for 13 weeks (0, 0.15, 0.77,
3.8, or 38 mg/kg/day for males; 0, 0.17, 0.86, 4.3, or 44 mg/kg/day for
females).  Slides of brain and cervical spinal cord from all control and
500 ppm rats were later re-examined to assess for vacuolation of the
white matter in the central nervous system (MRID 44807214).

No treatment-related mortalities, clinical signs of toxicity, changes in
body weights or body weight gains, differences in food or water
consumption, or ophthalmological findings were observed.  No
treatment-related effects in hematology, clinical chemistry, or
urinalyses parameters were noted.  Gross necropsies were negative.  At
termination, statistically significant treatment-related increases were
observed in the liver of 500 ppm males (absolute weights increased 8 %
(not significant) and relative liver/body weight ratios increased 11% in
comparison to controls), in the lungs of 500 ppm females (absolute
weights increased 18 % and relative lung/body weight ratios increased
25% in comparison to controls), and in the uterus of 500 ppm females
(absolute weights increased 36 % and relative uterus/body weight ratios
increased 43% in comparison to controls).  Statistically significant
compound-related histopathological lesions were observed in the livers
of 500 ppm males (increased incidences of periacinar hepatocyte
hypertrophy and sinusoidal chronic inflammation).  There was no effect
of treatment on the incidence or severity of vacuolation of the white
matter of the brain or cervical spinal cord in the 500 ppm rats as
compared with the controls.

The LOAEL is 500 ppm (38 mg/kg/day in males and 44 mg/kg/day in
females), based on increases in absolute and relative liver weights in
males, increases in absolute and relative lung and uterus weights in
females, and increases in histopathology in the liver of males
(increased incidences of periacinar hepatocyte hypertrophy and
sinusoidal chronic inflammation).  The NOAEL in this study is 50 ppm
(3.8 mg/kg/day in males and 4.3 mg/kg/day in females).

This subchronic oral toxicity study in rats is classified
Acceptable/Guideline and satisfies the Subdivision F guideline
requirement for a subchronic oral toxicity study [OPPTS 870.3100
((82-1a) in rats.

870.3100		90-Day Oral Toxicity – Mouse

Refer to carcinogenicity study.

870.3150		90-Day Oral Toxicity – Dog

Executive Summary: In a subchronic oral toxicity study (MRID 42248611,
44807215), fluazinam (98.5% a.i.) was administered to 4 beagle
dogs/sex/dose daily via gelatin capsule at doses of 0, 1, 10 or 100
mg/kg/day for 90 days.   Slides of brain tissue were later reexamined
(Addendum to the original report, MRID 44807215) to assess vacuolation
of white matter according to criteria for severity used in mouse
carcinogenicity studies.

At 100 mg/kg/day, retinal effects of slight hyper-reflection and
slight-to-moderate grey mottling of the tapetal fundus in all males and
females during at least two of the three ophthalmologic examinations (7,
10 and/or 13 weeks), increased serum alkaline phosphatase levels
((2-fold, largely due to 1 female), increased SGPT in 1 female ((2 to
3-fold), increased absolute/relative liver weights (males 31%/34% and
females 33%/36% above controls), hepatic coagulative necrosis (1 male,
focal; 2 females, multifocal, all slight, vs. 0, controls) and slight to
moderate bile duct hyperplasia with/without cholangiofibrosis (2 males
and 2 females) were observed.  A possible marginal increase in cerebral
white matter vacuolation was observed in the reevaluation reported in
the Addendum (1 male and 2 females, severity graded as trace vs. 0,
controls).   There were no treatment-related effects observed for
clinical signs of toxicity, body weight/weight gain, food consumption,
hematology or urinalysis parameters or gross pathology.  No
treatment-related findings were observed at 1 or 10 mg/kg/day.  The
LOAEL is 100 mg/kg/day, based on retinal effects, possible increased
serum alkaline phosphatase in females, increased relative liver weight
and liver histopathology and possible marginal increase in vacuolation
of the cerebral white matter.  The NOAEL is 10 mg/kg/day.

This study is classified Acceptable/Guideline and satisfies the
guideline requirement for a subchronic oral toxicity study [OPPTS
870.3150((82-1b) in the dog.

870.3200		21/28-Day Dermal Toxicity – Rat

Executive Summary: In a 21-day repeated dose dermal toxicity study (MRID
42270602), groups of 10 male and 10 female CD (Sprague-Dawley) rats were
treated with Fluazinam technical (98.0% a.i.; lot no. 8303-2) in 0.5%
methylcellulose in distilled water at doses of 0, 10, 100 or 1000
mg/kg/day.  Animals were treated by dermal occlusion for 6 hours per
day, 7 days per week, for 3 weeks.

No treatment-related mortalities occurred.  At 1000 mg/kg/day, decreased
body weight gain in males (19% compared to controls, p <0.05) was
observed.  Liver damage in both males and females was also evident at
1000 mg/kg/day as demonstrated by increased absolute liver weights
(17-26%), increased relative liver/body weight ratios (27-30%),
statistically significant increases in aspartate aminotransferase (AST)
and cholesterol levels, and highly increased incidences of periacinar
hepatocellular hypertrophy in males and females.  At 100 mg/kg/day,
statistically significant increases in AST and cholesterol levels were
observed in males.  The LOAEL for systemic toxicity is 100 mg/kg/day
based on increased AST and increased cholesterol levels in males.  The
NOAEL for systemic toxicity is 10 mg/kg/day.

At 1000 mg/kg/day, slight to severe erythema and atonia were observed
after 11-13 days and encrustation and/or staining at 21 days in males
and females.  At 100 mg/kg/day, slight erythema was observed after 14
days in males and females and encrustation and/or staining at 21 days in
females.  At 10 mg/kg/day, slight erythema was noted after 13 days in
one male.  Histologically, at 1000 mg/kg/day and 100 mg/kg/day,
acanthosis, dermatitis, scabs and ulceration were noted in males and
females.  At 10 mg/kg/day, acanthosis and dermatitis were observed in
males and females.  At 10 mg/kg/day, the test material was considered to
be a very mild irritant.  The LOAEL for dermal toxicity is <10 mg/kg/day
based on erythema, acanthosis and dermatitis in males and/or females.
No NOAEL for dermal toxicity was determined in this study (< 10
mg/kg/day).

This 21-day dermal toxicity study in rats is classified
Acceptable/Guideline and satisfies the Subdivision F guideline
requirement for a 21/28-day dermal toxicity study [OPPTS 870.3200 (OPP
82-2)].

870.3465	28- 	90-Day Inhalation – Rat

No subchronic inhalation study is available.  A 28-day study has been
requested.

A.3.2		Prenatal Developmental Toxicity

870.3700a 	Prenatal Developmental Toxicity Study – Rat

Executive Summary: In a developmental toxicity study (MRID 42248613), 20
presumed pregnant Sprague-Dawley CD rats per group were administered
Fluazinam (98.5% a.i, Lot No.: 8303-2) by gavage in corn oil at doses of
0, 10, 50 and 250 mg/kg/day on gestation days (GD) 6-15, inclusive.
Controls were treated with corn oil (vehicle).  On GD 20, all dams were
sacrificed and examined.  All fetuses were weighed, sexed and examined
for external malformations and variations.  Approximately half of the
fetuses from each litter were examined for soft tissue effects and half
were stained with Alizarin red S and examined for skeletal effects.

Maternal Toxicity.  At 250 mg/kg/day, statistically significant
reductions in body weight gain during treatment (30 gm vs. 51 gm for
controls on GD 6-15; p < 0.01; most pronounced during GD 6-8),
statistically significant reductions in food consumption during
treatment (13 mg/kg/day vs. 17 mg/kg/day for controls on GD 6-8; p
<0.01), increased water consumption (during GD 6-11) and an increased
incidence of urogenital staining (most pronounced during GD 6-8) were
considered to be treatment-related.  The maternal toxicity LOAEL is 250
mg/kg/day based on decreased body weight gain , decreased food
consumption, increased water consumption, and increased urogenital
staining during treatment.  The maternal toxicity NOAEL is 50 mg/kg/day.

Developmental Toxicity. At 250 mg/kg/day, statistically significant
decreased mean fetal body weights (2.81 gm vs. 3.19 gm for controls, p
<0.001, below historical control range), statistically significant
decreased placental weights (0.47 gm vs. 0.54 gm for controls, p <0.05,
within historical control range), increased fetal incidence of
facial/palate clefts (10 fetuses in 3 litters vs. none in controls),
increased fetal incidence of diaphragmatic hernia (7 fetuses in 2
litters vs. none in controls), delayed ossification in a number of bone
types, greenish amniotic fluid (10.5% fetal incidence vs. 0.0% in
controls) and possible increased late resorption/postimplantation loss
(0.55 late resorptions/dam vs. 0.05 late resorptions /dam for controls,
within historical control range;  and 11.0% postimplantation loss vs.
4.2% postimplantation loss for controls, within historical control
range) were considered to be treatment-related.  The developmental
toxicity LOAEL is 250 mg/kg/day based on decreased fetal body weights;
decreased placental weights; increased fetal incidences of facial/palate
clefts, diaphragmatic hernia and delayed ossification in several bone
types; greenish amniotic fluid and possible increased late resorptions
and postimplantation loss.  The developmental toxicity NOAEL is 50
mg/kg/day.

This developmental toxicity study in rats is classified
Acceptable/Guideline and satisfies the Subdivision F guideline
requirement for a developmental toxicity study in rats [OPPTS 870.3700
(OPP 83-3a)].  No major deficiencies were noted in this study.

Non-guideline  	Developmental Range-Finding Toxicity - Rat

Executive Summary: In a developmental range-finding toxicity study (MRID
42248612), 7 pregnant CD (Sprague-Dawley origin) rats per group were
administered B-1216 (98.5%; Lot No. 8303-2) by gavage in corn oil at
doses of 0, 1, 10, 100, and 1000 mg/kg/day on gestation days (GD) 6-15,
inclusive.  On GD 20, dams were sacrificed and necropsied, and the
number and location of viable and nonviable fetuses, early and late
resorptions, and the number of total implantations and corpora lutea
were recorded, as well as the weights of the ovaries, empty uteruses,
and adrenal and pituitary glands.  All fetuses were weighed, sexed and
examined externally, and approximately half of each litter was processed
for visceral examination, and the remaining half of each litter was
examined by fresh dissection then processed for skeletal examination.

Maternal toxicity was evident at 1000 mg/kg/day.  Two animals were found
dead on GD 13 and the remaining 5 were sacrificed in extremis between GD
12 and 14.  Prior to death the high-dose animals exhibited clinical
signs of stained and ungroomed coats, lethargy, hunched posture, ataxia,
flaccid muscles, and salivation. Post mortem findings included decreased
thymus size and gastrointestinal tract disturbances.  Marked weight loss
was observed at 1000 mg/kg/day after GD 7, and mean absolute body
weights were 74-86% of those of controls during GD 10-13.  Body weight
and survival were not affected in the 1, 10, and 100 mg/kg/day groups.

There were no differences between the control group and the 1, 10, or
100 mg/kg/day groups for number of corpora lutea, number of implantation
sites, live fetuses/dam, pre- and post-implantation losses, resorptions,
or fetal sex ratios.  At 100 mg/kg/day, mean fetal weight was marginally
decreased as compared with concurrent controls but fell within the range
of historical control data.  The incidence of incomplete ossification of
sternebrae was increased in the 100 mg/kg/day group as compared to
concurrent and historical controls (38.9% of fetuses and 7/7 litters vs.
11.8% and 3/7 litters for concurrent controls and a historical control
range of 1.1-28.3%); however, there was no evidence of delayed
ossification in any other bone types.  The incidence rate for litters
containing fetuses with additional (14th) rib(s) was 1/7, 2/7, 2/7, and
3/7 for the 0, 1, 10, and 100 mg/kg/day groups, respectively, with the
percentage of affected fetuses slightly increased in all treated groups
as compared with concurrent and historical controls.  Treatment with
B-1216 did not result in an increased incidence of fetal malformations.

Therefore, it was concluded that an appropriate high dose for the main
developmental toxicity study (MRID 42248613) would be greater than 100
mg/kg/day but less than 1000 mg/kg/day.  The dose levels chosen were 0,
10, 50, and 250 mg/kg/day.

This study is classified as Acceptable/Nonguideline and fulfills its
intent as a range finding study for a developmental toxicity study
[870.3700 ((83-3a)] in rats.  Despite numerous deficiencies in the
conduct of this study, an assessment of appropriate doses of the test
article for the main developmental toxicity study was possible.

870.3700b 	Prenatal Developmental Toxicity Study - Rabbit

Executive Summary: In a developmental toxicity study (MRID 42248616),
16-18 presumed pregnant New Zealand White rabbits per group were
administered Fluazinam (95.3% a.i, Lot No.: 8412-20) by gavage in 1% w/v
aqueous methyl cellulose at doses of 0, 2, 4, 7 and 12 mg/kg/day on
gestation days (GD) 6-19, inclusive.  Controls were treated with 1% w/v
aqueous methyl cellulose  (vehicle).  On GD 29, all surviving does were
sacrificed and necropsied and all fetuses were weighed, and examined for
external malformation/variations.  Each fetus was examined viscerally by
fresh dissection and the sex determined.  All carcasses were eviscerated
and processed for skeletal examination.

Maternal Toxicity.  At 12 mg/kg/day, statistically significant
reductions in body weight gain during treatment ( 0.0 kg vs. +0.25 kg
for controls on GD 10-20), decreased food consumption (268 g/animal/day
vs. 368 g/animal/day for controls on GD 6-19) and an increased incidence
of liver histopathology (cellular hypertrophy, single cell necrosis,
binucleate hepatocytes, increased brown pigment deposition, apoptosis)
were considered to be treatment-related.  At 7 mg/kg/day, slightly
depressed food consumption (139 g/animal/day vs. 186 g/animal/day for
controls on GD 13-19) and an increased incidence of liver histopathology
(cellular hypertrophy, single cell necrosis, binucleate hepatocytes,
increased brown pigment deposition, apoptosis) were also considered to
be treatment-related.  The maternal toxicity LOAEL is 7 mg/kg/day based
on decreased food consumption and increased liver histopathology.  The
maternal toxicity NOAEL is 4 mg/kg/day.

Developmental Toxicity. At 12 mg/kg/day, an increased incidence of total
litter resorptions (0, 0, 0, 1 and 5 for the 0, 2, 4, 7 and 12 mg/kg/day
groups respectively) was considered to be treatment-related.  Several
abortions were also observed (0, 0, 2, 2 and 1 for the 0, 2, 4, 7 and 12
mg/kg/day groups respectively).  The total number of litters lost was
such that the numbers of litters born were 15, 13, 10, 10 and 7 for the
0, 2, 4, 7 and 12 mg/kg/day groups respectively.  Also at 12 mg/kg/day,
there was an increased incidence of placental anomalies (0.7, 3.2, 0.0,
0.0 and 18.2 percent fetal incidence for the 0, 2, 4, 7 and 12 mg/kg/day
groups respectively) and a slight increase in some skeletal
abnormalities including kinked tail tip, fused or incompletely ossified
sternebrae, and abnormalities of head bones.  The developmental toxicity
LOAEL is 12 mg/kg/day based on an increased incidence of total litter
resorptions and a possibly increased incidence of fetal skeletal
abnormalities.  The developmental toxicity NOAEL is 7 mg/kg/day.

This developmental toxicity study in rabbits is classified
Acceptable/Guideline and satisfies the Subdivision F guideline
requirement for a developmental toxicity study in rabbits [OPPTS
870.3700 (OPP 83-3b)].  No major deficiencies were noted in this study.

870.3700b 	Prenatal Developmental Toxicity Study - Rabbit

Executive Summary: In another developmental toxicity study (MRID
42248615, 42248614, 42248617), 20-24 presumed pregnant New Zealand White
rabbits per group were administered Fluazinam (98.5% a.i, Lot No.:
8303-2) by gavage in 1% w/v aqueous methyl cellulose at doses of 0, 0.3,
1.0, and 3.0 mg/kg/day on gestation days (GD) 6-19, inclusive.  Controls
were treated with 1% w/v aqueous methyl cellulose (vehicle).  On GD 29,
all surviving does were sacrificed and all fetuses were weighed, and
examined for external malformation/variations.  Each fetus was examined
viscerally by fresh dissection and the sex determined.  All carcasses
were eviscerated and processed for skeletal examination.

Four control group animals and one each in the 1.0 and 3.0 mg/kg/day
groups died or were killed in extremis following observations of reduced
food intake, reduced fecal output, weight loss, nasal discharge,
tremors, inactivity, abdominal bloat, abnormal respiration, and/or
soiled, stained, wet, or matted fur.  Deaths were not considered to be
treatment-related and may be related to disease or intubation error. One
female each in the 0.3 and 3.0 mg/kg/day groups aborted and were
subsequently sacrificed on GD 21 and 28, respectively, following weight
loss.  An additional control group female that exhibited weight loss and
scouring was killed on GD 6 and categorized as (removed from study.  No
statistically or biologically significant differences in body weight or
food consumption were noted.  Therefore, the maternal toxicity LOAEL is
not identified, and the maternal toxicity NOAEL is greater than or equal
to 3.0 mg/kg/day.

There were no differences between the control group and the 0, 0.3, 1.0
or 3.0 mg/kg/day groups for number of corpora lutea, number of
implantation sites, live fetuses/dam, pre- and post-implantation losses,
or mean fetal weight.  The percentage of small fetuses (weighing less
than 32.0 g.) were 19.6% (8/16), 13.0% (4/15), and 20.8% (6/14 litters),
respectively, for the 0.3, 1.0, and 3.0 mg/kg/day groups as compared to
4.1% (5/18 litters) for concurrent controls.  However, these fetal
incidence rates did fall within the historical control range of 0.0 to
30.4% (mean 13.36%).  No other statistically or biologically significant
differences between treated and control groups were noted.

The overall incidence rates for litters containing grossly abnormal
fetuses in the 0, 0.3, 1.0 and 3.0 mg/kg/day groups were 2/18, 1/16,
0/15, and 1/14, respectively.  No single malformation apparently
occurred at a statistically significant incidence in the treated groups,
and the incidence of total major malformations was also not
significantly increased for any of the treated groups as compared to
controls.   However, this assessment may be flawed since data from two
separate studies were combined and disease and intubation errors were
clearly apparent in the data.   It is unclear how disease differed in
the two studies and how this may have impacted on study interpretation.
Therefore, the developmental toxicity LOAEL was not determined, and the
developmental toxicity NOAEL is greater than or equal to 3.0 mg/kg/day.

This study is classified as Unacceptable/Guideline and does not satisfy
the requirements for a developmental toxicity study 870.3700 ((83-3) in
rabbits.  At the high dose, significant maternal effects were not
reported, and developmental toxicity did not occur.   In addition, due
to the small number of animals available for examination due to disease
and intubation errors, an additional study was started at a later date
and these data were later combined and presented in this report. The use
of sick animals and the methods of combining different populations of
animals into a larger study as noted in this study are not acceptable.

A.3.3	Reproductive Toxicity

870.3800 Reproduction and Fertility Effects - Rat

Executive Summary: Technical grade fluazinam (95.3 % a.i.) was
administered to groups of 24 male and 24 female Sprague-Dawley rats at
dietary concentrations of 0, 20, 100, or 500 ppm for two generations
(MRID 42248619, 42208406, 42248618).  One litter was produced in each
generation.  Mean premating doses were 1.5, 7.3, and 36.6 mg/kg/day,
respectively for F0 males and 1.7, 8.4, and 42.1 mg/kg/day, respectively
for F0 females.  Mean premating doses were 1.9, 9.7, and 47.3 mg/kg/day
respectively, for F1 males and 2.2, 10.6, and 53.6 mg/kg/day,
respectively, for F1 females.  F1 adults were chosen from the F1 pups
and weaned onto the same diet as their parents.  Animals were given test
or control diet for 11 weeks before mating within the same dose group.

There were no deaths or clinical signs of toxicity that were
attributable to the presence of fluazinam in the diet.  Mean body
weight, body weight gain, food consumption and food efficiency among all
groups of F0 males and F0 females treated with 20 or 100 ppm and F0
males treated with 500 ppm were similar to the control group means.  The
F0 females treated with 500 ppm of the test diet had significantly
decreased (82% of control value, p< 0.001) overall body weight gain and
food consumption (96% of control value, p< 0.05) for the premating
period.  The F1 males and females treated with 20 or 100 ppm had mean
body weights, body weight gains, food consumption, and food efficiencies
that were similar to their respective control group means.  The F1
animals treated with 500 ppm had significantly decreased mean body
weight gain and food consumption values that were 88% and 92% (p< 0.001
and p< 0.01) and 85% and 93% (p< 0.001 and p< 0.01) of the control
values for males and females, respectively for the premating period.
The decreased body weights continued into gestation for females treated
with 500 ppm of both generations; some recovery was made during
lactation.  The relative liver weights of F0 and F1 males and F0 females
treated with 500 ppm were significantly increased compared to the
control group.  Histopathological findings included an increased
incidence of periacinar hepatocytic fatty changes and a decreased
incidence of hepatic glycogen pallor among F0 males treated with
500 ppm compared to the control group.  Males in the F1 generation
treated with 100 or 500 ppm also had significantly increased incidences
of periacinar hepatocytic fatty changes compared to the control groups.
The LOAEL for parental toxicity is 100 ppm (9.7 mg/kg/day), based on
liver pathology (increased incidences of periacinar hepatocytic fatty
changes) in F1 males.  The NOAEL is 20 ppm (1.9 mg/kg/day).

The fertility index for males and females treated with 500 ppm of the
test substance was slightly decreased (n.s.) for F1 parents compared to
the control group.  The number of implantation sites observed in F1 dams
was decreased significantly (p< 0.05) at 500 ppm (12.2 vs. 15.3 in
controls) and marginally (n.s.) at 100 ppm (13.1 vs. 15.3 in controls).
Mean litter size on day 1 was slightly decreased (n.s.) in the 500 ppm
groups compared to the control groups in both generations.  Mean litter
size on day 4 was slightly decreased (n.s.) in the 500 ppm group for F1
litters, but was significantly decreased (p<0.05) in the 500 ppm group
for F2 litters (9.8 + 3.7 for 500 ppm vs. 12.4 + 3.0 for controls).  Pup
survival was similar between the treated and control groups for both
generations.  The LOAEL for reproductive toxicity is 500 ppm (53.6
mg/kg/day), based on a decreased number of implantation sites and
decreased litter sizes to day 4 post partum for F1 females (F2 litters).
 The NOAEL is 100 ppm (10.6 mg/kg/day).

Mean overall body weight gain during lactation was significantly
decreased (10-13%), among pups in the 500 ppm groups in both
generations.  The most pronounced effect on pup weight gains occurred
between lactation days 7-21.  Absolute body weights, however, were not
significantly decreased compared to the control groups at any time point
during lactation.  A slightly decreased developmental time for pinna
unfolding, hair growth and eye opening, particularly in the F2 pups, was
observed.  The LOAEL for developmental toxicity is 500 ppm (42.1
mg/kg/day), based on decreased body weight gain during lactation for
both F1 and F2 pups.  The NOAEL is 100 ppm (8.4 mg/kg/day).

This study is classified as Acceptable/Guideline and satisfies the
requirements for a 2-generation reproduction study [OPPTS 870.3800
((83-4)] in rats.  No major deficiencies were noted in this study.

A.3.4	Chronic Toxicity

870.4100a (870.4300) Chronic Toxicity – Rat

Executive Summary: In a chronic oral toxicity study (MRID 44839901,
44807213), technical grade fluazinam (95.3%  a.i., Batch # 8412-20) was
administered to 25 Crl:CD((SD)BR rats/sex/dose in the diet at dose
levels of 0, 25, 50, or 100 ppm for 104 weeks (0, 1.0, 1.9, or 3.9
mg/kg/day for males; 0, 1.2, 2.4, or 4.9 mg/kg/day for females).  A
four-week range-finding study in rats was also conducted using 0, 10,
50, 250, or 3000 ppm (MRID 44807213).

No clinical signs of toxicity were observed, and survival rates were
unaffected by treatment.  At study termination, survival rates for the
0, 25, 50, or 100 ppm groups were  32, 52, 28, and 36% for the males,
respectively, and 72, 56, 72, and 52% for the females, respectively.  No
treatment-related effects on mean absolute body weights, body weight
gain, food consumption, or water consumption were noted.

No treatment-related differences were observed in hematology analysis or
urinalysis, and treatment-related clinical chemistry changes were
limited to a transient increase in total serum cholesterol in high-dose
females at Week 52 (154% of controls, p<0.01).  Relative liver weights
were increased in high-dose females at study termination (124% of
controls; p<0.01), but were not accompanied by any histopathological
correlates.  The increased relative liver weights and transient increase
in cholesterol were therefore not considered adverse.  Treatment with
fluazinam appeared to affect the testes.  High-dose decedent males
(those not surviving to study termination) had an increased incidence of
small and/or flaccid testes (11/16 or 69%, 8/16 or 50%, respectively;)
as compared with decedent controls (4/17 or 24% for both) during
macroscopic examination.   Microscopic examination revealed a
corresponding increased incidence of marked tubular atrophy in the
testes of these 100 ppm decedent males (8/16; 50%) as compared with
decedent controls (3/17; 18%); however, this increase was not
statistically significant and did not show a dose-response when
considering the average severity ranking for the decedent males (2.5,
3.4, 2.6, and 3.4 for the 0, 25, 50, and 100 ppm decedent males,
respectively).  When considering all 100 ppm males (decedent +
terminal), macroscopic examination did not reveal an increased incidence
of small and/or flaccid testes (16/25 vs. 16/25 controls), but the
overall severity of testicular tubular atrophy was increased in the
high-dose males (3.1) as compared with controls (2.6).  Because
testicular atrophy was also an effect noted in male rats following
dietary treatment with 100 or 1000 ppm in another chronic
toxicity/carcinogenicity study (MRID 42248620), it is considered an
effect of treatment in this study.

At the doses tested, there was not a treatment-related increase in tumor
incidences when compared to controls.

A LOAEL of 100 ppm (3.9 mg/kg/day) was identified for male rats based on
increased testicular atrophy, with a corresponding NOAEL of 50 ppm (1.9
mg/kg/day).  A LOAEL could not be identified for females. The NOAEL for
females was therefore 100 ppm (4.9 mg/kg/day).

This chronic oral toxicity study in the rat is classified as
Acceptable/Guideline and satisfies the Subdivision F guideline
requirement for a chronic oral toxicity study [OPPTS 870. 4100 ((83-1a)]
in rats.

Executive Summary: In a 4-week oral range-finding study (MRID 44807213),
technical grade fluazinam (96.3% a.i., Batch # 8203) was administered to
10 CD (Sprague-Dawley) rats/sex/dose in the diet at dose levels of 0,
10, 50, 250, or 3000 ppm for 4 weeks (0, 1.0, 5.1, 26.4, or 302
mg/kg/day for males; 0, 1.1, 5.3, 25.9, or 309 mg/kg/day for females).

The following treatment-related effects were observed at 3000 ppm in
both males and females: decreased body weight gain, decreased food
consumption, increased serum phospholipid, increased total cholesterol,
increased absolute liver weights, and increased relative liver/body
weight ratios.  In addition, increased incidences of  histopathological
findings were observed in the liver of males (increased periacinar
hypertrophy) and in the liver of females (increased single cell
necrosis).  The following treatment-related effects were also observed
at 250 ppm: decreased body weight gain (females), decreased food
consumption (females), increased serum phospholipid (females), increased
total cholesterol (males and females), increased relative liver/body
weight ratios (females), and increased histopathology in the liver of
males (increased periacinar hypertrophy).  In an addendum to this study,
there was no effect of treatment on the incidence or degree of white
matter vacuolation in the brain of male or female rats of the high dose
group (3000 ppm), compared with controls.

The LOAEL was 250 ppm (26.4 mg/kg/day for males; 25.9 mg/kg/day for
females), based on decreased body weight gain (females), decreased food
consumption (females), increased serum phospholipid (females), increased
total cholesterol (males and females), increased relative liver/body
weight ratios (females), and increased histopathology in the liver of
males (increased periacinar hypertrophy).  The NOAEL was 50 ppm (5.1
mg/kg/day for males; 5.3 mg/kg/day for females).

This range-finding study is classified as Acceptable/Nonguideline.  It
does not satisfy the Subdivision F guideline requirement for a
subchronic oral toxicity study [OPPTS 870.3100 (82-1)] in rats.

Executive Summary: In a combined chronic toxicity/carcinogenicity study
(MRID 42248620, 44807223, 45150201), fluazinam technical (95.3% a.i.,
lot number 8412-20) was administered to groups of 60 male and 60 female
Sprague-Dawley rats at dietary concentrations of 0, 1, 10, 100, or 1000
ppm (0, 0.04, 0.38, 3.8, or 40 mg/kg/day for males and 0, 0.05, 0.47,
4.9, or 53 mg/kg/day for females) for up to 104 weeks.  Groups of 10
rats of each sex per dose group were sacrificed at 52 weeks for interim
evaluations.

No treatment-related effects were observed in rats receiving the 1 ppm
or 10 ppm diets.  No treatment-related effect on mortality was observed
in rats receiving any dose of the test material.  The only clinical
signs observed were straw-discoloration of the fur in all rats receiving
the 1000 ppm diet and an increased incidence of alopecia in females
receiving the 1000 ppm diet.

Males receiving the 1000 ppm diet weighed 16% (p<0.01) less than
controls from week 8 to study termination, gained 15% less weight
overall, and consumed (8% less food than controls at each weekly
interval.  Females receiving the 1000 ppm diet weighed 24% (p<0.01) less
than controls from week 2 to termination, gained 35% less weight
overall, and consumed 18% less food than controls at each weekly
interval.  The food utilization factor or the food efficiency suggested
that reduced body weight gain was due in part to toxicity of the test
material.  No treatment-related effects were observed on body weights,
body weight gain, food consumption, or food utilization/efficiency in
male or female rats receiving the 1-, 10- or 100-ppm diets.  No
treatment-related effects were observed on the eyes at any dose at any
time during the study.  Clinical pathology evaluations showed only mild
anemia and elevated cholesterol in both sexes receiving the 1000 ppm
dose.  Treatment-related microscopic findings showed that the test
material was toxic to the following organs: liver, pancreas, lungs, and
possibly thyroid gland in both sexes, kidneys and testes in males, and
lymph nodes in females, but the primary target appeared to be the liver.
 Liver toxicity was manifested by increased organ weight and increased
incidences of gross and microscopic lesions.  Absolute liver weights
were increased by at least 7-27% and relative weights by 17-63% in both
sexes at 1000 ppm.  Gross lesions in the liver consisted of pale,
swollen or accentuated markings on the livers at 1000 ppm in males and
enlarged, pitted, or mottled livers at 1000 ppm in females.
Treatment-related microscopic liver lesions at 100 ppm consisted of
eosinophilic hepatocytes in 22% of females (8% in controls),
centrilobular hepatocyte rarefaction and vacuolation in 8% of each sex
(0% for controls), centrilobular sinusoidal dilatation in 10% of males
and 18% of females (0% for male and 2% for female control), and
pericholangitis in 18% of males and 14% of females (4% for male and 2%
for female controls).  Additional treatment-related liver lesions at
1000 ppm in main study group consisted of centrilobular hepatocyte
vacuolation in males, centrilobular hepatocyte necrosis in females, and
centrilobular fat and bile duct hyperplasia in both sexes.
Centrilobular hepatocyte vacuolation and centrilobular fat was also seen
in 1000 ppm group male and female rats at interim sacrifice.

The incidences of exocrine atrophy of the pancreas in both sexes and
acinal epithelial vacuolation or fat accumulation in females were
increased at 1000; the incidence of exocrine atrophy was also increased
at 100 ppm in females compared with that of control rats.  The incidence
of exocrine degranulation was increased in 1000 ppm group female rats at
interim sacrifice but not in the main study.  An increased incidence of
thyroid follicular hyperplasia was observed in males at 1000 ppm (8% vs.
2% for controls) and in females at 1000 ppm (10% vs. 2% in controls).
This  finding may possibly be related to treatment with the test
material.  In male rats, the incidence of cortical tubular basophilia in
the kidney was increased at 1000 ppm compared with that of the controls.
 Other treatment-related lesions included  pneumonitis, alveolar
adenomatosis, and alveolar epithelialization in 1000 ppm group males,
alveolar epithelialization and alveolar macrophage aggregates in 1000
ppm group females, testicular atrophy in 100 ppm and 1000 ppm group
males, and spermatocele granuloma also in 1000 ppm males.  The incidence
of sinus histiocytosis in the lymph nodes was increased in1000 ppm group
females.  Histopathologic assessment of the brain and spinal cord of
rats in the control and 1000 ppm dose groups showed no treatment-related
effect on vacuolation of white matter.

The lowest-observed-adverse-effect level (LOAEL) for fluazinam was 100
ppm (3.8 mg/kg/day for males and 4.9 mg/kg/day for females) based on
liver toxicity in both sexes, testicular atrophy in males and pancreatic
exocrine atrophy in females.  The corresponding
no-observed-adverse-effect level (NOAEL) was 10 ppm (0.38 mg/kg/day for
males and 0.47 mg/kg/day for females).

In this study, there were statistically significant positive trends for
thyroid gland follicular cell adenocarcinomas and combined follicular
cell adenomas/adenocarcinomas for the male rats.   There was also a
statistically significant increase by pair-wise comparison of the male
high dose group (1000 ppm) with the controls for combined follicular
cell adenomas/adenocarcinomas (23% vs. 8% in controls).  In addition to
an Exact Trend Test and a Fisher's Exact Test, a Peto's Prevalence Test
was also conducted (which excluded animals that died or were sacrificed
before observation of the first tumor at week 68).  For follicular cell
adenocarcinomas in males, results of the Peto's Prevalence Test showed a
statistically significant positive trend and a borderline statistically
significant (p= 0.056) increase by pair-wise comparison of the 1000 ppm
male group with the controls (7% vs. 0% in controls), indicating the
increased incidence of thyroid tumors had a malignant component to it.
For combined follicular cell adenomas/adenocarcinomas, Peto's Prevalence
Test also showed a statistically significant increase by pair-wise
comparison of the high dose male group with the controls (26% vs. 9% in
controls).  The incidences of thyroid gland  adenomas at 100 ppm and
1000 ppm (15% and 17%, respectively) and adenocarcinomas at 1000 ppm
(6%) were slightly outside  their respective ranges in the historical
control data (range: adenomas, 0%-13%; adenocarcinomas, 0%-5%).  Animals
in the lower dose groups were not microscopically examined for thyroid
lesions unless abnormalities were observed in that organ at gross
necropsy.  Therefore, percentage incidences of thyroid tumors in these
lower dose groups may have been somewhat misleading (too high).  The
highest dose level tested in this study was considered to be adequate
and not excessive because there were decreased  body weight gains (up to
15% and 35% in males and females, respectively), decreased food
consumption, decreased food efficiency, increased thyroid weights at 52
weeks, enlarged thyroids and a slightly increased incidence of thyroid
gland follicular cell hyperplasia  at 104 weeks in males. The survival
of the animals was not decreased by treatment with the test material.
There was no treatment-related increase in the thyroid tumor incidence
in the female rats in this study.  In addition, slightly increased
incidences of pancreatic islet cell adenomas were also observed in the
treated male rats, but these incidences were not dose-related and did
not exceed the upper range of historical control data for the same tumor
type (20 comparable studies at the same testing laboratory).  Further,
this type of tumor in this strain of rats is a common spontaneously
occurring neoplasm.  Therefore, the increased incidence of pancreatic
islet cell adenomas observed in the treated male rats in this study was
considered not likely to be related to treatment with the test material.
 High incidences of pituitary gland adenomas in males and females and of
mammary gland fibroadenomas in females were also observed in all groups,
including controls.  These neoplasms are not considered to be
treatment-related.  A toxicologically significant increase in tumors was
not observed in any other tissues in the treated male or female rats in
this study.

This combined chronic toxicity/carcinogenicity study in the rat is
Acceptable/Guideline and satisfies guideline requirements for a chronic
toxicity/carcinogenicity study [OPPTS 870.4300 ((83-5)] in rats.

870.4100b 	Chronic Toxicity – Dog

Executive Summary: In a chronic oral toxicity study (MRIDs 42270603,
main study and 44807219, addendum), Fluazinam (Lot No. 8412-20, 95.3%
purity) was administered to groups of six male and six female beagle
dogs/dose for 52 weeks at doses of 0, 1, 10, or 50 mg/kg/day in gelatin
capsules.

No animals died as a result of treatment. The most notable clinical
signs were increased incidence of salivation and nasal dryness, mainly
in the high-dose dogs but nasal dryness was also slightly increased in
females at 10 mg/kg/day. Body weight was mildly decreased at high dose
(-4%, males and -9%, females; not analyzed statistically), and total
body weight gain was significantly reduced (29%, p<0.05; -13% when
calculated as a percentage of initial body weight) only in females but
was also lower in males (-19%; -9% as a percentage of initial body
weight). Hematocrit, hemoglobin, and RBC counts of high-dose dogs were
consistently lower (8-17%; p<0.05, 0.01, or 0.001) than controls
throughout the treatment period, and WBC counts were elevated (32-64%,
p<0.05 or 0.001) at study end (these findings considered
treatment-related but not biologically significant). Alkaline
phosphatase was significantly increased (52-183%; p<0.05, 0.01, or
0.001) in high-dose dogs throughout the treatment period.

Absolute liver weight (males, 37%; females, 16%; p<0.05) and the
liver/body weight ratio (males, 45%; females, 47%; p<0.01) were
increased in high-dose dogs. In the reexamination of brain and spinal
cord tissues, incidence  of vacuolation of white matter in the brain was
increased in both sexes at the high dose (6/6 animals/sex affected vs.
2-4/6, controls) , along with increased severity (1.5-2.17 vs. 1.0,
controls).  In addition, vacuolation of the white matter of the spinal
cord was seen in high-dose females (4/6 affected vs. 0, controls). An
increase in liquefied GI tract contents and incidence/severity of
stomach mucosal lymphoid hyperplasia was seen in mid- and high-dose dogs
of both sexes, although in females, neither incidence nor mean severity
of the hyperplasia at these dose levels showed a dose-related increase.

The LOAEL is 10 mg/kg/day for both male and female dogs, based on
marginal increases in the incidence of nasal dryness in females and the
incidence/severity of gastric lymphoid hyperplasia in both sexes.  The
NOAEL is 1 mg/kg/day.

This chronic toxicity study is classified as Acceptable/guideline and
satisfies the guideline requirement for a chronic oral study [OPPTS
870.4100 ((83-1b)] in dogs. No major deficiencies were noted in this
study.

A.3.5		Carcinogenicity

870.4200b 	Carcinogenicity (feeding) – Mouse

Executive Summary: In a carcinogenicity study (MRID 42208405, 44807220,
44807212), Fluazinam (95.3% a.i., lot no. 8412-20) was administered to
groups of 52 male and 52 female CD(-1 mice in the diet at concentrations
of 0, 0, 1, 10, 100, or 1000 ppm.  There were 2 control groups.  The
test diets were given for 104 weeks.  These concentrations resulted in
mean daily compound intakes of 0.12, 1.1, 10.7, and 107 mg/kg/day for 1
ppm, 10 ppm, 100 ppm, and 1000 ppm, respectively, for males and 0.11,
1.2, 11.7, and 117 mg/kg/day, respectively, for females.  Additional
microscopic review of brain and spinal cord was presented in MRID
44807220.  A four-week-range finding study (MRID 44807212) using 0, 10,
50, 250, or 3000 ppm in the diet was also conducted.

Treatment with Fluazinam did not result in treatment-related changes in
survival, clinical signs, body weights, body weight gains, food
consumption or hematology parameters.  The group mean liver weights
adjusted for body weight were increased in males and females by 45% and
30%, respectively, at 1000 ppm compared to the controls, and by 15% in
females at 100 ppm after 104 weeks of treatment (p<0.01).  Microscopic
examination showed increased incidences of liver areas containing
basophilic hepatocytes (controls, 12%; 1000 ppm, 38%, p<0.01) and/or
eosinophilic vacuolated hepatocytes (controls, 1%; 100 ppm, 8%, p<0.05;
1000 ppm 19%, p<0.01) in treated males compared to the controls.
Increased incidences of granulomatous hepatitis of minimal severity were
seen in high-dose males (controls, 11%; 1000 ppm, 37%, p<0.01) and
females (controls, 11%; 1000 ppm, 21%, p<0.01).  Higher incidences of
aggregates of brown pigmented macrophages were seen in the livers of
treated males (controls, 13%; 100 ppm, 27%, p<0.05; 1000 ppm, 19%,
p<0.01) and females (controls, 15%; 1 ppm, 40%, p<0.01; 10 ppm, 21%, NS;
100 ppm, 38%; 1000 ppm, 50%, p<0.01).  Granulomatous hepatitis and brown
pigmented macrophage aggregates were most commonly seen in mice that
survived until the end of the study.  The only effects that were not
associated with the liver were an increased incidence of thymic
hyperplasia in high-dose females (controls, 5%; 1000 ppm, 21%, p <0.01),
and increased incidences of cystic thyroid follicles in high-dose males
(controls, 23%; 1000 ppm, 52%, p<0.01) and high-dose females (controls,
16%; 1000 ppm, 33%, p<0.01).

The central nervous systems of the animals were re-examined and the
results reported in an addendum to the main study (MRID 44807220).
Treatment-related increases in the incidences and severity of
vacuolation of white matter occurred in the brains of males at 1000 ppm
and increased severity of white matter vacuolation was seen in the
brains of females at 1000 ppm compared to the control groups.  No clear
effect of treatment on the incidence or severity of white matter
vacuolation in the spinal cord was seen in either sex, and no
treatment-related effects were seen at 1, 10, or 100 ppm.

The LOAEL is 100 ppm in the diet (10.7 mg/kg/day for males; 11.7
mg/kg/day for females), based on increased incidences of brown pigmented
macrophages in the liver of both sexes, increased incidences of
eosinophilic vacuolated hepatocytes in males, and increased liver
weights in females.  The NOAEL was 10 ppm (1.1 mg/kg/day for males; 1.2
mg/kg/day for females).

In this study, there were statistically significant positive trends for
hepatocellular adenomas, carcinomas and combined adenomas/carcinomas for
the male mice.  There were also statistically significant  increases by
pair-wise comparison of the male high dose group (1000 ppm) with the
controls for hepatocellular adenomas (34% vs. 16% in controls), for
hepatocellular carcinomas (34% vs. 19% in controls) and for combined
hepatocellular adenomas/carcinomas (62% vs. 33% in controls).  The
incidence of hepatocellular adenomas (34%) at the highest dose level for
males exceeded the highest incidence in the historical control data for
1981-1983 (4-27%) and for 1986-1988 (8-23%), and the incidence of
hepatocellular carcinomas (34%) for the highest dose level for males
exceeded the highest incidence in the historical control data for
1986-1988 (5-13%), but not for 1981-1983 (12-38%).  There were no
treatment-related tumors observed in the female mice in this study.
The highest dose level tested was considered to be adequate but not
excessive because liver and brain toxicity were observed in the male and
female mice at 1000 ppm.  Although there were no significant changes in
survival or body weight gain, mean liver weight gains were increased in
males and females and histopathological changes were observed in the
livers and brain (vacuolation of the white matter) of males and females.

This carcinogenicity study in the mouse is Acceptable/Guideline and does
satisfy the guideline requirement for an carcinogenicity study [OPPTS
870.4200 (83-2b)] in mice. An additional study has been done following
this study with higher concentrations of fluazinam (see MRID 44807222).

In a range-finding study (MRID 44807212), groups of 10 male and 10
female CD(1 mice were given concentrations of 0, 10, 50, 250, or 3000
ppm fluazinam (B-1216) in the diet for 4 weeks.  There were no
significant changes in body weights between treated and control animals;
however, group mean body weight gain was slightly less in both sexes at
250 and 3000 ppm.  The body weight change did not show a clear dose
dependency especially in females.  Platelet counts were marginally
elevated in males at 3000 ppm and total blood cholesterol was slightly
higher in both sexes at 3000 ppm compared to the controls.  Phospholipid
concentration was slightly increased in females and marginally increased
in males at 3000 ppm.  Blood glucose concentrations were increased in
females at 250 and 3000 ppm compared to the control group.  Absolute and
relative (to body weight) liver weights were increased at 3000 ppm in
both sexes and the absolute and relative kidney weights were increased
in females at 3000 ppm.  Hepatocyte periacinar hypertrophy incidences
and severity were increased in both sexes at 3000 ppm compared to the
control groups.

870.4200b 	Carcinogenicity (feeding) - Mouse

Executive Summary: In another carcinogenicity study (MRID 44807222,
44807221, 45201301, 44807211), technical grade Fluazinam (97.0% a.i.,
lot no. 1030/91) was administered to groups of 50 male and 50 female
Crl:CD(-1 mice in the diet at concentrations of 0, 1000, 3000, or 7000
ppm.  The test diets were given for 97 weeks to females and for 104
weeks to males.  These concentrations resulted in mean daily compound
intakes of 126, 377, and 964 mg/kg/day for males and 162, 453, and 1185
mg/kg/day for females for 1000 ppm, 3000 ppm, and 7000 ppm,
respectively.  Twenty mice were added to the control and 7000 ppm groups
for a satellite study in which the mice were killed and necropsied after
treatment for 78 weeks.  A Pathology Working Group (PWG) report
presenting revised incidences for hepatocellular tumors in the male mice
in this study was later submitted (MRID 45201301).  A four-week
range-finding study in mice was also conducted using 0, 3000, 5000, and
7000 ppm in the diet (MRID 44807211).

Treatment with fluazinam resulted in a significant decrease in survival
in females at 7000 ppm (control, 58%; 7000 ppm, 26%, p <0.01).  All
females were terminated after 97 weeks of treatment because of low
survival at the high-dose.  At 7000 ppm, body weight gain was decreased
in males during weeks 4-36 by 32% (p< 0.01) and food conversion ratios
over weeks 9-13 were increased by 86% compared to the controls
indicating decreased efficiency of food utilization.  At termination,
relative liver/body weight ratios were increased in males by 54%, 113%
and 182% and in females by 21%, 45%, and 109% at 1000, 3000, and 7000
ppm, respectively, compared to the controls (p<0.01).  Microscopic
examination showed increased incidences of altered hepatocyte foci at
all concentration levels in males and in high-dose females (males:
control, 12/50; 1000 ppm 24/50, p<0.05; 3000 ppm, 36/50; 7000 ppm,
33/50, p<0.01; females: control, 3/50; 7000 ppm 15/50, p<0.01).
Incidences of hepatocyte enlargement, pale or vacuolated hepatocyte
cytoplasm, and brown pigmented macrophage aggregates were increased in
all treated males and females compared to the control groups (p<0.01).
The pigmented macrophage aggregates also increased in severity from
0-22% of lesions in the controls to 41-58% of lesions at 7000 ppm graded
moderate or marked.  Incidences of brown pigmented centrilobular
hepatocytes and parenchymal inflammatory cells increased in males at
3000 ppm (both 6/50, p<0.05) and 7000 ppm (11-16/50, p<0.01) compared to
the controls (0-1/50).  Males were more sensitive to the hepatotoxic
effects of fluazinam than females.  Incidences of vacuolation of white
matter in the brains of all treated animals were increased compared to
the controls (p<0.01).  Vacuolation of white matter was also increased
in the cervical spinal cord of males at 3000 and 7000 ppm (control,
18/50; 3000 ppm, 37/50, p<0.05; 7000 ppm, 46/50, p<0.01) and marginally
in females (control, 37/50; 3000 and 7000 ppm, 45/50, NS).  The severity
of the vacuolation of white matter in the brain and spinal cord
increased with increasing dose from 0% graded moderate or marked in the
controls to 33-60% of lesions at 7000 ppm.  Incidences of left atrial
thrombus in the hearts of high-dose males and females were increased
compared to the controls and contributed to the unscheduled deaths of
about 46% of high-dose males and 30% of high-dose females during the
study.

The LOAEL is 1000 ppm in the diet (126 mg/kg/day for males; 162
mg/kg/day for females), based on increased liver weights in males and
females and on histopathological changes in the liver and brain in males
and females.  A NOAEL was not determined (<1000ppm).

In this study, there were no statistically significant positive trends
for hepatocellular adenomas, carcinomas or combined adenomas/carcinomas
for the male mice.  For the mid-dose male mice (3000 ppm), however,
there was a statistically significant increase by pair-wise comparison
with the controls for hepatocellular adenomas (47% vs. 17% in controls)
and for combined hepatocellular adenomas/carcinomas (49% vs. 18% in
controls).  For the high-dose male mice (7000 ppm), there was no
statistically significant increase by pair-wise comparison with controls
for hepatocellular adenomas or carcinomas when considered separately,
but there was a statistically significant increase for combined
adenomas/carcinomas (33% vs. 18% in controls).   The incidence of
hepatocellular adenomas at the mid-dose level for males (47%) exceeded
the highest incidence in the historical control data for 1991-1993
(8-34%) and for 1987-1993 (0-31%), but the incidence at the high-dose
level for males (30%) did not exceed the highest incidences in the
comparable historical control data.  Similarly, the incidence of
combined hepatocellular adenomas/carcinomas at the mid-dose level for
males (49%) exceeded the highest incidence in the historical control
data for 1987-1993 (4-42%), but the incidence at the high-dose level for
males (33%) did not exceed the highest incidences in the comparable
historical control data.  For the male mice in this study, the
tumorigenic response did not appear to be dose-related because the
response at 7000 ppm was less than that observed at 3000 ppm.  The
highest dose level tested for the male mice in this study was considered
to be adequate but not excessive.  In this study, hepatocellular tumors
were also observed in the female mice at the high-dose (7000 ppm).
These tumors, however, occurred at an excessively toxic dose which may
have resulted in indirect effects that may not have been present at
lower doses.  There was a treatment-related increased mortality for the
high-dose females in this study.  Although a statistically significant
positive trend was observed for combined hepatocellular
adenomas/carcinomas for the female mice in this study, this calculation
included the response at 7000 ppm.  At the next lower dose level (3000
ppm), there was no statistically significant pair-wise increase in
hepatocellular adenomas, carcinomas or combined adenomas/carcinomas for
the female mice in this study.

This carcinogenicity study in the mouse is Acceptable/Guideline and does
satisfy the guideline requirement for an carcinogenicity study [OPPTS
870.4200 (83-2b)] in mice.

Executive Summary: Treatment levels of 0, 3000, 5000, or 7000 ppm in the
diet were given to mice in a 4-week study (MRID 44807211).
Treatment-related changes were seen in the liver of mice at all
treatment levels and kidney effects were seen in males at 5000 and 7000
ppm.  Treatment-related increased incidences and severity of vacuolation
of white matter of the brain were seen at four weeks in males at 3000,
5000, and 7000 ppm and in females at 7000 ppm.  Treatment-related
increased incidences and severity of vacuolation of white matter of the
spinal cord were also seen in males at 5000 and 7000 ppm.

The LOAEL for vacuolation of white matter in the brain of male mice in
this four-week study was 3000 ppm (555 mg/kg/day; lowest dose level
tested).  A NOAEL was not demonstrated for this effect (i.e. NOAEL <555
mg/kg/day).

870.4300 	Combined Chronic Carcinogenicity Study – Rat

EXECUTIVE SUMMARY:  In a combined chronic toxicity/carcinogenicity study
(MRID 42248620, 44807223, 451450201), fluazinam technical (95.3% a.i.,
lot number 8412-20) was administered to groups of 60 male and 60 female
Sprague-Dawley rats at dietary concentrations of 0, 1, 10, 100, or 1000
ppm (0, 0.04, 0.38, 3.8, or 40 mg/kg/day for males and 0, 0.05, 0.47,
4.9, or 53 mg/kg/day for females) for up to 104 weeks.  Groups of 10
rats of each sex per dose group were sacrificed at 52 weeks for interim
evaluations.

No treatment-related effects were observed in rats receiving the 1 ppm
or 10 ppm diets.  No treatment-related effect on mortality was observed
in rats receiving any dose of the test material.  The only clinical
signs observed were straw-discoloration of the fur in all rats receiving
the 1000 ppm diet and an increased incidence of alopecia in females
receiving the 1000 ppm diet.

8% less food than controls at each weekly interval.  Females
receiving the 1000 ppm diet weighed 7–24% (p<0.01) less than controls
from week 2 to termination, gained 35% less weight overall, and consumed
18% less food than controls at each weekly interval.  The food
utilization factor or the food efficiency suggested that reduced body
weight gain was due in part to toxicity of the test material.  No
treatment-related effects were observed on body weights, body weight
gain, food consumption, or food utilization/efficiency in male or female
rats receiving the 1-, 10- or 100-ppm diets.  No treatment-related
effects were observed on the eyes at any dose at any time during the
study.  Clinical pathology evaluations showed only mild anemia and
elevated cholesterol in both sexes receiving the 1000 ppm dose.

Treatment-related microscopic findings showed that the test material was
toxic to the following organs: liver, pancreas, lungs, and possibly
thyroid gland in both sexes, kidneys and testes in males, and lymph
nodes in females, but the primary target appeared to be the liver.
Liver toxicity was manifested by increased organ weight and increased
incidences of gross and microscopic lesions.  Absolute liver weights
were increased by at least 7-27% and relative weights by 17-63% in both
sexes at 1000 ppm.  Gross lesions in the liver consisted of pale,
swollen or accentuated markings on the livers at 1000 ppm in males and
enlarged, pitted, or mottled livers at 1000 ppm in females.
Treatment-related microscopic liver lesions at 100 ppm consisted of
eosinophilic hepatocytes in 22% of females (8% in controls),
centrilobular hepatocyte rarefaction and vacuolation in 8% of each sex
(0% for controls), centrilobular sinusoidal dilatation in 10% of males
and 18% of females (0% for male and 2% for female control), and
pericholangitis in 18% of males and 14% of females (4% for male and 2%
for female controls).  Additional treatment- related liver lesions at
1000 ppm in main study group consisted of centrilobular hepatocyte
vacuolation in males, centrilobular hepatocyte necrosis in females, and
centrilobular fat and bile duct hyperplasia in both sexes.
Centrilobular hepatocyte vacuolation and centrilobular fat was also seen
in 1000 ppm group male and female rats at interim sacrifice.

The incidences of exocrine atrophy of the pancreas in both sexes and
acinal epithelial vacuolation or fat accumulation in females were
increased at 1000; the incidence of exocrine atrophy was also increased
at 100 ppm in females compared with that of control rats.  The incidence
of exocrine degranulation was increased in 1000 ppm group females rats
at interim sacrifice but not in the main study.  An increased incidence
of thyroid follicular hyperplasia was observed in males at 1000 ppm (8%
vs 2% for controls) and in females at 1000 ppm (10% vs 2% in controls).
This  finding may possibly be related to treatment with the test
material.  In male rats, the incidence of cortical tubular basophilia in
the kidney was increased at 1000 ppm compared with that of the controls.
 Other treatment-related lesions included  pneumonitis, alveolar
adenomatosis, and alveolar epithelialization in 1000 ppm group males,
alveolar epithelialization and alveolar macrophage aggregates in 1000
ppm group females, testicular atrophy in 100 ppm and 1000 ppm group
males, and spermatocele granuloma also in 1000 ppm males.  The incidence
of sinus histiocytosis in the lymph nodes was increased in1000ppm group
females.  Histopathologic assessment of the brain and spinal cord of
rats in the control and 1000 ppm dose groups showed no treatment-related
effect on vacuolation of white matter.

The lowest-observed-adverse-effect level (LOAEL) for fluazinam was 100
ppm (3.8 mg/kg/day for males and 4.9 mg/kg/day for females) based on
liver toxicity in both sexes, testicular atrophy in males and pancreatic
exocrine atrophy in females.  The corresponding
no-observed-adverse-effect level (NOAEL) was 10 ppm (0.38 mg/kg/day for
males and 0.47 mg/kg/day for females).

CARC Comments: In this study, there were statistically significant
positive trends for thyroid gland follicular cell adenocarcinomas and
combined follicular cell adenomas/adenocarcinomas for the male rats.
There was also a statistically significant increase by pair-wise
comparison of the male high dose group (1000 ppm) with the controls for
combined follicular cell adenomas/adenocarcinomas (23% vs 8% in
controls).  In addition to an Exact Trend Test and a Fisher’s Exact
Test, a Peto’s Prevalence Test was also conducted (which excluded
animals that died or were sacrificed before observation of the first
tumor at week 68).  For follicular cell adenocarcinomas in males,
results of the Peto’s Prevalence Test showed a statistically
significant positive trend and a borderline statistically significant
(p= 0.056) increase by pair-wise comparison of the 1000 ppm male group
with the controls (7% vs 0% in controls), indicating the increased
incidence of thyroid tumors had a malignant component to it.  For
combined follicular cell adenomas/adenocarcinomas, Peto’s Prevalence
Test also showed a statistically significant increase by pair-wise
comparison of the high dose male group with the controls (26% vs 9% in
controls).  The incidences of thyroid gland  adenomas at 100 ppm and
1000 ppm (15% and 17%, respectively) and adenocarcinomas at 1000 ppm
(6%) were slightly outside  their respective ranges in the historical
control data (range: adenomas, 0%-13%; adenocarcinomas, 0%-5%).  Animals
in the lower dose groups were not microscopically examined for thyroid
lesions unless abnormalities were observed in that organ at gross
necropsy.  Therefore, percentage incidences of thyroid tumors in these
lower dose groups may have been somewhat misleading (too high).  The
highest dose level tested in this study was considered to be adequate
and not excessive because there were decreased body weight gains (up to
15% and 35% in males and females, respectively), decreased food
consumption, decreased food efficiency, increased thyroid weights at 52
weeks, enlarged thyroids and a slightly increased incidence of thyroid
gland follicular cell hyperplasia at 104 weeks in males. The survival of
the animals was not decreased by treatment with the test material.
There was no treatment-related increase in the thyroid tumor incidence
in the female rats in this study.  In addition, slightly increased
incidences of pancreatic islet cell adenomas were also observed in the
treated male rats, but these incidences were not dose-related and did
not exceed the upper range of historical control data for the same tumor
type (20 comparable studies at the same testing laboratory).  Further,
this type of tumor in this strain of rats is a common spontaneously
occurring neoplasm.  Therefore, the increased incidence of pancreatic
islet cell adenomas observed in the treated male rats in this study was
considered not likely to be related to treatment with the test material.
 High incidences of pituitary gland adenomas in males and females and of
mammary gland fibroadenomas in females were also observed in all groups,
including controls.  These neoplasms are not considered to be
treatment-related.  A toxicologically significant increase in tumors was
not observed in any other tissues in the treated male or female rats in
this study.

This combined chronic toxicity/carcinogenicity study in the rat is
Acceptable/Guideline and satisfies guideline requirements for a chronic
toxicity/carcinogenicity study [OPPTS 870.4300 (§83-5)] in rats.

A.3.6	Mutagenicity

Gene Mutation

Guideline 870.5100

Bacterial reverse mutation assay (Ames Test) with S. typhimurium and E.
coli

MRID 42270605

Acceptable

ut S9 up to cytotoxic concentrations.  Compound was tested up 2μg/plate
for S. typhimurium strains and up to 250 μg/plate for E. coli in the
absence of S9, and up to 100 μg/plate for S. typhimurium strains and up
to 500 μg/plate for E. coli in the presence of S9.

Guideline 870.5100

Bacterial reverse mutation assay (Ames Test) with S. typhimurium and E.
coli

MRID 42270604

Acceptable

Negative with and without S9 up to cytotoxic concentrations.  Compound
was tested up 1μg/plate for S. typhimurium strains and up to 250
μg/plate for E. coli in the absence of S9, and up to 100 μg/plate for
S. typhimurium strains and up to 500 μg/plate for E. coli in the
presence of S9.

Guideline 870.5300

Mammalian cells in culture forward gene mutation assay with mouse
lymphoma L5178Y/TK +/- cells

MRID 45156902

Acceptable

Negative with and without S9 up to 5 μg/mL.  Compound was tested up to
cytotoxic concentrations.

Guideline 870.5300

Mammalian cells in culture forward gene mutation assay with mouse
lymphoma L5178Y/TK +/- cells

MRID 45261801

Acceptable

Negative with S9 up to 9 μg/mL and without S9 up to 0.3 μg/mL.
Compound was tested up to cytotoxic concentrations.

Cytogenetics

Guideline 870.5375

In vitro mammalian chromosome aberration (CHL cells)

MRID 42270606

Acceptable

Negative with and without S9 up to cytotoxic concentrations.  Compound
was tested up 4 μg/mL in the absence of S9, and up to 9.5 μg/mL in the
presence of S9.  Cells harvested at 24 and 48 hours in nonactivated
studies and at 24 hours in activated studies.

Guideline 870.5395

Mammalian erythrocyte micronucleus test (mouse)

MRID 44807224

Acceptable

Negative.  Test compound at 500, 1000, or 2000 mg/kg (oral gavage) with
a 24 hour sacrifice or at 2000 mg/kg with 24, 48, and 72 hour sacrifices
did not induce the formation of micronuclei in polychromatic
erythrocytes from bone marrow.  Clinical signs included piloerection,
decreased motor activity, loose stools, and/or soiled fur.

Other Genotoxicity

Guideline 870.5550

Unscheduled DNA synthesis in primary rat hepatocytes

MRID 45156901

Unacceptable	Negative; however, there were several serious study
deficiencies: treatment time shorter than recommended, no data
supporting the claim of cytotoxicity, and data variability for major
endpoints.

Guideline 870.5500

Differential Killing/Growth Inhibition in B. subtilis

MRID 42270607

Negative without S9 up to 0.3 μg/disk and with S9 up to 30 μg/disk.
Deficiency: only one replicate per plate dose was used.

A.3.7	Neurotoxicity

870.6200 	Acute Neurotoxicity Screening Battery

Executive Summary: In an acute oral neurotoxicity study (MRID 44807210),
single gavage doses of 0, 50, 1000 or 2000 mg/kg fluazinam (96.8%, Lot
No.: 1030/91) in 1.5% (w/v) aqueous methylcellulose were administered to
groups of fasted Sprague-Dawley rats (10/sex/dose).   Functional
Observational Battery (FOB) and Motor Activity (MA) assessments were
performed before test substance administration, between 5 and 7 hours
post-administration (time of peak effect) and on days 7 and 14.  Body
weights were measured weekly and clinical signs were recorded daily.
The animals were sacrificed and grossly examined 14 days after
administration of the test material.  Five rats/dose/sex were perfused
in situ for neuropathological evaluation.

There were no treatment-related deaths, clinical signs, or body weight
effects during the study.  Treatment-related soft stools were observed
in mid- and high-dose males and females, but only on the day of
treatment.  Treatment-related mean motor activity values were
significantly decreased (23-65%) in mid- and high-dose females compared
to controls (not dose-related), but only on the day of treatment.   No
other treatment-related effects of any kind were observed at any time
during the study.  No treatment-related gross effects or histopathology
were observed. Since the decreased mean motor activity values were
observed in one sex only (females), on one occasion only (on the day of
dosing), at high doses only (>1000 mg/kg), and were not dose-related,
and were observed in one study only (not observed in the subchronic
neurotoxicity study in rats, MRID 44807217, 44807218), it is likely that
this treatment-related effect (decreased mean motor activity values in
females only on the day of dosing only) is a manifestation of acute
general systemic toxicity and not a direct neurotoxic response to the
administration of the test material.

Under the conditions of this study, the acute general systemic toxicity
LOAEL is 1000 mg/kg for male and  female rats based on soft stools and
decreased motor activity.  The acute general systemic toxicity NOAEL is
50 mg/kg for male and female rats.  The LOAEL for neurotoxic effects is
not identified (> 2000 mg/kg).  The NOAEL for neurotoxic effects is 2000
mg/kg.

This acute oral neurotoxicity study is classified Acceptable/ Guideline.
This study does satisfy the guideline requirement for an acute oral
neurotoxicity study [OPPTS 870.6200 (81-8ss)] in rats.

870.6200 	Subchronic Neurotoxicity Screening Battery

Executive Summary: In two subchronic oral neurotoxicity studies (MRID
44807217 & MRID 44807218), groups of 10 male and 10 female Crl:CD BR
rats were fed diets containing 0, 300, or 1000 ppm fluazinam (MRID
44807217, 96.9%, Lot No. 6109) or 0, 1000, 2000, or 3000 ppm fluazinam
(MRID 44807218, 98.4%, Lot No. 9601-2) for 13 weeks.   Achieved doses
were 20.7, 69-74, 149, and 233 mg/kg/day for males in the 300, 1000,
2000, and 3000 ppm groups, respectively; and 23.4, 81-89, 175, and 280
mg/kg/day for females in the 300, 1000, 2000, and 3000 ppm groups,
respectively.  Functional Observational Battery (FOB) and Motor Activity
(MA) assessments were performed prior to treatment and during weeks 4,
8, and 13 of treatment.  Body weights, food consumption, and clinical
signs were monitored throughout the study.   At the end of the treatment
period, all rats were perfused in situ.  The brain from all rats was
removed, weighed, and measured and 5 males and 5 females from the
control and high-dose groups of each study were subjected to
neuropathological evaluation.

There were no treatment-related deaths or clinical signs.  At the end of
the study, group mean body weight gains were significantly (p<0.01)
decreased in females in and above the 1000 ppm groups and in males in
the 2000 and 3000 ppm groups.  Similarly, cumulative food consumption
was decreased in males (p<0.01) and females (p<0.05) fed 2000 and 3000
ppm fluazinam.  Food efficiency was decreased in males at 3000 ppm and a
dose-related decrease in food efficiency was observed in females in all
treatment groups.

No treatment-related FOB or MA effects were observed.  Brain weights of
females in the 3000 ppm group were 8% lower (p<0.01) than controls;
however, no supporting pathology was observed.  No treatment-related
gross effects or histopathology were observed.

Under the conditions of these studies, the neurotoxicity NOAEL is 3000
ppm (233 mg/kg/day) for male rats and 3000 ppm (280 mg/kg/day) for
female rats.  A neurotoxicity LOAEL was not identified.

These subchronic neurotoxicity studies are classified as
Acceptable/Guideline. When considered together, these studies satisfy
the guideline requirement for a subchronic neurotoxicity study [OPPTS
870.6200 (82-7ss)] in rats.

870.6300 	Developmental Neurotoxicity Study

EXECUTIVE SUMMARY: In a developmental neurotoxicity study (2005, MRID
46534401), Fluazinam (97.8% a.i.; lot # A629/1995, impurity #5 0.09%)
was administered by gavage to 24 Crl:CD® (SD) IGS BR rats/sex/dose at
0, 2, 10 or 50 mg/kg/day from gestation day (GD) 6 through lactation day
(LD) 20.  The pups were administered the same doses by gavage from
postnatal days (PND) 7 to 20 or 21. Maternal evaluation consisted of a
Functional Operational Battery (FOB) was performed on GDs 12 and 18, and
on post partum days (PPDs) 35, 45 and 60. Additional behavioral
assessments included: motor activity on GD 15 and PPD 60; auditory
startle habituation on GD 19 and PPD 58; and learning and memory on LD
16 and PPD 61.  From each maternal group, 12 were sacrificed on both LD
21 and PPD 66; of these, 10/group were selected for neuropathology
procedures. The additional assessment of the dams is related to trying
to further characterizing neurotoxicity that is attributed to impurity
#5. On postnatal day (PND) 4, litters were culled to yield four males
and four females (as closely as possible).  Offspring were allocated for
detailed clinical observations (FOB) and assessment of motor activity,
auditory startle reflex habituation, learning and memory (watermaze
testing), and neuropathology at days 23/24 and on PND day 66.   On PND
21, the whole brain was collected from 10 pups/sex/dose group for
micropathologic examination and morphometric analysis.  Pup physical
development was evaluated by body weight.  The age of sexual maturation
(vaginal opening in females and preputial separation in males) was
assessed.

Maternal parameters.  No effects on absolute body weight were noted.
Mean body weight gain for GDs 6-14 was significantly decreased (14%) at
10 and 50 mg/kg/day and during GDs 6-20 at 50 mg/kg/day (10%).  Mean
body weight gain during lactation was not affected.  Food consumption
during gestation was comparable to controls but during lactation was
significantly decreased at 10 (10 to 14%) and 50 mg/kg/day (7 to 11%).
Mean body weight and body weight gain post weaning (days 28-63) were not
affected. The weight gain and food consumption data that occurs in the
absence of absolute weight differences for the dams are not considered
to be of sufficient magnitude to be included as a true toxic response.
Reproductive parameters and behavioral assessments, including FOB, motor
activity, auditory startle reflex habituation and learning and memory,
were not affected by treatment.  No treatment-related changes were
observed at either the necropsy on LD 21 or PPD 66.  The characteristic
grey matter vacuolation attributed to fluazinam in previous studies was
not seen at LD 21 or PPD 66.  The maternal LOAEL for Fluazinam was not
established.  The maternal NOAEL is > 50 mg/kg/day. The weight gain and
food consumption data that occurs in the absence of absolute weight
differences for the dams are not considered to be sufficient of
sufficient magnitude to be included as a true toxic response.

Offspring parameters. No treatment-related effects were observed on
litter size at birth or survival to weaning.  Birth weight was lower in
females at 10 and 50 mg/kg/day (both 6%, p < 0.01) but not in males.
Mean offspring body weight was significantly decreased in males and
females at 10 (6-11%) and 50 mg/kg/day (6-16%) during lactation.  Mean
body weight gain was significantly decreased in male and female pups at
10 (4-24%) and 50 mg/kg/day (16-35%) during lactation. During the
post-weaning period (PNDs 28-63), mean body weight was significantly
decreased in males and females at 10 (3-7%) and 50 mg/kg/day (7-15%).
However, post weaning body weight gain was essentially comparable to the
control group.  The mean age of completion of balano-preputial
separation was significantly delayed at 10 and 50 mg/kg/day.   Rearing
counts in female pups were decreased on day 21 in the 10 (to a mean of
3.5 vs. 8.1 in the controls) and 50 (mean 3.7) mg/kg/day dose groups.
Dark and/or distended abdomens observed in a total of 12 offspring from
4 litters at 50 mg/kg/day were considered treatment-related since
similar signs were observed in the preliminary study. The peak amplitude
in the auditory startle response was affected in males in the high dose
group at day 23/24.  Absolute brain weight for high dose males was 6.1%
decreased on PND 21 and slight changes in brain width were noted.  No
treatment-related effects were observed on other behavioral assessments,
including FOB, motor activity or learning and memory.  Grey matter
vacuolation was not seen in the LD21 neuropathology assessment.  A
single isolated incident of grey matter vacuolation in the high dose
group at day 66 was not considered related to treatment.  The offspring
LOAEL is 10 mg/kg/day based on decreased body weight and body weight
gain and delay in completion of balano-preputial separation.  The
offspring NOAEL is 2 mg/kg/day.

This developmental neurotoxicity is classified as
Acceptable/Non-Guideline and may be used for regulatory purposes.  It
does not, however, satisfy the guideline requirement for a developmental
neurotoxicity study in rats (OPPTS 870.6300, §83-6): OECD 426 (draft)
due to the pending review of the positive control data.

A.3.8		Metabolism

870.7485		Metabolism – Rat

Executive Summary: In a metabolism characterization study (MRID No.
44807233), Fluazinam (IKF-1216) was administered by gavage at single
doses of 0.5 mg/kg or 50 mg/kg, or 14-day repeated doses of 0.5
mg/kg/day.  In addition to nonlabeled IKF-1216 (lot no. T9002, 99.6%
purity), [14C]-IKF-1216 labeled on the phenyl moiety (lot. No. 93-5,
purity 98%, sp. act. 57.3 mCi/mmol) or pyridyl moiety (lot. No. 93-90,
98% purity; sp. act. 66.2 mCi/mmol)  were also administered in some
studies to assess metabolic cleavage of the phenyl or pyridyl ring of
the test material.  Experimental groups were established for overall
distribution/excretion assessment and for analysis of biliary secretion.
The metabolite profiles of urine, feces, and bile were examined and
major metabolites were identified.

There were no treatment-related deaths in the rats.  Overall recovery of
the administered radioactivity (reported in MRID Nos. 43521006,
43521007, and 43521008 and evaluated in a separate DER) was acceptable
(93.10-103.55%).  Excretion via the urine was minor.  AMPA mercapturate
and DAPA, the major urinary metabolites, represented only 0.05-0.39% of
the administered dose.  Radioactivity in the feces represented most of
the administered dose (88.78-100.03%) as determined by review of MRID
Nos. 43521006, 43521007, and 43521008 and evaluated in a separate DER.
Identified fecal metabolites, however, represented only 11.20-68.59% of
the administered dose. For all dose groups, most of the fecal
radioactivity appeared to reside with unextractable components in the
post-extraction solids (PES).  Further analysis of the PES components
using base hydrolysis indicated that most of this radioactivity could be
attributed to hydrolysis products of AMPA and DAPA.   PES radioactivity
was also greatest for the low-dose group which was consistent with the
lower overall accounting of identified metabolites for this group.
Approximately 20-25% of the aqueous phase of the fecal extraction was
identified as a cysteine conjugate of DAPA and represented <1% of the
administered dose. With the exception of the low-dose group, parent
compound represented most of the identified radioactivity in the feces.
AMPA and DAPA were identified in the feces from all dose groups but
these metabolites never represented more than 5% of the administered
dose (except for high-dose female rats where AMPA accounted for 10.22%).

DAPA glucuronide  and AMPA mercapturate were the major biliary
metabolites but represented  <4% of the administered dose.  Total
biliary radioactivity, however represented 25-34% of the administered
dose (MRID Nos. 43521006, 43521007, and 43521008 evaluated in a separate
DER).  Analysis of chromatograms indicated that numerous other
metabolites were present in the bile but were individually of
insufficient quantity to allow for characterization.

Metabolite profiles from administration of different label positions
(pyridyl and phenyl) indicated that there was no metabolic cleavage of
the ring structures.  Minor quantitative differences in metabolite
recovery were observed between genders but not of sufficient magnitude
to suggest biologically relevant differences in the metabolism of
IKF-1216.

This metabolism study is Acceptable/Guideline.  When considered together
with the previously submitted general metabolism studies on IKF-1216
(MRIDs 43521004 through 43521008 and MRID 43553001), the requirement for
a general metabolism study in rats [OPPTS 870.7485 ((85-1)] is
satisfied.

870.7600	Dermal Absorption

There is no dermal absorption study with fluazinam and no study has been
requested.

A.3.9			Special/Other Studies

Nonguideline  	90-Day Oral Toxicity – Rat (Special Liver Toxicity
Study)

Executive Summary: In a 90-day oral toxicity study (special liver
toxicity study) (MRID 42248609), groups of 10 male and 10 female CD
rats were given Fluazinam (a.i. 98.5%, Lot/Batch # 8303-2) administered
at 0 or 500 ppm (equivalent to average mg/kg/day levels of 0 and 37.63
in males and 0 and 44.71 in females) in the diet.  An additional group
of 10 male and 10 female rats were given Fluazinam at 0 or 500 ppm in
the diet for 90 days, then given no Fluazinam for 4 weeks in a
reversibility study.

≤ 0.01) and females (+15%, p≤ 0.01), and periacinar hepatocytic
hypertrophy in 10/10 males (0/10 controls).  These effects  resolved
after the 4-week reversibility phase.

This special liver toxicity study in rats is classified as Acceptable
/Nonguideline.  The study did not fully meet its objective of assessing
the hepatotoxic effects of the test material or determine their
reversibility because only one dose (500 ppm) of Fluazinam was utilized,
and the modest liver changes observed were of questionable toxicological
significance.

Nonguideline  	11-Week Oral Toxicity – Dog (Special Retinal Toxicity
Study)

Executive Summary: In an 11-week oral toxicity study (special retinal
toxicity study) (MRID 44807216), Fluazinam (2 batches from Lot no.
8303-2: 98.0% and 98.1% a.i.) was administered in gelatin capsules for
11 weeks to 6 male beagle dogs/group at 0 or 200 mg/kg/day (dose
reduced to 150 mg/kg/day in two animals at week 3, and in the remaining
four animals at week 5, due to excessive toxicity).  Three
animals/dose group were terminated on study day 77 (Group II;
corresponding controls Group I), and the remaining 3 animals/group
remained on study without treatment for 5 weeks before termination on
study day 112 (Group IIW; corresponding controls Group IW).  This study
was designed to evaluate retinal changes seen at 100-150 mg/kg/day in
previous 28- and 90-day studies, follow the time course of any changes,
assess retinal function and assess reversibility.  In addition to
ophthalmologic evaluation, fundic photography and electroretinography
were performed pretest, weekly during treatment and at weeks 2 and 4 of
compound withdrawal.  Eyes were examined microscopically and by electron
microscopy.

At 150 mg/kg/day (maximum tolerance level), dogs experienced frequent
loose/liquid feces, vomiting, inappetence, excess salivation,
vasodilation (as noted by pink or reddened ears and/or abdomens); these
findings were observed sporadically or not at all in controls.  Unusual
behavioral (2 males) and motor changes (1 male)were observed on the
first day of dosing only.  Decreased mean body weight/weight gains
during treatment (at termination of treatment, -15%/-89% less than
corresponding control group, Group II; -6%/weight loss of -0.61 kg,
Group IIW), were also observed.  Food consumption was also decreased
during treatment (mean estimated at >10%, Group II and >6%, Group IIW;
see Results for details) but not withdrawal (measurements were not
possible for several weeks due to mixing with unspecified amounts of
supplemental diet).  Mean ALP, ALT, AST and blood urea levels were
elevated due to increases above the provided reference range in 1-2
animals/dose group.  Instead of grey mottling of the retinal tapetal
fundus noted in previous studies, increased brown granularity of the
tapetal fundus was observed in the tapetal fundus of one main study
animal and in one withdrawal group treated animal, although this effect
was too subtle to be demonstrated photographically.  Electroretinography
(ERG) demonstrated that dosing was associated with decreased a- and
b-wave amplitudes of (50%, but waveform was not altered. Other
measurements of scotopic (dark or dim light) vision were not
significantly altered and there was no evidence of neural damage. The
ERG amplitudes in 2/3 treated animals recovered to some degree during
withdrawal.

This eleven-week oral toxicity in the dog is Unacceptable/Nonguideline
(not upgradeable).   This study did not repeat the ophthalmologic
findings observed in the previous 28- and 90-day studies (grey mottling
of the retina), although this did not necessarily render this special
study results invalid and brown granularity in the retina was observed
in 2 treated dogs. However, several factors compromised the integrity of
the study:  unexplained incongruity in the ERG data between control
groups and between the control and treated animals and other technical
problems in the ERG evaluation, double-penning of the animals and the
possibility of illness due to an infectious agent, unspecified technical
problems, errors and uncertainties in food consumption data, a striking
parallelism of mean body weights for control and treated animals and
unexplained decreased gain in the withdrawal control animals.  Despite
these problems, the study does appear to support the conclusion that the
retina is a target tissue for fluazinam and that functional as well as
morphological alterations may result from exposure.

Nonguideline  	7-Day Inhalation Toxicity (Range-Finding)- Rat

                              (Test Material:  Frowncide( WP (51.9%
fluazinam, a.i.)

Executive Summary: In a 7-day inhalation toxicity (range-finding) study
(MRID 42248621), groups of five male and five female young adult CD rats
 were exposed nose-only to Frowncide( WP (51.9% Fluazinam, a.i., Batch
No. 004) for two 3-hour periods per day for 7 days at concentrations of
0, 0.003, 0.011, 0.032, or 0.110 mg/L. The estimated achieved dosages of
Frowncide( WP over the 7 days of treatment were calculated to be 0.72,
2.76, 7.93 and 27.43 mg/kg/day for males and 0.75, 2.97, 8.50 and 29.23
mg/kg/day for females for the concentrations of 0, 0.003, 0.011, 0.032,
and 0.110 mg/L, respectively.  The mass median aerodynamic diameter
(MMAD) was estimated to be 3.22-3.98 (m and the geometric standard
deviation was 2.04-2.69 (m.  Approximately 60-70% of particles had an
aerodynamic diameter < 6.0 (m.  The animals were observed daily.
Hematology, clinical chemistries and urinalyses were performed.  All
animals were necropsied after completion of exposure, but no
histopathology was performed.

No rats died during the study.  No clinical signs of toxicity were noted
from any rat.  The body weight changes of all groups were similar to
that of the control group.  No toxicologically significant effects of
the test material were noted on food consumption, water consumption,
food efficiency, hematology, clinical chemistries, or urinalyses.  At
0.110 mg/L, slightly increased lung weights (males and females),
slightly increased testes weights (males), and slightly increased liver
weights (females) were observed.  At 0.032 mg/L, slightly increased
testes weights (males), and slightly increased liver weights (females)
were also observed.  No macroscopic changes attributed to treatment with
test material were noted at necropsy.  Histopathological examination of
tissues was not performed.

The LOAEL is 0.032 mg/L (7.93 mg/kg/day in males and 8.50 mg/kg/day in
females), based on slightly increased testes weights (males) and
slightly increased liver weights (females).  The NOAEL is 0.011 mg/L
(2.76 mg/kg/day in males and 2.97 mg/kg/day in females).

This inhalation study is classified as Acceptable/Nonguideline.  It does
not satisfy the subdivision F guideline requirements for a repeated dose
inhalation study in the rat because histopathological examination of
tissues was not performed.  The study was conducted as a range-finding
study (for a four-week inhalation study with Frowncide( WP in rats) and
is acceptable for that purpose.

Nonguideline:  A Review of 8 Special Mechanistic Studies Conducted to
Assess                                                  the CNS White
Matter Vacuolation Produced by Impurity-5 Present In
               Fluazinam Technical

Background:  A neurotoxic lesion described as vacuolation of the white
matter of the central nervous system (CNS) was observed initially in
long-term (1-2 year) guideline chronic studies on mice and dogs and
later, upon careful re-examination of the CNS, also in shorter-term
(4-week to 90-day) subchronic studies on mice and dogs.  This lesion was
observed during the (light) microscopic examination of several tissues
of the CNS, occurring most frequently in brain (sections of cerebrum
and/or sections of cerebellum, pons, medulla, and midbrain) and less
frequently in cervical spinal cord.  Although this lesion was also
observed in control animals, the increased incidence and/or severity of
the lesion in test animals was clearly treatment-related and
dose-related.  It is noteworthy that the lesion was not observed in any
guideline studies on rats, even though a careful re-evaluation of the
CNS was performed for all critical studies, including the major chronic
(MRID 42248620) and subchronic (MRID 42248610) studies.  Further
investigation of this lesion in a series of 8 additional special
mechanistic studies, however, demonstrated the lesion could also be
induced in rats at higher dose levels than used in the guideline
studies.  These 8 additional special mechanistic studies were designed
to determine, if possible, the etiology of the vacuolation of the white
matter of the CNS observed in the guideline studies on fluazinam and to
further evaluate several additional characteristics of the lesion.

Summary:  Eight special studies (MRIDs 44807225, 44807226, 44807227,
44807228, 44807229, 44807230, 44807231, and 44807232)  conducted on the
etiology of an impurity in Fluazinam Technical which produced
vacuolation of the white matter in the CNS of mice, rats, and dogs have
revealed several important toxicological features.  In the mouse, rat,
and dog, vacuolation of the white matter in the brains (and optic nerves
of mice) was observed only when high doses of fluazinam technical were
administered.  Fluazinam technical itself was not responsible for the
induction of this aberration.  An analysis of the effects of nine
impurities present in Fluazinam Technical revealed that one impurity,
Impurity-5, is responsible for the appearance of white matter
vacuolation.  With respect to the ability of Impurity-5 to produce white
matter vacuolation, there seems to be a non-linear dose-response with a
clear threshold below which no effect occurs.  No significant
differences between species/sex susceptibility were observed.  White
matter vacuolation in the CNS was reversible, and no progression of this
abnormality was observed with time.  An age-related increased
sensitivity was identified in mice and rats at 10 weeks compared to 3
weeks of age.  Electron microscopy of the white matter (cerebellum) of
mice treated with fluazinam technical indicated that treatment-related
effects were confined to the myelin sheaths.  Large vacuoles were
observed in the intramyelin sheaths due to the accumulation of fluid
between the sheaths.  The nucleus and mitochondria in oligodendroglia
were observed to remain intact, suggesting no damage to these cells. The
myelin sheaths appeared to recover completely during a recovery period
of up to 56 days.  Macroscopic changes in the liver of animals treated
with fluazinam technical and the analytical standard of fluazinam
revealed that liver effects are due to fluazinam itself and not to the
presence of Impurity-5.

Review: A brief synopsis of the results in the special mechanistic
studies is presented below.

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e for inducing the lesion in mice, dogs

             and rats

Fluazinam, per se, was not responsible for the induction of this lesion.
 An analysis of the effects of nine impurities present in fluazinam
technical revealed that one single impurity, Impurity-5, is solely
responsible for the appearance of white matter vacuolation.  Impurity-5
was present in the various lots of fluazinam technical used for toxicity
testing at concentrations ranging from <0.005% to 0.2% w/w.  With
respect to the ability of Impurity-5 to produce white matter
vacuolation, there seems to be a non-linear dose-response with a clear
threshold below which no effect occurs.

Single oral (gavage) doses of 5 mg/kg of Impurity-5 (99.5% purity) given
to fasted mice  caused course fur, staggering gait, sedation for 20
hours and decreased body weight.  All mice were sacrificed in extremis
at 24 hours.  Increased brain weights, edema of the brain and
vacuolation of the brain were observed in the treated animals.

In rats, single oral (gavage) doses of 5000 mg/kg of the analytical
standard of fluazinam (containing <0.0005% Impurity-5)  caused no
vacuolation of the white matter of the brain whereas similar doses of
fluazinam technical did cause vacuolation of the white matter of the
brain.

2.   Determination of dose-response relationships for fluazinam
technical

In the mouse and rat, vacuolation of the white matter in the brains (and
optic nerves of mice) was observed only when high doses of fluazinam
technical were administered.

In mice, single oral (gavage) doses of 3000 mg/kg of fluazinam technical
(95.3% purity) caused decreased locomotor activity, prone position,
paralysis of hind legs, tremors,  staggering gait and moribundity.
Edema of the brain and vacuolation of the white matter of the brain were
observed in the treated animals.  Assuming a concentration of 0.1 %
Impurity-5 in fluazinam technical, this dose (3000 mg/kg) is
approximately equal to a dose of 3 mg/kg of Impurity-5.

In mice, administration of fluazinam technical (containing 0.12%
Impurity-5) in the diet for 4 days at 20,000 ppm resulted in abnormal
behavior on day 4 and trace edema of the brain.   In mice,
administration of fluazinam technical (containing 0.12% Impurity-5) in
the diet for 4 days at 7,000 ppm resulted in trace edema of the brain.
In mice, administration of fluazinam technical (containing 0.12%
Impurity-5) in the diet for 28 days at 7,000 ppm resulted in vacuolation
of the white matter of the brain.  Vacuolation of white matter was
observed in the brains of all treated animals sacrificed at the end of
treatment.

In rats, single oral (gavage) doses of 5000 mg/kg of fluazinam technical
(containing 0.12% to 0.20% Impurity-5) caused decreased locomotor
activity, prone position, paralysis of hind legs, tremors, staggering
gait and moribundity.  Edema of the brain and vacuolation of the white
matter of the brain were observed in the treated animals.  Assuming a
concentration of 0.12 % Impurity-5 in fluazinam technical, this dose
(5000 mg/kg) is approximately equal to a dose of 6 mg/kg of Impurity-5.
In rats, administration of fluazinam technical (containing 0.12%
Impurity-5) in the diet for 14 days at 30,000 ppm (1742 mg/kg/day) and
at 10,000 ppm (714 mg/kg/day) resulted in edema of the brain and minimal
to moderate vacuolation of the white matter of the brain at 30,000 ppm,
and trace vacuolation of the white matter of the brain at 10,000 ppm.

3.   Reversibility of the CNS lesion

White matter vacuolation in the CNS was  reversible, and no progression
of this abnormality was observed with time.  In the 14-day dietary study
in rats described above, recovery from the CNS lesion was also studied.
Some rats were allowed to recover for an additional 25 days (no
fluazinam in the diet) and then examined.  For 30,000 ppm animals, only
trace vacuolation of the white matter of the brain was observed.  For
10,000 ppm animals, no vacuolation of the white matter of the brain was
observed.

In the mouse study described above in which fluazinam technical was
administered in the diet for 4 days at 20,000 ppm, or for 4 days at
7,000 ppm, or for 28 days at 7,000 ppm, vacuolation of white matter was
observed in the brains of all treated animals sacrificed at the end of
treatment.  This abnormality was not observed after a 24-day recovery
period among animals treated at 7,000 ppm for 4 days or after 56 days
among those treated at 7.000 ppm for 28 days or at 20,000 ppm for 4
days.

4.  Differences in species and sex susceptibility

In a series of studies, no significant differences in susceptibility or
in incidence or severity of vacuolation of the white matter of the CNS
were observed between species (mice, dogs, or rats).  Similarly, no
significant differences were attributed to sex.

5.   Differences in age-related susceptibility

An age-related increased sensitivity was identified in mice and rats at
10 weeks compared to 3 weeks of age.

Impurity-5 was administered to groups of male mice aged 3, 10, or 24
weeks by a single oral gavage at 2.5 mg/kg.  The severity of white
matter vacuolation increased with age until about ten weeks, then
plateaued at 24 weeks as observed under the limitations of this study.

The difference in age sensitivity was comparable between rats and mice
as displayed in another oral gavage study using 3 and 10 week-old mice
and rats dosed with 0 or 0.5 mg/kg of Impurity-5.  Microscopic
observation of the brains of treated animals revealed white matter
vacuolation with slightly different severity between the respective age
groups.  The severity of these lesions was similar for both species of
the same age, but greater in 10 week old animals as compared to 3 week
old animals.

	6.  Electron Microscopy of the CNS lesion

Electron microscopy of the white matter (cerebellum) of mice treated
with fluazinam technical indicated that treatment-related effects were
confined to the myelin sheaths.  Large vacuoles were observed in the
intramyelin sheaths due to the accumulation of fluid between the
sheaths.  The nucleus and mitochondria in oligodendroglia were observed
to remain intact, suggesting no damage to these cells. The myelin
sheaths appeared to recover completely during a recovery period of up to
56 days.

Comments on Threshold Dose for Vacuolation of the White Matter of the
CNS:  There appears to be a non-linear dose-response with a clear
threshold below which no effect occurs.  When the guideline data were
analyzed and presented graphically [see Figure 1 (page 100) in the
Overview Document (MRID 44807207) prepared by the applicant], it was
apparent that no white matter vacuolation occurred when the dose of
Impurity-5 was below about 0.1 mg/kg/day.  The lowest effect level for
white matter vacuolation was observed in the dog chronic study at 0.1
mg/kg/day of Impurity-5 (equivalent in that study to 50 mg/kg/day of
fluazinam technical containing 0.2% of Impurity-5).

Based on a consideration of all the available data and information
relating to this treatment-related neurotoxic lesion, the HIARC
concluded that a LOAEL of 0.1 mg/kg/day and a NOAEL of 0.02 mg/kg/day
for CNS effects could be established for Impurity-5.

NOAEL (for CNS effects)  =  0.02 mg/kg/day of Impurity-5

At the current maximum concentration of Impurity-5 in technical grade
fluazinam of 0.1% w/w [see memorandum from Indira Gairola, Technical
Review Branch, RD (7505C) to Cynthia Giles-Parker, Fungicide Branch, RD
(7505C), dated May 18, 2001, DP Barcode D272455], this is equivalent to:

     NOAEL (for CNS effects) = 20.0 mg/kg/day of technical grade
fluazinam

Calculation:   0.02 mg/kg/day   =   x  mg/kg/day

                   	0.1%		      	100%		          x  = 20.0

This NOAEL (for CNS effects) of 20.0 mg/kg/day for technical grade
fluazinam is to be compared to:

NOAEL (for chronic effects for chronic RfD)  =  1.1 mg/kg/day of
technical grade fluazinam

The chronic RfD of 0.011 mg/kg/day for (all populations(, including
infants and children, is therefore protective of the CNS effects caused
by Impurity-5 present in technical grade fluazinam at levels up to 0.1%
w/w.

A.4	Additional Toxicology Study

Fulcher, S. (2005) Technical Fluazinam: Developmental Neurotoxicity
Study in the Rat by Oral (Gavage) Administration. Project Number:
ISK/272/042019. Unpublished study prepared by Huntingdon Life Sciences,
Ltd. 1471 p.

Appendix B:  Metabolism Assessment

B.1. Metabolism Guidance and Considerations

 TC \l1 "Appendix C:  Tolerance Reassessment Summary and Table

Chemical Name (other names in parenthesis)	Matrix

Structure

Parent

Fluazinam	Primary crop, Ruminant, Rat, and Drinking water

*AMGT	Primary crop

(AMGT data only available for blueberries)	Not available

AMPA 	Ruminant, Rat (+ AMPA hydrolysis products)	Not available

DAPA	Ruminant, Rat (+ DAPA hydrolysis,conjugation products), Drinking
water

CAPA

	Drinking water

DCPA	Drinking water

Appendix C:  Tolerance Reassessment Summary and Table

Table C.1	Tolerance Summary for Fluazinam.

Commodity	Proposed Tolerance (ppm)	Recommended Tolerance (ppm)	Comments

[Correct Commodity Definition]

Ginseng	3.00	4.5

	Bean, dry	0.01	0.02	[Pea and bean, dried shelled, except soybean,
subgroup 6-C (except peas)]

Succulent-shelled legume vegetables subgroup 6B, except pea	0.02	0.04
[Pea and bean, succulent shelled, subgroup 6-B (except peas)]

Edible-podded legume vegetables subgroup 6A, except peas	0.15	0.10
[Vegetable, legume, edible-podded, subgroup 6-A (except peas)]

Leafy Brassica greens subgroup	0.02	0.01	Crop group tolerance is
appropriate. [Vegetable, Brassica leafy, group 5]

Head and stem Brassica subgroup	0.01

Turnip, leaves	0.02	0.01	[Turnip, tops]

Bushberry subgroup 13B	4.5	7.0	[Bushberry subgroup 13-B]

Aronia berry	4.5	7.0

	Blueberry, lowbush	4.5	Not needed	Low bush blueberry is a member of the
bushberries  subgroup 13-B

Buffalo currant	4.5	7.0

	Chilean guava	4.5	7.0

	European barberry	4.5	7.0

	Highbush cranberry	4.5	7.0

	Honeysuckle	4.5	7.0

	Jostaberry	4.5	7.0

	Juneberry	4.5	7.0

	Lingonberry	4.5	7.0

	Native currant	4.5	7.0

	Salal	4.5	7.0

	Sea Buckthorn	4.5	7.0

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