Interregional Research Project No. 4 (IR-4)

PP# 3E8211

EPA has received a pesticide petition (3E8211) from IR-4, Rutgers, The State 

University of New Jersey, 500 College Road East, Suite 201W, Princeton, NJ 08540

proposing, pursuant to section 408(d) of the Federal Food, Drug, and Cosmetic Act (FFDCA), 21 

U.S.C. 346a(d), to amend 40 CFR part 180, by establishing a tolerance for residues of

metrafenone, (3-bromo-6-methoxy-2-methylphenyl)(2,3,4-trimethoxy-6-

methylphenyl)methanone] in or on the raw agricultural commodity apricot at 0.7 parts per 

million (ppm); cherry, subgroup 12-12A at 2.0 ppm; fruit, small, vine climbing, except fuzzy 

kiwifruit, subgroup 13-07F at 4.5 ppm; hop, dried cones at 70.0 ppm; peach, subgroup 12-

12B at 0.7 ppm; and vegetable, cucurbit, group 9 at 0.5 ppm. IR-4 also proposed, upon the 

approval of the aforementioned tolerances, to remove established tolerance for grapes. EPA has 

determined that the petition contains data or information regarding the elements set forth in 

section 408 (d)(2) of FDDCA; however, EPA has not fully evaluated the sufficiency of the 

submitted data at this time or whether the data supports granting of the petition. Additional data 

may be needed before EPA rules on the petition.

A. Residue Chemistry

1. Plant metabolism. In plant metabolism studies conducted in grapevines, cucumber, 

and wheat with metrafenone, the parent compound was the principal residue in almost all 

matrices. Metrafenone is metabolized through methyl and ring oxidative bio-transformations, 

followed by enzymatic glucoside conjugation reactions to yield the conjugates from their 

corresponding aglycons. All major metabolites, which were present at very low levels, are 

considered non-relevant as all were detected in the rat metabolism study as aglycons or 

conjugates, and can therefore be considered as not toxicologically significant. Thus, only 

parent metrafenone is considered relevant to define the residue in plants.

2. Analytical method. The residues of parent metrafenone in/on Cherry, Hops, Peach, 

Cucumber, Cantaloupe, and Squash RAC samples were quantitated using an LC/MS/MS 

multi-residue QuEChERS method (BASF Study No. 398340). The method was successfully 

validated on all the relevant commodities in conjunction with these studies prior to analysis 

of the field samples. Acceptable concurrent method recovery data for all the RAC samples 

were also obtained for metrafenone. The validated limit of quantitation (LOQ) for residues 

of metrafenone in/on all the commodities was 0.01 ppm. The method limit of detection 

(LOD) was 0.001 ppm, or approximately 10% of the LOQ. An independent laboratory 

validation demonstrated good performance of the QuEChERS method.

3. Magnitude of residues. IR-4 has submitted field trial data for metrafenone (BAS 

560 F) on cherry, hops, peach, cucumber, cantaloupe, and squash. The number and 

locations of field trials are in accordance with OPPTS Guideline 860.1500. Field trials were 

carried out using the maximum label rates, the maximum number of applications, and the 

minimum pre-harvest interval (PHI) for all the crops.

B. Toxicological Profile
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1. Acute toxicity. In an acute toxicity study with rats, metrafenone was practically 

non-toxic (EPA Tox. Category IV). In an acute neurotoxicity study with rats, no signs of 

neurotoxicity were detected up to 2,000 mg/kg b.w., the highest dose tested.

2. Genotoxicty. Metrafenone was evaluated for its potential genotoxicity in vitro using 

bacterial and mammalian cell mutagenicity tests and a chromosome damage 

(clastogenicity) test. The results of these studies demonstrated the absence of a genotoxic 

effect. In vivo, the test substance was assessed for the induction of micronuclei in mice. The 

result of this study showed that metrafenone has no chromosome-damaging potential.

It is therefore concluded, that metrafenone has no mutagenic or genotoxic properties both 

in vitro and in vivo.

3. Reproductive and developmental toxicity. The reproductive and developmental 

toxicity of metrafenone was investigated in a 2-generation reproduction study in rats as well 

as in prenatal toxicity studies in rats and rabbits.

In the definitive 2-generation reproduction study in rats, the NOAEL for fertility and 

reproduction was 10,000 ppm (the highest concentration tested), equivalent to 

approximately 811 mg/kg b.w./day based on food consumption data during the premating 

periods. The parental NOAEL of metrafenone is 500 ppm (approximately 39 mg/kg 

b.w./day based on food consumption data). The 1,000 ppm treatment showed statistically 

significant decreases in body weight and weight gain in F1 parental males and increased 

absolute and relative liver weights in females. The NOAEL for pup (offspring) toxicity was 

1,000 ppm (approximately 79 mg/kg b.w./day based on food consumption data); the 10,000 

ppm treatment showed a statistically significant decrease in pup body weight (31%) during 

lactation, an increase in relative liver weights at weaning, and a delay in vaginal opening 

(F1 only).

In the prenatal toxicity study in rats, there was no treatment-related maternal or 

developmental toxicity observed, even at the highest dose tested (1,000 mg/kg b.w./day). On 

the basis of these data, the maternal NOAEL of metrafenone is 1,000 mg/kg b.w./day 

(highest dose tested). The 1,000 mg/kg b.w./day dose showed a statistically significantly 

increased ratio of liver weight to terminal body weights. However, no correlating 

microscopic changes were diagnosed, indicating that the increased relative liver weight was 

not considered toxicologically significant. The developmental NOAEL for metrafenone is 

1,000 mg/kg b.w./day (no effects were observed at the highest dose tested). Based on the 

above results, metrafenone is not selectively toxic to the fetus. In addition, metrafenone was 

not teratogenic in rats.

In the rabbit prenatal toxicity study, developmental toxicity was observed in the presence 

of maternal toxicity. Specifically, the maternal NOAEL is 50 mg/kg b.w./day based on the 

following: reduced body weight gains and reduced feed consumption (absolute and 

relative) at both 350 and 700 mg/kg b.w./day. In addition, at these two dose levels, a doserelated, statistically significant increase in absolute and relative (to body weight) liver 

weights occurred, as compared to controls. Also, increased incidences and/or severities of 
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periportal hepatocellular hypertrophy and diffuse and/or periportal hepatocellular 

vacuolation were observed at 350 and 700 mg/kg b.w./day. In this prenatal toxicity study 

in rabbits, the developmental NOAEL of metrafenone is 350 mg/kg b.w./day, as based on 

the slight but statistically significant reduction in fetal body weights at 700 mg/kg b.w./day. 

Therefore, because developmental toxicity was only observed at dose levels that were 

maternally toxic, metrafenone is not selectively toxic to the fetus. In addition, metrafenone

was not teratogenic in rabbits.

Overall, for the reproductive and developmental toxicity studies, the lowest NOAEL for 

maternal/parental toxicity was 39 mg/kg b.w./day (two-generation reproduction study) and 

for pup toxicity was 79 mg/kg b.w./day (two-generation reproduction study).

4. Subchronic toxicity. The subchronic toxicity of metrafenone was investigated in a 

dietary 28-day study in rats, a 28-day oral toxicity study in dogs, and 3-month toxicity 

studies in rats, mice, and dogs. In addition, a 28-day neurotoxicity study as well as a 28-

day immunotoxicity study, were conducted with rats. 

The signs of toxicity observed in the three species tested were overall similar and consisted 

of reduced body weight gain at the highest tested dose levels. The observed effects on 

clinical chemistry in rats and mice can be linked to the microscopic effects observed in the 

liver. The effects observed typically included increased cholesterol, total protein, and total 

bilirubin (mice only). Organ weights and/or pathology indicated the liver to be the only 

consistent target organ in all three species tested. The changes included, periportal 

cytoplasmic vacuolation in rats, centrilobular hepatocellular hypertrophy in mice, and 

increased liver weights in rats, mice, and dogs.

In the 28-day neurotoxicity study with rats, there was no evidence of neurotoxicity in all 

treatment groups, with a No-Observed-Adverse-Effect Level (NOAEL) for neurotoxicity of 

15,000 in the diet (equivalent to 1,371 mg/kg b.w./day for both sexes, as based on food 

consumption data, which exceeds a limit dose of 1,000 mg/kg b.w./day). These results, 

coupled with the lack of neurotoxic effects observed in acute, sub-chronic and chronic 

studies with other species, indicate that metrafenone is not potentially neurotoxic. 

In the 28-day immunotoxicity study with female rats, there was no evidence of 

immunotoxicity in all treatment groups, with a No-Observed-Adverse-Effect Level

(NOAEL) for immunotoxicity of 12,000 in the diet (equivalent to 1,086 mg/kg b.w./day, as

based on food consumption data, which exceeds a limit dose of 1,000 mg/kg b.w./day). 

These results, coupled with the lack of immunotoxic effects observed in acute, sub-chronic 

and chronic studies with other species, indicate that metrafenone is not potentially 

immunotoxic.

5. Chronic toxicity. The chronic toxicity of metrafenone was evaluated in a 1-year 

oral administration study in dogs, a 24-month feeding study with rats and an 18-month 

feeding study with mice.

Administration of metrafenone to dogs for one year resulted in mild increases in mean 

alkaline phosphatase in males after 9 and 12 months of dosing at 500 mg/kg b.w./day 
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(highest dose tested). Mean absolute and relative liver weights were both statistically 

significantly increased in females at 500 mg/kg b.w./day and relative liver weights were 

statistically significantly increased in males at 500 mg/kg b.w./day. However, there were no 

associated macroscopic or microscopic findings in the livers of these animals. Therefore, 

the slight but statistically significant differences in liver weights and alkaline phosphatase 

(in males) were not considered to be of toxicological significance. All other parameters 

evaluated including mortality, clinical observations, body weights and food consumption 

data, ophthalmology, hematology, coagulation and urinalysis data, and macroscopic and 

microscopic examinations revealed no adverse effects due to administration of 

metrafenone. Therefore, the NOAEL for oral administration of metrafenone to pure-bred 

Beagle Dogs was 500 mg/kg b.w./day, the highest dose tested.

Treatment of Sprague-Dawley rats with metrafenone for 24 months resulted in markedly 

reduced body weight gains in females at 5,000 (26%) and 20,000/10,000 ppm (42%) and a 

lesser reduction in body weight gain for males at 20,000 ppm (14%). Microscopic effects in 

the liver and kidney were associated with clinical chemistry changes in males and females 

at doses of 5,000 ppm and greater. Slight anemia was observed in females at 5,000 ppm 

and higher, which was reversed at 24 months. An increase in the incidence of 

hepatocellular necrosis after 12 months and benign hepatocellular adenomas was observed 

in females at 5,000 ppm and greater which are dietary concentrations that exceeded the 

MTD. Therefore, the NOAEL for systemic toxicity and for oncogenic effects following 

dietary administration of metrafenone to Sprague-Dawley rats for 24 months, was 500 ppm 

(25 mg/kg b.w./day as based on food consumption data).

Treatment of albino mice with metrafenone for 18 months resulted in statistically 

significant increases in the following: liver weights (absolute and relative, in females), 

hepatocellular hypertrophy in males, renal chronic nephropathy in males and 

extramedullary hematopoiesis in the spleen of females at 1,000 ppm, the next highest 

concentration tested. Therefore, the NOAEL for systemic toxicity following dietary 

administration of metrafenone to albino mice for 18 months, was 250 ppm (46 mg/kg 

b.w./day as based on food consumption data). 

A statistically significant (P<0.01) increase in the incidence of hepatocellular neoplasms 

(adenomas and carcinomas) was observed in the livers of male mice receiving 7,000 ppm 

(19/66, 28.8%), as compared to controls (6/65, 9.2%). There was also a marginal increase 

in the incidence of hepatocellular neoplasms in females receiving 7,000 ppm (4/65), as 

compared to the control mice (2/65). The incidence of 14/66 (21.2%) hepatocellular 

adenoma for males at 7,000 ppm is slightly above the maximum range of historical control 

data at the testing laboratory (14.8%) and at Charles River Laboratories (19.2%). The 

incidence of 5/66 (7.6%) hepatocellular carcinoma in males at 7,000 ppm is within the 

maximum range of historical control data at both the testing facility (9.2%) and Charles 

River Laboratories (11.5%). Although the number of hepatocellular neoplasms in males 

receiving 1,000 ppm was also slightly increased (9/65, 13.8%) compared to the untreated 

controls (6/65, 9.2%), this slight increase was not statistically significant (p<0.05) and was 

within the range of the historical controls (9.2-18.5%) at the testing laboratory. The slight 

increase in hepatocellular neoplasms in male mice at 1,000 ppm may have resulted from 
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increased survival observed at termination in male mice at 1,000 ppm as compared to 

controls (55 versus 45 in the surviving male controls). The incidences of hepatocellular 

neoplasms in terminally sacrificed male mice were 5/45 or 11%, 3/53 or 6%, 8/55 or 15% 

and 18/54 or 33%, at respective treatment levels of 0, 250, 1,000 and 7,000 ppm.

For females, whereas the incidence of hepatocellular adenoma was the same (2/65 or 3.1%) 

at 7,000 ppm and for controls, the incidence of hepatocellular carcinoma was slightly 

increased at 7,000 ppm (2/65 or 3.1%) versus control (0/65 or 0%). Since this incidence of 

hepatocellular carcinoma (3.1%) for females at 7,000 ppm is slightly above the maximum 

range of historical control data at both the testing facility (1.5%) and Charles River 

Laboratories (2.0%), the incidence represents an equivocal finding. However, because this 

increase was very small, was not statistically significant, and did not demonstrate decreased 

latency compared to the controls, this finding was considered to represent only equivocal 

toxicologic significance.

Therefore, based on the weight-of-the-evidence analysis, the NOAEL for oncogenicity is 

1,000 ppm (approximately 190 mg/kg of b.w./day, calculated from food consumption data 

and nominal concentrations) based on the increased incidence of hepatocellular neoplasms 

observed in male mice at 7,000 ppm, as compared to controls.

In summary, long-term exposure studies with metrafenone in dogs, rats and mice resulted 

in increased liver weights with corresponding changes in clinico-chemical parameters. 

Microscopic pathological changes were identified in the liver, kidney, and spleen for rats 

and/or mice. The main target organ was identified as the liver in all species. Additionally, 

increased incidences of primary hepatocellular neoplasms were observed at the highest 

doses in rats and mice.

6. Animal metabolism. Metrafenone was rapidly absorbed and efficiently excreted 

following oral administration in the rat. The amount of absorption ranged from 

approximately 89 to 92% of the administered dose of 10 mg/kg b.w. Most of the 

administered dose was eliminated via bile within 72 hours (85.2 to 90.1%) and feces within 

7 days (84.1 to 98.8%), with a small amount (0.69 to 6.6%) in urine. metrafenone-related 

residues showed no potential for accumulation as <1.2% of the applied dose was retained 

over seven days, with highest levels of residues found in the liver, GI tract, plasma, blood, 

kidney, and fat. The tissue retention of the orally administered dose was less than 2% at 

48-72 hours and less than 1.2% at 168 hours. metrafenone was extensively metabolized in 

the rat to multiple metabolites formed via demethylation of the aromatic methoxy-group(s) 

followed by mono-O-glycosidation, hydroxylation of the bromophenyl ring, and 

hydroxylation of the methyl substituent to hydroxymethyl followed by O-glycosidation or 

further oxidation to aldehyde or lactone.

7. Metabolite toxicology. In plant metabolism studies conducted in grapevines with 

metrafenone, the parent compound was the principal residue in grapes. metrafenone is 

metabolized through methyl and ring oxidative biotransformations, followed by enzymatic 

glucoside conjugation reactions to yield the conjugates from their corresponding aglycons. 

All major metabolites, which were present at very low levels, are considered non-relevant 
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as all were detected in the rat metabolism study as aglycons or conjugates, and can 

therefore be considered as not toxicologically significant. Thus, only parent metrafenone is 

considered relevant to define the residue in plants.

The results from a hydrolysis study designed to simulate different industrial and household 

processes (pasteurization, brewing, boiling and sterilization) showed that metrafenone is 

stable. Therefore, metrafenone as unchanged parent is proposed as the relevant residue in 

grapes and industrial or household preparations of grapes.

Since metrafenone is not registered for use in the United States, the toxicity of metabolites 

formed in matrices other than the raw or processed treated commodity (e.g., water, soil) is 

not relevant.

8. Endocrine disruption. Data from the reproduction / developmental toxicity and 

short- and long-term repeated dose toxicity studies with metrafenone in the rat, rabbit, 

mouse, or dog, do not suggest any endocrine disruption activity. This information is based 

on the absence of any treatment-related effects from the histopathological examination of 

endocrine organs as well as the low level of concern for possible effects on fertility, 

reproductive performance, or any other aspect of reproductive function, or on growth and 

development of the offspring.

C. Aggregate Exposure

1. Dietary exposure. Assessments were conducted to evaluate the potential risk due to 

chronic dietary exposure of the U.S. population to residues of metrafenone. The U.S. EPA 

has not established an acute reference dose for either the general population or females 

ages 13 – 49 years because no study demonstrated evidence of toxicity from a single dose 

exposure for these populations. Therefore, an acute exposure risk evaluation is not 

required. The chronic assessment was conducted using all currently established tolerances 

for metrafenone listed in 40CFR180.624, the pending tolerance for the Pome Fruit Crop 

Group 11-10 of 1.5 ppm, and the proposed new tolerances for crop group 8 – fruiting 

vegetables at 1.0 ppm, crop group 9 – cucurbit vegetables at 0.5 ppm, crop subgroup 12-

12A – cherry subgroup at 2.0 ppm, crop subgroup 12-12B – peach subgroup at 0.7 ppm, 

hops at 70 ppm, and apricots at 0.7 ppm, and crop subgroup 13-07F – small fruit vine 

climbing except fuzzy kiwifruit at 4.5 ppm.

i. Food. The dietary assessment analysis followed an initial tier approach using 

100% crop treated and current or proposed tolerance values for all commodities. Dietary 

exposure assessments were conducted using Dietary Exposure Evaluation Module (DEEMFCID Ver 3.18). 

Acute Dietary Exposure Assessment

An acute assessment was not conducted because there are no relevant toxicological 

endpoints for acute dietary exposure and a risk assessment is not required. 

Chronic Dietary Exposure Assessment
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Results of the chronic exposure estimates were compared against the chronic Population 

Adjusted Dose (cPAD) of 0.25 mg/kg b.w./day that was based on the NOAEL from the rat 

chronic toxicity study, a safety factor of 100, and no additional FQPA safety factor is 

required. 

Results of the chronic dietary exposure assessments are listed in Table 1. The highest 

chronic dietary exposure of 11.8% of the cPAD was for children 1-2 years old. 

Table 1. Chronic Dietary Food Exposure Assessment for Metrafenone.

Population

Exposure 

Estimate %cPAD

Subgroups

(mg/kg 

b.w./day)

US Population 0.005349 2.1

All infants (< 1 year) 0.021824 8.7

Children 1-2 0.029566 11.8

Children 3-5 0.017028 6.8

Children 6-12 0.007348 2.9

Youth 13-19 0.002893 1.2

Adults 20-49 0.003558 1.4

Adults 50+ yrs 0.003375 1.4

Females 13 - 49 yrs 0.003025 1.2

The results in Table 1 demonstrate there are no unreasonable risk concerns for any 

subpopulation based on established and new uses, and that the results clearly meet the 

FQPA standard of no unreasonable adverse effect.

ii. Drinking water. The drinking water exposure values used in this assessment are 

from the most recent U.S. EPA risk assessment for Grapes (U.S. EPA, March 1, 2010 

“Metrafenone: Human Health Risk Assessment for Foliar Use on Grape”). The water 

values calculated by EPA are the higher values than the pending and proposed new uses. 

The chronic surface water concentration used in the human exposure assessment was 22.82

ug/L. 

Drinking water chronic exposure was assessed based on a metrafenone water concentration 

of 22.82 ug/L using drinking water consumption, and body weights reported in CSFII, 

using DEEM-FCID Ver 3.18 software. The chronic estimated water exposure values are 

summarized in Table 2. 

Table 2. Summary of chronic dietary exposure assessment for drinking water considering 

direct and indirect sources. 

Population

Subgroups

Exposure Estimate

(mg/kg b.w./day) %cPAD
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US Population 0.000478 0.2

All infants (< 1 year) 0.001232 0.5

Children 1-2 0.000689 0.3

Children 3-5 0.000581 0.2

Children 6-12 0.000419 0.2

Youth 13-19 0.000348 0.1

Adults 20-49 0.000477 0.2

Adults 50+ yrs 0.000471 0.2

Females 13 - 49 yrs 0.000475 0.2

%cPAD = percent of chronic population adjusted dose 

Exposure based on the maximum estimated water concentration of 22.82 ug/L

Acute Aggregate Exposure (Food + Water)

An acute assessment was not needed because there are no relevant toxicological endpoints 

for acute dietary exposure and an aggregate risk assessment is not required. 

Short- and Intermediate Aggregate Exposure (Food + Water + Residential)

Metrafenone is not registered for any use which will result in residential exposure. 

Therefore, a short- or intermediate-term exposure and aggregate risk evaluation is not 

required. 

Chronic Aggregate Exposure (Food + Water)

The aggregate chronic risk includes residues of metrafenone from food and water (Table 

3). The results demonstrate there are no unreasonable risk concerns for any subpopulation 

based on established and new uses, and that the results clearly meet the FQPA standard of 

no unreasonable adverse effect.

Table 3. Estimated chronic aggregate exposure and risk of Metrafenone

Population Subgroup cPAD 

(mg/kg/day)

Total 

Exposure, 

Food and 

Water 

(mg/kg/day)

% 

cPAD

US Population 0.25 0.005827 2.3

All infants (< 1 year) 0.25 0.023056 9.2

Children 1-2 0.25 0.030255 12.1

Children 3-5 0.25 0.017609 7.0

Children 6-12 0.25 0.007767 3.1

Youth 13-19 0.25 0.003241 1.3

Adults 20-49 0.25 0.004035 1.6

Adults 50+ yrs 0.25 0.003846 1.5

Females 13 - 49 yrs 0.25 0.003500 1.4

]
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2. Non-dietary exposure. Metrafenone is not registered for any use which will result 

in non-dietary residential exposure.

D. Cumulative Effects

At this time, there is no available information to indicate that metrafenone or its 

metabolites have a common mechanism of toxicity with other substances. Therefore, there 

is no reason to include this pesticide or its metabolites in a cumulative risk assessment. For 

the purposes of this tolerance action EPA has not assumed that metrafenone and its 

metabolites have a common mechanism of toxicity with other substances.

E. Safety Determination

1. U.S. population. Results of the analysis demonstrate no unreasonable risk 

concerns for the U.S. population based on established and new proposed uses, and that the 

results clearly meet the FQPA standard of no unreasonable adverse effect.

2. Infants and children. Results of the analysis demonstrate no unreasonable risk 

concerns for any U.S. population including infants and children based on established and 

new proposed uses, and that the results clearly meet the FQPA standard of no 

unreasonable adverse effect.

F. International Tolerances

No maximum residue levels (MRLs) have been established for metrafenone by the 

Codex Alimentarius Commission (CODEX) or in Mexico.