EPA REGISTRATION DIVISION COMPANY NOTICE OF FILING FOR PESTICIDE 

PETITIONS PUBLISHED IN THE FEDERAL REGISTER 

Interregional Research Project Number 4 (IR-4)

Pesticide Petition (PP) #4E8264

EPA has received a pesticide petition, PP# 4E8264, from Interregional Research 

Project Number 4 (IR-4), IR-4 Project Headquarters, Rutgers, The State University of 

New Jersey, 500 College Road East, Suite 201 W, Princeton, NJ 08450 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

prohexadione calcium in or on the raw agricultural commodities strawberry at 0.3 parts 

per million (ppm) and watercress at 2.0 ppm. EPA has determined that the petition 

contains data or information regarding the elements set forth in section 408 (d)(2) of 

FDDCA; however, EPA has not fully evaluated the sufficiency of the submitted data at 

this time or whether the data supports granting of the petition. Additional data may be 

needed before EPA rules on the petition.

A. Residue Chemistry

1. Plant metabolism. The metabolism of prohexadione calcium in plants peanuts 

and apples) is understood, the residue of concern is the parent compound, prohexadione 

calcium.

2. Analytical method. [The method of analysis for strawberry included extraction 

and LC/MS/MS quantitation. The limit of quantitation (LOQ) is 0.01 ppm. The method 

of analysis for watercress included extraction and GC-MS quantitation. The limit of 

quantitation (LOQ) is 0.1 ppm.]

3. Magnitude of residues. [A total of nine residue trials were conducted on 

strawberries grown in British Columbia, California, Florida, Michigan, New Jersey, Nova 

Scotia, Ontario, and Quebec in 2010. Strawberries were treated three times at a rate of 

0.032 – 0.036 lbs prohexadione calcium per acre per application and 8 – 15 days between 

applications. Whole strawberries were collected 16 to 25 days after the last application 

for analysis and analyzed by LC/MS/MS. Residues ranged from 0.03 to 0.16 ppm. A 

total of four residue trials were conducted on watercress grown in Florida and Maryland 

in 2009. Watercress was treated twice at a rate of 0.135 – 0.143 lbs prohexadione 

calcium per acre per application and 6 – 7 days between applications per crop. 

Watercress leaves and stems were collected 3 days after the second application for 

analysis and analyzed by GC-MS. Residues ranged from 0.167 to 1.12 ppm. ]

B. Toxicological Profile
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1. Acute toxicity. [EPA has evaluated the toxicity database for prohexadione 

calcium and published assessments in the Federal Register on May 3, 2000, FR Vol. 65, 

No. 86 pages 25655-25660 and on June 1, 2001, FR Vol. 66, No. 106 pages 29705

through 29712. The Agency determined that prohexadione calcium is of low acute 

toxicity. All tests showed the compound to toxicity category III or IV]

2. Genotoxicty. [Genotoxicity. Ames Test (1 Study; point mutation): Negative; in

vitro V79 Cells CH/HGPRT Locus Mammalian Cell Mutation Assay (1 Study; point 

mutation): Negative; in vitro CHO Cytogenetic Assay (1 Study; Chromosome Damage): 

Negative; in vivo Mouse Micronucleus (1 Study; Chromosome Damage): Negative; in 

vivo Rat Bone Marrow Cytogenetic Assay (1 Study; Chromosomal Damage): Negative; 

Rec Assay (1 Study; DNA damage and repair): Negative; in vitro Rat Hepatocyte (1 

Study; DNA damage and repair): Negative. 

Prohexadione calcium has been tested in a total of 7 genetic toxicology assays 

consisting of in vitro and in vivo studies. Based on the results described above, it can be 

stated in summary that prohexadione calcium did not show any mutagenic activity when 

tested under the conditions of the studies mentioned above. Therefore,

prohexadione calcium does not pose a mutagenic hazard to humans.]

3. Reproductive and developmental toxicity. [The reproductive and developmental 

toxicity of prohexadione calcium was investigated in a 2-generation rat reproduction 

study as well as in rat and rabbit teratology studies. The 2-generation rat reproduction 

study was conducted at dose levels of 0, 500, 5,000 and 50,000 ppm. There were no 

adverse effects on reproduction parameters seen even at the dose level of 50,000 ppm 

(5164 mg/kg bw for males and 5,600 mg/kg bw for females). The No Observed Adverse 

Effect Level (NOAEL) for parental systemic toxicity was 500 ppm (48 mg/kg bw for 

males and 51 mg/kg bw for females) and the NOAEL for developmental toxicity was 

5,000 ppm (270 mg/kg bw for females). Stomach lesions were observed at 5,000 ppm.

Two mid-dose males and two males and one female of the high-dose from the F0 died. 

Body weight and food consumption changes and slight transient reduction in offspring 

growth were noted at 50,000 ppm. No impairment of reproductive function was observed 

at any of the dose levels tested. 

The reproductive and developmental studies are summarized below. A developmental study was conducted via oral gavage in rats at dose levels of 0, 100, 300, and the 

1,000 highest dose tested (HDT) mg/kg bw. The No Observed Adverse Effect Level 

(NOAEL) for developmental and maternal toxicity was 1,000 mg/kg bw, HDT. This was 

based on the fact that there were no signs of maternal toxicity, fetotoxicity or teratogenic 

effects. A developmental study was conducted via oral gavage in rabbits at dose levels of 

0, 40, 200, and 750 (HDT) mg/kg bw. The NOAEL for development toxicity was 40 

mg/kg bw and the NOAEL for maternal toxicity was 40 mg/kg bw based on the 

following findings. Toxicity in the form of maternal mortality with values 16/20 and 4/20 

was excessive in the mid- and high-dose group, respectively. Fetal deaths also occurred. 

Dose levels believed to exceed MTD; NOAELs for maternal and developmental effects 

are not considered reliable and useful for risk characterization. No teratogenic effects 

were noted in this study.

i. Teratogenicity. Prohexadione calcium had no teratogenic potential at dose levels 

as high as 1,000 mg/kg bw in the rat and 350 mg/kg bw in the rabbit. The NOAEL for 

maternal toxicity in the teratogenicity studies is 100 mg/kg bw (rabbit) and 1,000 mg/kg 
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bw (rat), and the NOAEL for fetotoxicity in the teratogenicity studies is 350 mg/kg bw 

(rabbit) and 1000 mg/kg bw (rat). An additional teratology study in the same strain of 

rabbits was conducted at dose levels of 0, 30, 75, and 150 mg/kg bw. The NOAEL for 

development toxicity was 150 mg/kg bw and the NOAEL for maternal toxicity was 30 

mg/kg bw based on the following findings. One low-, two mid-, and three high-dose 

animals died prior to day 29, however, at the high dose group one died of gavage error 

and another pneumonia, and the reason for the other deaths could not be determined. No 

teratogenic or fetoxtoxic effects were noted in this study.

ii. Oral teratology study. An oral range-finding gavage teratology study in the same 

strain of rabbits (5 animals/dose level) was conducted in another independent laboratory. 

The dose levels selected were 0, 20, 100, 250, 500, and 1,000 mg/kg bw. This range 

finding study was conducted with a limited number of animals and a limited scope of 

examination. Based on these results the dose levels selected for the main study at this 

independent laboratory were 0, 30, 100, and 350 mg/kg bw. The NOAEL for 

development toxicity was 350 mg/kg bw and the NOAEL for maternal toxicity was 100 

mg/kg bw based on the following findings. At the 350 mg/kg bw dose group transient 

body weight decreases and two abortions were observed. No teratogenic or fetotoxic 

effects were noted in this study.

iii. Conclusions from teratology studies. More than one definitive rabbit 

teratology study was conducted because issues associated with exceeding the maximum 

tolerated dose (MTD) in the first study and spurious deaths, apparently not compoundrelated, in the second study confounded the determination of a NOAEL for maternal 

toxicity. There were no signs of teratogenic or fetotoxic effects in any study other than 

the first definitive study in which maternal deaths above the MTD apparently occurred. It 

is BASF's opinion based on a thorough review of the teratology studies that the following 

overall NOAELs can be derived for the teratology studies:

a. NOAEL maternal toxicity. 100 mg/kg body weight (rabbit) and 1,000 mg/kg 

body weight (rat). 

b. NOAEL prenatal toxicity. 350 mg/kg body weight (rabbit) and 1,000 mg/kg body

weight (rat). 

The overall NOAEL of 100 mg/kg bw for maternal toxicity in rabbits is based on 

the last rabbit study, and is based on reduction of body weight gain and food intake at 

dose levels of 250 mg/kg body weight onwards. The NOAEL of 350 mg/kg bw for 

fetotoxic effects in the rabbit is also based on a reduction in body weight gain. Based on 

the overall study results, it is concluded that there are no developmental effects of 

concern. Based on preliminary discussions with EPA concerning the rabbit teratology

studies, EPA concluded that the definitive NOAEL for maternal toxicity considering all 

of the studies ranges from 30 to 100 mg/kg/bw. Agency scientists further stated that they

needed to review the studies in detail to ultimately determine the definitive NOAEL for 

maternal toxicity. This uncertainty associated with maternal toxicity in the rabbit 

teratology studies does not impact risk considerations since the risk assessment is based 

on a lower NOAEL (20 mg/kg bw) in the chronic dog study.]

4. Subchronic toxicity. [The subchronic toxicity of prohexadione calcium was 

investigated in 90-day feeding studies with rats, mice and dogs. In all these studies, 

prohexadione calcium displayed low toxicity. Prohexadione calcium showed no signs of

neurotoxicity in a 90-day neurotoxicity rat study. Additionally, the results seen in four 
4

week feeding range-finding studies for rats and dogs were similar to the findings 

observed in the 90-day studies in the same animals.]

5. Chronic toxicity. [Based on review of the available data, the Reference Dose 

(RfD) for prohexadione calcium will be based on a 1-year feeding study in dogs with a 

threshold No Adverse Effect Level (NOAEL) of 20 mg/kg/day. Using an uncertainty 

factor of 100, the RfD is calculated to be 0.2 mg/kg/day. The following are summaries of 

studies submitted to EPA. 

Prohexadione calcium was administered to Beagle dogs at dietary concentrations of 

0, 20, 200, and 1,000 mg/kg bw for 12 months. Slight changes were observed for 

hematological and clinical chemical parameters and dilated basophilic renal tubules 

(without histopathological concurrence) at dose levels greater than 200 mg/kg bw. The 

NOAEL was 20 mg/kg bw for the males and female dogs. 

The 24-month Fisher 344 rat chronic/carcinogenic feeding study was conducted at 

dose levels of 0, 400, 2,000, 10,000, and 20,000 ppm with 80 male and 80 female animals 

per dose group. After 26, 52, and 78 weeks, 10 animals were sacrificed (satellite groups). 

The remaining animals were autopsied after 104 weeks of diet administration. The 

NOAEL for chronic toxicity was 2,000 ppm for males (93.9 mg/kg bw) and 2,000 ppm 

for females (114 mg/kg bw). The following effects were observed in the 10,000 and 20, 

000 ppm groups: 

i. Decreased body weights were observed in both male and female rats at the 20,000 

ppm dose level;

ii. Clinical chemical effects (i.e., lower potassium, bilirubin, and glucose levels) 

were observed in male and female rats at the 20,000 ppm dose level, in the 10,000 ppm 

dose level, reduced glucose levels were only seen in the males, and increased 

albumin/globulin ratios,

sodium, chloride and calcium levels were observed only in the females; 

iii. Increased urine volumes and lower specific gravity were observed in the midhigh and high-dose groups for both male and female rats;

iv. Minor changes in organ weights were noted for animals of the high dose group 

only, which consisted of increased relative liver, adrenal and kidney weights, the latter 

also absolute in females only, at week 26; at the end of the study decreased liver weights 

and increased relative brain and testis weights were noted and these changes were 

considered to be associated with the decreased body weights;

v. Macroscopic findings revealed an increase of pituitary nodules in the high dose 

group for both male and female rats which was not confirmed histopathologically and 

submucosal ectopic tissue in the glandular stomach was found in both male and female 

rats in the highest dose levels that was confirmed by histopathology which showed an 

increase of squamous cell hyperplasia in males and of basal cell hyperplasia in the 

forestomach;

vi. A higher incidence of cellular hyperplasia was observed in the thyroid in the 

mid-high and high dose levels for male and female rats; and

vii. No increased incidence of neoplasms occurred at any dose levels tested in this 

study.

In the 24-month B6C3F1 mouse feeding study, conducted at dose levels of 0, 400, 

2,000, 20,000, and 40,000 ppm with interim sacrifices at 52 and 78 weeks, prohexadione 

calcium was negative for oncogenicity. The NOAEL for chronic toxicity was 2,000 ppm 
5

for males (279 mg/kg bw) and 2,000 ppm for females (351 mg/kg bw). The following 

effects were observed in the 20,000 and 40,000 ppm groups:

i. Statistically significant decreases in body weights were observed in male mice at 

the 20,000 ppm dose level and in female mice at the 40,000 ppm dose level;

ii. A variety of changes in hematological parameters were noted in the respective 

investigations at weeks 52, 78, and 104, however, most of the changes were not dose 

related or consistent over time;

iii. Increased absolute and/or relative heart, brain, testes, liver, ovary, and kidney

weights were observed in the mid-high and highest dose groups with a slight progression 

of severity to the highest dose group;

iv. A higher incidence of splenomegaly was observed only in the male mice of the 

highest dose group;

v. Histopathological examinations revealed an ectopic proliferation of the mucosal 

and glandular epithelium in the submucosal layer of the glandular stomach in male and 

female mice in the highest dose group tested, these changes were assessed to represent 

heteroplastic, ectopic proliferative changes accompanied by lumen dilatation and 

cytological degeneration;

vi. A higher incidence of hyperkeratosis of the forestomach was observed in both 

male and female mice and hyperplasia of the squamous epithelium of the forestomach of 

female male mice was observed in the highest dose group tested;

vii. Vacuolic changes in the exocrine pancreas of the high dose female were 

observed; and

viii. No increased incidence of neoplasms occurred at any dose levels tested in this 

study.

a. Threshold effects. Based on review of the available chronic toxicity data, BASF 

believes EPA will establish the Reference Dose(RfD) for prohexadione calcium at 0.20 

mg/kg/day. This RfD for prohexadione calcium is based on the 1-year feeding study in 

dogs with a threshold NOEL of 20 mg/kg/day in male and female dogs. Using an 

uncertainty factor of 100, the RfD is calculated to be 0.20 mg/kg/day. Based on the acute 

toxicity data BASF believes that prohexadione calcium does not pose any dietary risks. 

b. Non-threshold effects. Based on EPA Proposed Guidelines For Carcinogen Risk 

Assessment, BASF believes that prohexadione calcium will be classified as ``Not Likely

a Human Carcinogen''. Under the current assessment method BASF believes that EPA 

will classify prohexadione calcium as Group E, no evidence of carcinogenicity based on 

studies in two species. There was no evidence of carcinogenicity in mice and rat 24-

month feeding studies at the dosage levels tested. The doses tested were adequate for 

identifying a cancer risk.]

6. Animal metabolism. [The metabolism in animals (goats and poultry)

is adequately understood.]

7. Metabolite toxicology. []

8. Endocrine disruption. [No specific tests have been conducted with prohexadione 

calcium to determine whether the chemical may have an effect in humans that is similar 

to an effect produced by a naturally occurring estrogen or other endocrine effects. 

However, there were no significant findings in other relevant toxicity studies (i.e., 

subchronic and chronic toxicity, teratology and multi-generation reproductive studies) 

which would suggest that prohexadione calcium produces endocrine related effects.]
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C. Aggregate Exposure

1. Dietary exposure. [An assessment was conducted to evaluate the potential risk 

due to chronic dietary exposure of the U.S. population and sub-populations to 

residues of prohexadione calcium. Tolerance values for prohexadione calcium 

have previously been established and are listed in U.S. 40 CFR § 180.547. ]

i. Food. [BASF has conducted a prohexadione calcium dietary risk assessment for 

all currently registered crops and the proposed new tolerances on strawberry at 0.3 ppm 

and watercress at 2.0 ppm. The assessment was conducted using 100 % crop treatment 

factors, tolerance level residues, and default processing factors of 1. The assessment 

included all food commodities and water at a concentration of 9.1 ppb. This water value 

was determined by the US EPA during the most recent evaluation of a turf use for 

prohexadione calcium (US EPA, March 30, 2010, “Prohexadione Calcium. Proposed use 

on commercial and residential turf, golf courses, sod farms, and ornamental bedding 

plants and bedding plant plugs.”). 

Acute Dietary Exposure Assessment 

The EPA has determined that an acute assessment of prohexadione calcium is not 

required because there are no toxicological effects attributable to a single oral exposure. 

Chronic Dietary Exposure Assessment

The most highly exposed subpopulation was children 1-2 years of age. The % Chronic 

Population Adjusted Dose ( cPAD) utilized was 16.6 %. Table 1 shows the exposure 

and % cPAD utilized by all sub-populations. 

Table 1. Results for Prohexadione Calcium Chronic Dietary Exposure (Food + 

Water) Analysis Considering all Current Tolerances and the New 

Proposed Tolerances for Strawberry and Watercress.

Population Exposure Estimate %cPAD

Subgroups (mg/kg b.w./day)

US Population 0.00423 2.1

All infants (< 1 year) 0.02110 10.5

Children 1-2 years old 0.03317 16.6

Children 3-5 years old 0.01833 9.2

Children 6-12 years old 0.00711 3.6

Youth 13-19 years old 0.00256 1.3

Adults 20-49 years old 0.00177 0.9

Adults 50+ yrs 0.00184 0.9

Females 13-49 years old 0.00194 1.0

cPAD = 0.2 mg/kg bw/day. 

* Exposure estimates based on tolerance values, default processing factor, and 100% crop treated values 

Results of the analysis show that for the U.S. population and all sub-populations, the 

exposures are below a level of concern (< 100% cPAD). Additional refinements in the 
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chronic dietary risk assessment (i.e. utilizing anticipated residue values, process factors, 

percent crop treated values) would further reduce the estimated exposure values]

ii. Drinking water. [The human exposure from drinking water was included in the 

dietary exposure. 

Acute Aggregate Exposure

Acute aggregate exposure was not determined because the EPA has determined that there 

is no toxicological effect resulting from a single oral dose of prohexadione calcium. 

Short- and Intermediate Term Aggregate Exposure and Risk (food,water, and residential)

Short- and intermediate-term aggregate exposure takes into account residential exposure 

plus chronic exposure from food and water. Residential exposure is used to refer to nonoccupational and non-dietary exposure. Prohexadione calcium was registered for uses in 

2010 which can result in post-application exposure to the general population and 

children. The residential exposure value used in this risk assessment was previously

determined by the EPA (US EPA, March 30, 2010, “Prohexadione Calcium. Proposed 

use on commercial and residential turf, golf courses, sod farms, and ornamental bedding 

plants and bedding plant plugs.”). The short-term aggregate risk assessment is shown in 

Table 3. The MOE values indicate that the aggregate risk from the use of prohexadione 

calcium is acceptable. 

Table 3. Short-Term Aggregate Risk from the use of Prohexadione Calcium. 

SubPopulations Scenario

Short term Aggregate Risk

Dietary

MOE

Dermal 

MOE

Oral hand 

to mouth 

MOE

Total 

Aggregate 

MOE

General 

Population

Res -post 

app exp, 9466 11000 NA 5088

children 1 -2

Res -post 

app exp, 1206 14000 180000 1104

youth 13-19

Res -post 

app exp 15645 7400 5024
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Chronic Aggregate Exposure:

Chronic aggregate exposure includes exposure from food and drinking water. The 

dietary assessment conducted above includes food and water and therefore, is an 

aggregate exposure. The aggregate exposure to prohexadione calcium utilized less than 

17% of the cPAD for all subpopulations. ]

2. Non-dietary exposure. [Residential exposure is used to refer to nonoccupational and non-dietary exposure. Prohexadione calcium was registered 

for uses in 2010 which can result in post-application exposure to the general 

population and children. The residential exposure value used in this risk 

assessment was previously determined by the EPA (US EPA, March 30, 2010, 

“Prohexadione Calcium. Proposed use on commercial and residential turf, golf 

courses, sod farms, and ornamental bedding plants and bedding plant plugs.”). 

The MOE values from the assessment are presented in the Table 3. 

Table 3. Residential Exposure and Risk Estimates for Post-Application Uses 

for Prohexadione Calcium. 

Population 

subgroup Scenario

Dermal exp 

(mg/kg bwday)

Dermal 

MOE

Oral exp -

hand to 

mouth 

(mg/kg bw 

- day)

Oral 

MOE

Adult

Post-App 

golfer 0.00364 11000 NA

youth

Post-App 

golfer 0.00541 7400 NA

Toddler

Post-App 

Turf 0.00286 14000 0.00044 180001

]

D. Cumulative Effects

[BASF is aware of only one other registered compound, trinexapac-ethyl 4-

(cyclopropyl-a-hydroxymethylene)-3,5-dioxocyclohexanecarboxylic acid ethyl ester, that 

has a structure similar to prohexadione calcium. However, BASF has no information that 

would indicate that the two compounds have a common mechanism of toxicity. Furthermore, trinexapac is registered for use only on turf. Therefore, even if the compounds 

were considered similar there would be no cumulative dietary exposure issue because of 

the differences in use patterns. In summary, dietary exposure to prohexadione calcium 

should not result in cumulative toxicity with other known chemical compounds.]
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E. Safety Determination

1. U.S. population. [Based on this risk assessment, BASF concludes that there is a 

reasonable certainty that no harm will result to the general population from the aggregate 

exposure to prohexadione calcium residues. ]

2. Infants and children. [Based on this risk assessment, BASF concludes that there 

is a reasonable certainty that no harm will result to infants or children from the aggregate 

exposure to prohexadione calcium residues]

F. International Tolerances

[A maximum residue level (MRL) has not been established for prohexadione 

calcium in strawberries or watercress by the Codex Alimentarius Commission.]