UNITED STATES ENVIRONMENTAL PROTECTION AGENCY

WASHINGTON D.C., 20460

										

	MEMORANDUM							DATE:  03/16/2011

	SUBJECT:	Revised Non-target Organism and Endangered Species Screening
Risk Assessment for the N-6-Benzyladenine Registration Review
Preliminary Work Plan

		Chemical Class: 			Biochemical	

		PC Code: 				116901

		CAS Number:			1214-39-7

		Tolerance Exemptions: 	40 CFR 180.			

FROM:	Russell S. Jones, Ph.D., Senior Biologist   /s/ 02/09/2011

	Biochemical Pesticides Branch

	Biopesticides & Pollution Prevention Division

TO:	Chris Pfeifer, Regulatory Action Leader

	Biochemical Pesticides Branch

	Biopesticides & Pollution Prevention Division

	

	EXECUTIVE SUMMARY

The plant regulator, N-6-Benzyladenine, is a naturally-occurring
cytokinin found in plants (van Staden & Crouch, 1996).  Available
non-target organism hazard and exposure information as well as Agency
risk assessments on N6-benzyladenine were evaluated against current
safety standards established by the Agency’s scientific policies and
regulations and it was determined that there is no need to conduct
additional risk assessments.  The active ingredient is a
naturally-occurring plant hormone, has a non-toxic mode of action, and
is of low toxicity.  There is reasonable certainty of no harm to
non-target organisms when N-6-Benzyladenine is applied in accordance
with EPA-approved product labeling.  A screening level assessment was
conducted to evaluate the existing non-target organism data base.  

Based on the fact that N-6-Benzyladenine (N-6-BA) is relatively
non-toxic to non-target organisms and on its use pattern and use
instructions, EPA has determined that N-6-BA will have "No Effect" on
any currently listed threatened or endangered species or any designated
critical habitat. 

		1.	Description of the Active Ingredient

			Common Name:	N6-Benzyladenine

			Chemical Name:	N-(phenymethyl)-1H-purine-6-amine

			Synonyms:		6-Benzylaminopurine

			Empirical Formula:	C12H11N5

			

					

						Figure 1.  Structure of N-6-Benzyladenine

N-6-Benzyladenine is a plant growth regulator used to enhance flowering,
fruit size and shape, lateral and basal bud break and shoot growth.  It
is also used as a thinning agent.  The active ingredient is employed in
pesticide products used on apples, pears, pistachios, nonbearing sweet
cherries and ornamental plants.  Historically, the Agency has classified
the chemical as a synthetic cytokinin; however, more recent data
indicate that the chemical (EPA, 1994; van Staden & Crouch, 1996) is
naturally-occurring. 

N-6-Benzyladenine was classified as a Biochemical Pesticide Active
Ingredient by the OPP Biotechnology Workgroup in 1990 (EPA, 1990).

		2.	Registered End-Use Products and Maximum Use Rates

There are 10 currently registered End-Use Products (EPs) containing
N-6-Benzyladenine (N-6-BA) as their active ingredient (see Appendix
Table 1).  Five products contain N-6-Benzyladenine (1.8% to 2% by weight
of product) as their sole active ingredient.  The remaining six products
contain Gibberellins A4A7 as a second active ingredient at
concentrations of 0.18% or 1.8% by weight of product.  Most of the
end-use product labels state that no more than 182 g N-6-BA may be
applied per acre per year.  All labels specify that no more than two
applications be made in a growing season, with either a 3- or 7-day
interval between applications.  The highest single application rate is
for Promalin Plant Growth Regulator (EPA Reg. No. 73049-41) and Perlan
(EPA Reg. No. 62097-6) in apple orchards at 0.039 lb active
ingredient/A.  The highest split application rate with a 7-day interval
is for Riteway (EPA Reg. No. 71368-60) and MaxCel Plant Growth Regulator
(EPA Reg No. 73049-407) in apple orchards at 0.165 lb active
ingredient/A/application.  The highest split application rate with a
3-day interval is for Riteway (EPA Reg. No. 71368-60) at 0.041 lbs
active ingredient/A/application.  The aforementioned maximum
applications rates will be used in the non-target organism exposure and
hazard assessments and endangered species risk assessments.  

	3.	Ecological Hazards

Adequate non-target toxicology data/information are available to support
registration of N-6-Benzyladenine (N-6-BA).  All non-target organism
toxicology data requirements for N-6-BA have been satisfied.  No
additional data are required.  

There are no concerns for any non-target organisms when N-6-BA is
applied in accordance with EPA-approved label use directions.  Toxic
endpoints have not been identified for non-target birds (dietary basis)
in laboratory testing.  There are no reports available indicating any
phytoxicity to plants resulting from direct application or from drift.  
The active ingredient is slightly toxic to mammals, birds, and
freshwater fish on a acute basis in laboratory testing (see Appendix
Table 2).  A review of the ECOTOX database did not reveal any additional
toxicity data or endpoints that were lower than those previously
evaluated by the Agency.  

	

	4.  Environmental Fate and Ground Water Data

The need for environmental fate and groundwater data was not triggered
because results of the acute toxicity assessment did not trigger any
additional Tier I studies.  Some data, however, are available from
studies submitted in support of an Experimental Use Permit (EUP) and
from external technical sources.  Residues of N-6-BA applied to food
crops are expected to dissipate to background levels before they are
distributed for consumption.  Data from soil metabolism studies
submitted in support of an Experimental Use Permit in 1977 (EPA RED,
dated 1994; and MRID 00120680) confirm the rapid dissipation of N-6-BA
in soil with an approximate half-life of 7 weeks in La Porte loamy sand
and 9 weeks in Elliot silt clay.  Other data suggest a range of
degradation rates as rapid as 1 to 3 days in aerobic soil, 12.1 days in
aqueous sediments, and an aqueous photolysis half life of 2.5 days at pH
7.  

 	5.  Ecological Exposure and Risk Characterization

Terrestrial Residues:  Maximum residues on plant foliage and insects
present on the foliage following a single application, or two
consecutive applications of N-6-BA at the maximum label use rates, were
estimated using the Terrestrial Exposure Model (T-REX; EPA, 2005).   For
all application timings and rates, residues ranged from 0.59 to 4.77 ppm
on fruits, pods, seeds, and large insects (present on foliage at the
time of applications), to 5.27 to 42.9 ppm on broadleaf plants and small
insects, and up to 9.36 to 76.26 ppm on short and tall grasses.  The
model assumed a conservative environmental half-life of 9 weeks (EPA
RED, 1994).  The greatest residues occurred following the application of
Riteway (EPA Reg. No. 71368-60) (Appendix Table 3).  In evaluating
N-6-Benzyladenine foliar application to a crop the Agency has assumed a
worst case scenario of  5% drift from the target site onto adjacent
areas that can result in expected residues with significant decreases
farther from the target site.  Based on the data, foliar residues
resulting from unintentional spray drift would not exceed approximately
3.8 ppm

Aquatic Residues:  Maximum estimated residues in water following a
single application, or two consecutive applications of N-6-BA at the
maximum label use rates, were estimated using the Generic Estimated
Environmental Concentration Model , ver.2 (GENEEC2: EPA, 2001).  Both
foliar and airblast methods of application were modeled, although most
label application sites were for “standard spray equipment” in
orchards.  Non-orchard sites were labeled for ornamental spot sprays and
greenhouse use that would result in little or no environmental exposure
to the active ingredient.  

The airblast method of application resulted in the highest estimated
residues.  For all airblast application timings and rates, aquatic
residues ranged from a peak EEC of 1.06 to 8.55 ppb, declining to a
90-day average EEC ranging from 0.33 to 2.66 ppb.  The model assumed a
conservative aerobic terrestrial soil half-life of 9 weeks (EPA RED,
1994), an aqueous photolysis half-life of 2.5 days at pH 7 (PPDB, 2011)
and an aerobic aquatic soil half-life of 12.1 days (PPDB, 2011).  The
greatest residues occurred following two applications of Riteway (EPA
Reg. No. 71368-60) at 0.165 lb a.i./A/application with a 7-day
application interval (Appendix Table 4).  

	6.  Threatened and Endangered Species Assessment

	

Terrestrial residues on plant foliage and insects present on the foliage
following a single application, or two consecutive applications of
N-6-BA at the maximum label use rates, were estimated using the
Terrestrial Exposure Model (T-REX; EPA, 2005).   Maximum terrestrial 
residues were attained following two applications of Riteway (EPA Reg.
No. 71368-60) at 0.165 lb a.i./A/application with a 7-day application
interval.  Residues ranged from 4.77 ppm on fruit, pods, seedlings, and
large insects, to 42.90 ppm on broadleaf plants and small insects, to
34.95 ppm on tall grass and 76.26 ppm on short grass.  The
aforementioned values were used by the T-REX model to calculate Risk
Quotients (RQs) for mammals and birds.  

Aquatic residues following a single application, or two consecutive
applications of N-6-BA at the maximum label use rates, were estimated
using the Generic Estimated Environmental Concentration Model, Version 2
(GENEEC 2; EPA, 2001).  Maximum aquatic residues were attained following
two applications of Riteway (EPA Reg. No. 71368-60) at 0.165 lb
a.i./A/application with a 7-day application interval.  Peak aquatic
Estimated Environmental Concentrations (EEC) = 8.55 parts per billion
(ppb) of N-6-BA; EECs declined from a 4-day average of 8.15 ppb to a
90-day average of 2.66 ppb (Appendix Table 4).  The aforementioned
values were used by the GENEEC 2  model to calculate Risk Quotients
(RQs) for fish and aquatic invertebrates.  

Mammals:  Mammalian (rat) acute oral toxicity ranged from 1300 mg/kg to
1584 mg/kg (Toxicity Category III; Appendix Table 1).  The subchronic
(90-day) dietary No Observable Effects Level (NOEL) was 1500 ppm.  Both
acute dose-based-, acute dietary, and chronic dietary-based RQs ranged
from <0.01 to 0.05 for all mammalian classes and body weights.  The RQs
did not exceed any Levels of Concern (LOCs) for non-target mammals,
including threatened and endangered species. 

Birds:  Avian acute oral toxicity ranged from 1599 mg/kg to 2875 mg/kg
(Slightly Toxic to Practically Non-toxic; Appendix Table 1).  Avian
dietary toxicity was >5620 ppm (Practically Non-toxic).  Both acute
dose-based-, acute dietary, and chronic dietary-based RQs ranged from
<0.01 to 0.08 for all avian classes and body weights.  The RQs did not
exceed any Levels of Concern for non-target birds, including threatened
and endangered species. 

Fish:  Freshwater fish LC50s ranged from 21.4 ppm (rainbow trout) to
42.0 ppm (Brachydanio rerio) which are categorized as slightly toxic
(Appendix Table 4).  Using the most conservative endpoint for fish (21.4
ppm) in laboratory testing and the maximum EEC of N-6-BA in water, the
RQ was calculated as:

			RQ		=	0.00855 ppm	=	0.0004

				   			21.4 ppm

The RQ is well below any LOC for non-target fish, including threatened
and endangered species.  

Aquatic Invertebrates:  Aquatic Invertebrate EC50s for daphnids ranged
from 4.52 ppm to >20.5  ppm which are categorized as moderately to
slightly toxic (Appendix Table 4).  Using the most conservative endpoint
for aquatic invertebrates (4.52 ppm) in laboratory testing and the
maximum EEC of N-6-BA in water (8.55 ppb or 0.00855 ppm), the RQ was
calculated as:

			RQ		=	0.00855 ppm	=	0.002

				   		4.52 ppm

Plants:  N-6-Benzyladenine is a naturally-occurring plant hormone that
has no known toxic effects when applied exogenously to plants at the
proposed rates of application.  It has a non-toxic mode of action and is
intended for use on plants for the purpose of increasing yield,
appearance, and/or quality of plant foliage and fruits.  

Plant testing of biological pest control agents is conditionally
required if incidents have occurred or if there is published information
that the compound is toxic to plants. Therefore, to the best of our
knowledge N-6-Benzyladenine (N-6-BA) is not known to be phytotoxic.  
Positive effects, however, are problematic when it comes to measurement
or observation in the field and thresholds will be different for various
plant species relative to environmental conditions.  Since
N-6-Benzyladenine is intended for use as a plant growth enhancer and
will be applied foliarly to orchard crops, there is a possibility of
drift to adjacent vegetative areas. 

This indirect exposure, however, is minimal and not efficacious. The
Agency has made this conclusion by noting that the direct application
scenario for target plants can result in maximum expected foliar
residues of up to 76.26 ppm (T-REX; EPA, 2005).  Numerous field trials
have been conducted in orchards using N-6-BA and the minimum
concentrations needed to elicit a a statistically significant positive
response ranged from 100 to 200 ppm for the control of canopy formation
in Christmas trees and fruit trees, and 25 to 200 ppm for fruit thinning
in apple orchards (see review by Buban, 2000).   Thinning of pears and
blueberries required minimum concentrations of 100 ppm and 200 ppm,
respectively (Bound and Mitchell, 2000; Koran and Stopar, 2004).  

In evaluating N-6-Benzyladenine foliar application to a crop the Agency
has assumed a worst case scenario of  5% drift from the target site onto
adjacent areas that can result in expected residues with significant
decreases farther from the target site. These values are approximately
20X below the minimum efficacy levels reported by Buban (2007),  Bound
and Mitchell (2000), and Koran and Stopar (2004).  Based on these data,
the Agency has no concerns for non-target plants.  

Insects:  The N-6-Benzyladenine RED (EPA, 1994) concluded that studies
and data on non-target Insects would not be required “...based on a
lack of exposure and any documented  effects of plant growth regulators
on beneficial non-target insects.”  Subsequent to the publication of
the RED, non-target insect studies were reported demonstrating that the
contact toxicity LD50 for insects (honey bee) was >58.73 ug/bee (see
Appendix Table 3), which is categorized as practically non-toxic.  No
toxic effects were observed at the highest rate used in laboratory
testing.  Based on these data, and the earlier conclusions made by the
N-6-Benzyladenine RED (EPA, 1994), the Agency has no concerns for
non-target insects.  

CONCLUSIONS:  There are no concerns for any non-target organism when
N—6-Benzyladenine is applied in accordance with EPA-approved product
labeling.  

Based on the fact that N-6-Benzyladenine (N-6-BA) is relatively
non-toxic to non-target organisms and on its use pattern and use
instructions, EPA has determined that N-6-BA will have "No Effect" on
any currently listed threatened or endangered species or any designated
critical habitat. 

	REFERENCES

	Bound, S. A. and L. Mitchell.  2000.  A new post-bloom thinning agent
for Packam’s T	riumph pear.  ISHS Acta Horticulturae 596:  VIII
International Symposium on Pear.  

Buban, T.  2000.  The use of benzyladenine in orchard fruit growing:  a
mini review.  Plant Growth Regulation 32:  381-390.  

EPA,  1990.  Classification of N6-Benzyladenine (Chem. No. 116901) as a
Biochemical.  Memorandum from J. T. McClintock to R. Engler

EPA.  1994.  Registration Eligibility Decision.  N6-Benzyladenine.  List
B.  Case 2040.  Environmental Protection Agency, Office of Pesticide
Programs, Special Review and Registration Division.  188 p.    HYPERLINK
"http://www.epa.gov/oppsrrd1/REDs/old_reds/n6benzyladenine.pdf" 
http://www.epa.gov/oppsrrd1/REDs/old_reds/n6benzyladenine.pdf 

EPA.  2001.  GENEEC 2.0 (Generic Estimated Environmental Concentration
Model, Version 2).  Environmental Fate and Exposure Division, Office of
Pesticide Programs.

EPA.  2008.  T-REX (Terrestrial Exposure Model) Version 1.4.1. 
Terrestrial Biology and Exposure Technical Teams, Environmental Fate and
Exposure Division, Office of Pesticide Programs.

Koran, D. and M. Stopar.  2004.  Effects of thinners on yield, fruit
size, and ripening time of highbush blueberry.  ISHS Acta Horticulturae
715:  VIII International Symposium on Vaccinium Culture.

Van Staden, J. and N. R. Crouch.  1996.  Benzyladenine and derivatives
– their significance and interconversion in plants.  Plant Growth
Regulation 19:  153-175.  

Pesticide Product Data Base (PPDB).  2011.  6-Benzyladenine (Ref: 
ABG-3191).  Agricultural and Environmental Research Unit, University of
Hertfordshire, UNITED KINGDOM.  January 17, 2011 update,  

	cc:	C. Pfeifer, R.S. Jones, BPPD Subject File/IHAD

	R. S. Jones, Ph.D., Sr. Scientist, FT, OPY:  03/16/2011

APPENDIX

Appendix Table 1.  Currently registered products and maximum label use
rates for each product label.  Highest use rates are boldfaced.  

Product 	EPA Reg. No.	Active Ingredient(s)	Maximum Use Rate per Acre per
Application (app) 3	No. of apps and app Intervals (int)

Exilis Plus	62097-9	2.0% N-6-BA 1	0.083 lb/A/app

0.020 lbs/A/app	2 app @ 7 d int

2 app @ 3 d int

Configure	62097-19	2.0%  N-6-BA 	No field applications

Riteway	71368-60	1.9%  N-6-BA 	0.165 lb/A/app

0.041 lb/A/app	2 apps @ 7 d int

2 app @ 3 d int

MaxCel Plant Growth Regulator	73049-407	1.9%  N-6-BA 	0.165 lb/A/app 	2
apps @ 7 d int

Promalin Plant Growth Regulator	73049-41	1.8%  N-6-BA 

1.8% GA4/7 2	0.039 lb/A

0.0195 lb/A/app 	1 app

2 apps @ 3 d int

Chrysal BVB

100% re-pack of above	72992-10	1.8%  N-6-BA 

1.8% GA4/7 	No field applications

Perlan	62097-6	1.8%  N-6-BA 

1.8% GA4/7 	0.039 lb/A

0.030 + 0.020 lb/A/app 	1 app

2 apps @ 3 d int

Typy	55146-78	1.8%  N-6-BA 

1.8% GA4/7 	0.022 lb/A

0.011 lb/A/app	1 app

2 apps @ 3 d int

Agtrol 6-BA	55146-86	1.8%  N-6-BA 

0.18% GA4/7 	0.066 lb/A/app	2 apps @ 7 d int

Accel Plant Growth Regulator	73049-29	1.8%  N-6-BA 

0.18% GA4/7 	0.066 lb/A/app	2 apps @ 7 d int

1	N-6-Benzyladenine

2	Gibberellins A4/A7

3	Rate for N-6-BA only



Appendix Table 2.  Summary of Non-Target Organism Data

Study Type/OPPTS Guideline	

LD50/LC50/EC50 Results	

Toxicity Category	MRID



 Acute Oral Toxicity

/OPPTS 870.1100  	1300 mg/kg (rat)	Tox Category III	00120681

	1584 mg/kg (rat)	Tox Category III	PPDB, 2011



 Subchronic Oral Toxicity (rat), 90-day

/OPPTS 870.3100  	NOEL = 1500 ppm

LOEL = 5000 ppm	

Practically non-toxic	42329201



 Avian Acute Oral Toxicity

/OPPTS 850.2100  	1599 ppm	

Slightly toxic 1	41895204

	2875 ppm	

Practically non-toxic	PPDB, 2011



 Avian Dietary Toxicity

/OPPTS 850.	>5620 ppm	

Practically non-toxic	41895205



Freshwater Fish Acute Toxicity, 96 hr

/OPPTS 850.1075  	21.4 ppm 2	Slightly toxic	41895206

	42.0 ppm 3	Slightly toxic	PPDB, 2011



Aquatic Freshwater Invertebrate Toxicity, 48-hr /OPPTS 850.1010 	

17.0 mg/L 4	Slightly toxic	41895207

	

>20.5 mg/L 4	No toxicity at highest tested dose	PPDB, 2011

	4.52 mg/L 5	Moderately toxic	PPDB, 2011



Other Arthropods (non-Guideline)	80 g/hectare 6	Not applicable	PPDB,
2011

	36.2 g/hectare 7	Not applicable	PPDB, 2011



Earthworms (non-Guideline) (14-day)	>500 mg/kg	No toxicity at highest
dose tested	PPDB, 2011



Non-target Plants

	Waiver	

No expected adverse effects	N-6-BA RED (1994)



Non-target Insects (Honey Bee Contact Toxicity, 48-hr)/OPPTS 850.3020	
>25 ng/bee	

Practically non-toxic	41623704

	>58.73 ng/bee	

Practically non-toxic	PPDB, 2011

1	The Benzyladenine RED incorrectly categorized avian acute oral
toxicity as practically non-toxic and avian dietary toxicity as slightly
toxic, when the reverse was true based on the data (see EPA, 1994).

2	Onchorhyncus mykiss

3	Brachydanio rerio

4	Daphnia

5	Chironomus riparius (96-hr)

6	Typhlodromus pyri

7	Aphidius rhopalosiphi



Appendix Table 3.  Maximum N-6-BA residues in terrestrial environments
following a single application, or two consecutive airblast applications
at the maximum label use rates (airblast/orchard application method). 1

Environmental Matrix	Residues (ppm) following  single application at 
0.039lb a.i./A 2	Residues (ppm) following  two applications at 0.041 lb
a.i./A with a 3-day interval 3	Residues (ppm) following  two
applications at 0.165 lb a.i./A with a 7-day interval 3

Short Grass	9.36	19.36	76.26

Tall Grass	4.29	8.87	34.95

Broadleaf Plants/Small Insects	5.27	10.89	42.9

Fruits/Pods/Seedlings/Large insects	0.59	1.21	4.77

1	Dietary-based Estimated Environmental Concentration (EEC) calculated
using T-REX model (EPA, 2005).  

2	Promalin Plant Growth Regulator (EPA Reg. No. 73049-1)

3	Riteway (EPA Reg. No. 71368-60)

Appendix Table 4.  Maximum N-6-BA residues in aquatic environments
following a single application, or two consecutive airblast applications
at the maximum label use rates (airblast/orchard application method). 1

Environmental Matrix	Residues (ppb) following  single application at 
0.039lb a.i./A 2	Residues (ppb) following  two applications at 0.041 lb
a.i./A with a 3-day interval 3	Residues (ppb) following  two
applications at 0.165 lb a.i./A with a 7-day interval 3

Peak GEEC	1.06	2.18	8.55

Maximum 4-Day Avg. GEEC	1.01	2.08	8.15

Maximum 21-Day Avg. GEEC	0.77	1.58	6.19

Maximum 60-Day Avg. GEEC	0.45	0.93	3.65

Maximum 90-Day Avg. GEEC	0.33	0.68	2.66

1	Estimated Environmental Concentration (EEC) calculated using GENEEC2
model (EPA, 2001).  

2	Promalin Plant Growth Regulator (EPA Reg. No. 73049-1)

3	Riteway (EPA Reg. No. 71368-60)

N-6-Benzyladenine		

PC Code:  116901	

Non-Target Organism Hazard Assessment and Endangered Species Screening
Risk Assessment for Registration Review Preliminary Work Plan	

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