.
ENVIRONMENTAL ASSESSMENT
OF THE USE OF SEMDURAMICIN SODIUM PREMIX
IN THE FEED OF BROILER CHICKENS
FOR THE PREVENTION OFCOCCIDIOSIS
I
PFIZER INC.
APRIL 1993
ENVIRONMENTALASSESSMENT
TABLE OF CONTENTS
.
Page
1
TITLE PAGE
2
TABLE OF CONTENTS
4
1,2,3. DATE, APPLICANT, ADDRESS
4
DESCRIPTION OF THE PROPOSED ACTION
4.
4
lDENTlFlCATlON.OF CHEMICAL SUBSTANCES
5.
a) Semduramicin Sodium
b) Other Premix Ingredients
6
6. INTRODUCTION OF SUBSTANCES INTO
THE ENVIRONMENT
a) From the Site where Bulk Drug is Produced
b) From the Site where the Premix is Produced
c) Introduction of Substances as i llesult of Use
7. 18
FATE OF EMITTED SUBSTANCES INTHE ENVIRONMENT
a) Air
b) Terrestrial Ecosystems
c) Aquatic Ecosystems
21
ENVIRONMENTAL EFFECTS OF RELEASED SUBSTANCES
8.
a) On Terrestrial Species
b) On Aquatic Species
USE OF RESOURCES AND ENERGY 23
9.
MITIGATION MEASURES
10. 23
ALTERNATIVES TO THE PROPOSED ACTION 23
11.
LIST OF PREPARERS
12. 24
CERTIFICATION
13. 25
REFERENCES
14. 26
Page
APPENDICES
15.
a) Manufacturing and WorkerSafety
Safety Material 28 Data Sheets
1.
2 NPDES permit explanation 33
.
of Compliance - Bulk Manufacturing Sites
Certification
3. 35
4. Certification of Compliance - Premix Manufacturing Site
38
40 Chartsb) Data Summary
c) Summaries of Study Reports
(1) Excretion of semduramicin and its metabotites by
broilers.
45
Physical-chemical of
(2) properties semduramicin sodium.
54
(3) octanol-water
The partition coefficient of semduramicinsodium.
60
(4) Soil sorption and
desorption of semduramicin sodium.
62
.(5) Hydrolysis of semduramicin sodium. 69
Photodegradation of semduramicin sodium in
aqueous
solution. 72
(6)
(7) Biodegradation of semduramicin sodium in soil. 76
(8) Effect of semduramicin sodium on soil microbes. 80
Effect of semduramicin sodium on seed germination
(9) 83
and root elongation.
(10) Effect of semduramicin sodiumon seedling growth. 88
(11) Effect of sodium
semduramicin on algae.' 94
(12) toxicity
Acute study with semduramicin sodium in
Daphnia.
97
(13) toxicity
Acute study with semduramicin sodium in
bluegill. 100
(14)Acutetoxicitystudy with semduramicin sodium inrainbowtrout. 103
(1 5) Acute dermal.and ocular irritation studies with sernduramicin
sodium in rabbits.' 106
ENVIRONMENTAL ASSESSMENT
OF
THE USE OF SEMDURAMICIN SODIUM PREMIX IN THE FEED
OF BROILER CHICKENS FOR THE PREVENTION OF COCCIDIOSIS
April 5 , 1993
1. DATE:
APPLICANT: Pfizer Inc.
2.
(Sponsor W00069)
ADDRESS: 235 East 42nd Street
3.
New York, N.Y. 10017
4. DESCRIBE THE PROPOSED ACTION:
Pfizer Inc. is filing a
New Animal Drug Application requesting approvalfor the use of a
premix containing semduramicin sodium in broiler feeds. Feed containing
semduramicin activity at the recommended use level 25 ppm (corresponding to a
of
concentration of 25.6 ppm of semduramicin sodium) would be fed continuously to
broilers for the prevention of coccidiosis.
The bulk drug will be produced at one or more of Pfizer's existing manufacturing plants
in Groton, Connecticut and Taketoyo, Japan. The premix, a 5.13% Type A Medicated
Article, will be formulated and packaged at P-fizer's existing Lee's Summit, Missouri
plant and will be blendedto Type C medicated broiler feeds at feedmills in broiler-
producing areas. The finished feeds will be used in commercial broiler houses. These
are located primarily in rural areasof the states of Alabama, Arkansas, California,
Delaware, Georgia, Maine, Maryland, Mississippi,North Carolina, Pennsylvania, Texas,
and Virginia.
IDENTIFICATION OF CHEMICAL SUBSTANCES THAT ARETHE SUBJECT OF THE
5.
PROPOSED ACTION:
A. Semduramicin Sodium
Semduramicin sodium is an antibiotic produced by a strain of Actinomadura
roseorufa var. Huang. It belongs to aclass of antibiotics knownas ionophores or
polyether carboxylic acids. It will be produced as a crystalline sodium salt.
Generic Name:
Semduramicin
sodium
Trade AVIAX
Name:
Chemical
Name: (2R,3S,4SI5E,6S)-tetrahydro-2,4-dihydroxy-
6-[( 1FlJ-1-[(2Sl5E,7S,8R,9S)-9-hydroxy-
2,8-dimethyl-2-[(2E,5S)-tetrahydro-5-methyl-
. 5-[(2E,3SI5E)-tetrahydro-5-[2S,3S,5FJ,6S)-
tetrahydro-6-hydroxy-3,5,6-trimethyl-2H-
pyran-2-yl]-3-[[(2S,5S,6FlJ-tetrahydro-
5-methoxy-6-methyl-2~-pyran-2-yl]
oxy]-2-furyl]-2-furyl]-l,6-dioxaspiro[4.5]
dec-7-yl]ethyl]-5-methoxy-3-methyl-2H-
pyran-2-acetic acid- sodium
CAS Registry Number (sodium salt): 119068-77-8
Pfizer Code Number: UK-61,689-2
MolecularWeight: 895
Structural Formula:
Physical Description: White Solid, m.p. 17OoC
B. Other
Premix
Ingredients:
In addition to semduramicin sodium, premixes may contain diluents commonly
used in feed premixes, such soybean mill run, rice hulls, calcium carbonate,
as
sodium carbonate, sodium aluminosilicate, and mineral oil.
6. INTRODUCTIONOFSUBSTANCESINTO THE ENVIRONMENT:
A. From the sites where bulk drug is produced:
The-manufacture of semduramicin sodium willbe carried out in one two general
or
purpose fermentation and recovery plants designed to have minimal
environmental impact and be in compliance with all applicable emissions
requirements. Either of these sites would operate in accordance with local
environmental regulations. The plants are located in Groton, Connecticut and
Taketoyo, Japan.
1. Production/Processing Overview
Semduramicin sodium is a fermentation-produced ionophore that is primarily
associated withthe solid mycelial portion ofthe fermentation broth.
Recovery of semduramicin sodium from the fermentation broth involves standard
unit operations including: separation of broth solids, solvent extraction, carbon
treatment, evaporativeconcentration, crystallization, drying, and milling.
The aqueous portion of the fermentation broth (i.e. the broth filtrate) contains a
small percentage of semduramicin sodium, and is treated as a waste stream
during recovery processing.
All effluent/waste streams, regardless of production site, will be treated in a
to
manner to reduce residual semduramicin sodium content in discharged waste
less than 0.1 ppm.
2. Manufacturing and Occupational Safety
a.
Material Safety Data Sheets
The manufacturing site(s) will make available to employees the appropriate
detailed Material Safety DataSheets (MSDS) essentially similar to OSHA
Form 20. The MSDS for semduramicin sodium and semduramicin sodium
5.13% premix will contain the informationshown in the attached examples
(Appendix a-1), though the format and local language will vary from one site
to another.
b. Hazard
Evaluation Studies
Dermal and occular irritation studieswere conducted in rabbits (Appendix C-
15). Only mild dermal irritation wasobserved following a 24 hour exposure
to 0.5 g semduramicin sodium applied to intact skin. Abraded skin exposed
to the same dose for 24 hours showed well defined erythema which
subsided completely within 4-5 days. Instillation of 21.5 mg semduramicin
sodium to the conjunctival sac caused slight reddening of the conjunctivae,
slight chemosis and/or slight discharge as well as circumcorneal injection
and a small, localized area of iritis in one.rabbit. Within 24 hours of dosing,
treated eyes appeared normal.
Steps have been taken to minimize occupational and user exposure to
semduramicin at Pfizer bulk drug and premix manufacturing sites. Facilities
for production of semduramicin bulk are equipped with appropriate physical
isolation and air handling facilities to minimize worker exposure. Many of the
production operations are automated. Pfizer workers will also wear
appropriate protective equipment including gowns, gloves and protective
masks as circumstances require. The 5% semduramicin sodium premix
product (Aviax) will be manufactured in a new, automated premix plant
located in Lee's Summit, Missouri, which has been specifically designed to
minimize worker exposure. Exposure to semduramicin will be minimized by
means of both the physical isolation of operators from automated drum
dumping stations, mixers and packaging operations, and by the design of
the air handling systems. Furthermore, workers will wear appropriate
protective clothing, as required.
To determine exposure to airborne semduramicin sodium, air samplers were
placed at key manufacturing stations, either adjacent to equipment or fitted
to operators to sample air close to the breathing zone. Membrane filters
from the air samplers were analyzedfor the presence of semduramicin
sodium and expressed as mg per cubic meter of air. The results (below)
show that airborne semduramicin was either not detectedor was present
only in extremely minute quantities.
mg semduramicin sodium/
Testinq Area Sampling Period cubic meter of
air
Bulk drug dump station 29 minutes
0.08
Vrieco Conical Premix 9 minutes non detected
Blender (15 min. mix)
Blend Sifter 9 minutes non detected
Premix Bag Filler 18 minutes 0.07
Semduramicin premix has been specifically formulated to protect feed mill
operators from exposure to airborne semduramicin. The low dusting
characteristics of semduramicin premix were demonstrated by results
obtained with a Heubach dustmeter. In this test, a sample of premix in a
-rotating drum is dropped repeatedly through a moving air stream, which
carries any dislodged respirable dust to a capture filter for analysis. The
method is useful for comparing therelative dustiness of premix formulations.
In this test, semduramicin commercial premix showed <2 micrograms
semduramicin dust/g premix. The results are also significantly lower than
those obtained for four other contemporary, commerically available poultry
premix products, which gave results of 30 (Coban), 12 (Cygro), 24
(Monteban) and 50 (Biocox) rncg active/g under the same conditions.
3. Emissions
The substances which could be emitted and/or discharged from specific
production units, and the respective exposure limits (when available) for the
Groton site, are as follows:
Abstracts
Chemical TWAl
ppm
Substance Reqistry No. mg/m3
.
113378-31-7(acid form)
Semduramicin sodium
--
Extracted mycelium
Unconsumed fermentation nutri-
ents (i.e. hydrolyzed starch,
molasses, yeast extracts,
soybean oil, soybean meal,
blood meal, cotton seed meal,
corn starch liquor, etc.)
Solvents
67-56-1
Methanol 260 200
67-63-0
Isopropanol 980 400
67-64-1
Acetone 1800 750
108-1
Methyl isobutyl ketone 0-1 205 50
141 -78-6
Ethyl acetate 1400 400
123-86-4
n-Butyl acetate 71150 0
75-09-2
Methylene chloride 175 50
Chloroform 67-66-3 9.78 2
142-82-5
Heptane 1600 400
1 10-54-3
Hexane 180 50
8032-32-4
Petroleum ether 3002
108-88-3
Toluene 375 100
71 -55-6
1,1,1 -trichloroethane 350 1900
108-20-3
Isopropyl ether 1050 250
AcidslBases
7647-01
Hydrochloric acid [ceiling]7 5
-0
Sulfuric acid 7664-93-9 1
131 0-73-2
Sodium hydroxide 2 [ceiling]
Calcium hydroxide 1305-62-0 5
Calcium oxide 1305-78-8 5
Potassium hydroxide 1310-58-3 2 [ceiling]
Ammonium hydroxide 1336-21 -6
Other
Filteraid (silica) 68855-54-9 15
Carbon 7440-44-0 3.5
Sodium chloride 7647-14-5
Ammonium carbonate 8000-73-5
Calcium carbonate 471 -34-1 15 total dust
Ammonium nitrate 6484-52-2
Calcium nitrate 13477-34-4
7722-76-1
Ammonium phosphate, monobasic
Ammonium phosphate, dibasic 7783-28-0
Potassium phosphate, monobasic 7778-77-0
7758-1 1-4
Potassium phosphate, dibasic
Page 9
TWA鈥? Chemical
Abstracts
!%.!m
ppm
Substance Registry No.
10049-21-5
Sodium phosphate, monobasic
Sodium phosphate, dibasic 7782-85-6
Sodium sulfate 7727-73-3
Ammonium sulfate 7783-20-2
Magnesium sulfate 7487-88-9
Manganese sulfate 10034-96-5
Ferrous sulfate 7782-63-0
Cobalt chloride 7791-13-1
7447-40-7
Potassium chloride
Urea 57-13-6
1 .o
Formaldehyde 50-00-0
1 .o 1.4
7722-84-1
Hydrogen peroxide
Allowable 8-hour time-weighted average exposure according OSHA Air
to
Contaminants 29 CFR 1910.1000
Limit set by ACGIH.
Page 10
4. Groton Site
The Groton plant site is a large, multi-product, pharmaceutical and specialty
chemical manufacturing facility. Through its Environmental Control Department,
the ptant maintains an integrated program for management of solid, liquid and air-
borne wastes. The plant has available a number of waste disposal systems and,
depending upon the magnitudeand concentration of each stream as well as the
mix, the most efficient treatment system is selected for each
overall plant product
stream. Where more than one treatment is possible for a particular waste stream,
each alternative treatment planis described below. Each treatment alternative is
capable of insuring that the plant remains in compliance with all emissions
requirements.
Solid Wastes
Broth solids will be disposed by incineration/pyrolysis:
of
Incineration/pyroiysis facilities will operate under ResourceConservation and
Recovery Act permits andemploy technology sufficient to destroy more than
99.99% of the waste.
These broth solids willbe handled incompliance with Federal requirements
of U.S.Environmental Protection Agency Regulations 40 CFRParts 260-267
and with Connecticut Department of Environmental Protection Section25-
54cc(c).
Activated carbon solids will be either incinerated/pyrolyzedas above or sent
to a contracted disposalfirm in compliance with Federal requirements of 40
CFR Parts 260-267 and Connecticut Department of Environmental
Protection Section 25-54cc(c).
Liquid Wastes
Broth Filtrate and Aqueous Crystallization Mother Liquors: These primary
.
aqueous streamswill be disposed of by one ofthe following methods:
1) The stream will be treated as necessary in a chemical pre-treatment
operation (acid treatment) for degradation of semduramicin sodium. The
treated filtrate stream will be sent to the Pfizer siteeffluent treatment facility
and discharges will have semduramicin sodium concentration below 0.1
ppm. Effluent is disposed of from this facility into the Thames River underall
limitations in plant site NPDES permit CT 00000957 issued in accordance
with U.S. Environmental Protection Agency Regulation 40 CFR Parts 124
and 125 and administered by the Connecticut Department of Environmental
Protection. This permit is being extended administratively by the
Connecticut DEP until it issues the renewal permit, for which a timely
application has been submitted by Pfizer Inc. (Appendix a-2).
The limitations include a pH between 5.0 and 9.0, a BOD not to exceed
21,500 kg/day, absence of a visible oil sheen, foam or floating solids, no
discoloration of the receiving waters and a rise in the temperature of the
receiving streamof no more than 4OF and to no higher than83OF.
2) The stream will be incinerated/pyrolyzed as described above for the broth
solids.
Other aqueous waste streams, such as tank and floor washings, will be chemically
pre-treated (acidtreatment) as needed for degradation of semduramicin sodium,
and, where necessary, stripped of residual organic solvent in appropriate
evaporative distillation equipment. The treated aqueousstreams will be sent to
the Pfizer site effluent treatment facility (as above) prior to disposal under all
limitations specified in plant site NPDES permit CT00000957.
Solvent streams will be recovered/recycledas much as practicable via distillation
waste heat recoveryor
systems. Disposal of unusable solvent streams will be via
pyrolysis in compliance with the following regulations administered by the
Connecticut Department of Environmental Protection: Connecticut General
Statutes Air Regulations Title 22a, Environmental Protection, Chapter 439 and
U.S. Environmental Protection Agency Regulations 40 CFR Parts 264 and 265.
These regulations require that Resource Conservation and Recovery Act permits
be obtainedand that the technology employed ensuresdestruction of more than
99.99% of the applicable waste.
Air Emissions
Evaporation of organic solvents will be controlled as appropriate with condensers
or scrubbers. Air emissions from process vessels will be controlled by vent
condensers andlor conservation vents. All air emissions will be in compliance
with Connecticut General Statutes Air Regulations Title 22a, Environmental
Protection, Chapter 439.
Occupational exposure to air contaminants during the bulk manufacturing process
will be limited, since most of the operations will be contained within a closed
system. Emission of particulate matter during bulk drying and milling will be
.
controlled bylocal exhaust ventilation and filter: dust collectors.
Monitoring of the work area to ascertain occupational exposure will be regularly
carried out and all exposure limits (see table under 6A3, above) will be in
000
compliance according to OSHA Air Contaminants 29 CFR 191 0.1
The attachedstatement (Appendix a-3) certifies compliance with all Federal, State
and local emissions requirements.
5. Taketoyo, Japan Site
SolidWastes .
Broth solids willbe disposed of by incineration of solids:
Incineration facilities will operate under the permits of the Agreement with
Taketoyo Townand employ technology sufficient to destroy more than 99.99% of
the waste.
These broth solids will be handled in compliance with National requirementsof
Environmental Protection Agency Regulations, Article 12 ofthe Industrial Waste
Disposal Control Law and with the Taketoyo Town Environmental Protection
Regulations, Articles 20-30.
Activated carbon solids will be sent to a contracted disposal in compliance
firm
with Prefectural requirements of Environmental Protection Regulations, Article 19.
Liquid Wastes
Broth Filtrate: The aqueous broth filtrate stream, which contains a small
percentage of total broth semduramicin sodium content, will be treated as
necessary in a chemical pre-treatment operation (acid treatment) for degradation
of semduramicin sodium. The treated filtrate stream will be sent to the Pfizer site
effluent treatment facility and discharges will have semduramicin sodium
concentration below 0.1 ppm. Effluent is disposed of from this facility into Kinuura
Bay under all limitations imposedby the Environmental Protection Agreementwith
Taketoyo Town, Articles 16-20 and by the National Water Pollution Prevention
Law, Article 3.
Other aqueous waste streams, such as tank and floor washings, will be chemically
as
pre-treated (acid treatment) needed for degradation of semduramicin sodium,
and, where necessary, stripped of residual organic solvent in appropriate
evaporative distillation equipment. The treated aqueous streams will sent to
be
the Pfizer site effluent treatment facility (as above) prior to disposal under all
limitations imposedby the Agreement with Taketoyo Town and the National Water
Pollution Prevention Law (described above).
Disposal of other solvent streams will beby an outside contracted firm certifiedby
the Prefectural Government in compliance with the Environmental Protection
Regulations, article 19.
These regulations require thatthe technology employed ensures destructionof
more than 99.99% of the waste.
Air Emissions
Evaporation of organic solvents will be controlled as appropriate with condensers.
Air emissions generated during evaporation will be controlled vent condensers
by
and/or conservation vents. All air emissions will be in compliance with Air
Pollution Prevention Law, Article 3, Prefectural Environmental Protection
Regulations, Article 19 and the Agreement with Taketoyo Town, Article 16.
Occupational exposure to air contaminants during the bulk manufacturing process
will be limited, since most of the operations will be contained within a closed
system. Emission of particulate matter during bulk drying and milling will be
controlled by localexhaust ventilation and filter dust collectors.
Monitoring of the work area to ascertain occupational exposure will beregularly
carried out and the exposure limits (see table under 6A2, above) will be in
Compliance with Industrial Safety and Health Law,
Article 28.
The attached statement (appendix a-3) certifies compliance with all National,
Prefectural and Local emissions requirements.
B. From the site where premix is produced:
packaged for
Semduramicin sodium willbe incorporated into feed premixes and
sale at Pfizer Inc.鈥檚 general purpose plant for the manufacture of animal health
products located at 1107 SE Missouri 291, in Lee鈥檚 Summit, Missouri. The plant
is designed to have minimal environmental impact and be in compliancewith all
Federal, State, and local emission requirements.
The premix manufacturing operation will involve only the blending of
semduramicin sodiumbulk with inert ingredients in equipment constructedof non-
reactive product contact parts.
Particulate emissions from the manufacturing process will be controlled via
cyclones and baghouse filters. While the particulate emissions will result
principally from the transfer and processing of soybean mill run, there will be
emission controls on all manufacturing areas. Particulate emissions will be
controlled in compliance with the Missouri Air Pollution Control Regulations
1OCBR10.2.
Particulate concentrations in the workplace will be monitored and maintained
below the Permissible ExposureLimits (PEL鈥楽) accordingto the OSHA standard in
29CFR1910.1000.
Wastewater discharges from the manufacturing process will consist of wash water
from equipment clean-out operations. Since the manufacturing processes will be
dry operations, equipment clean-outs will typically be done by scouring with dry
feed. Components of the equipment will be cleaned with water infrequently. This
wastewater discharge may contain semduramicin sodium, sodium carbonate,
mineral oil, sodium aluminosilicate and soybean dust. The quantity of product
present inthe wastewater discharge will beminimal because of the nature of the
process and ordinary cleaning procedures. Wastewater from the site is
discharged to the Little Blue Valley Sewer District publicly owned wastewater
treatment plant. This discharge is authorized by an operating permitissued to the
Pfizer Lee鈥檚 Summit plant by the Missouri Water Pollution Control Board. All
discharges will be in compliance with the standardsset by this Board.
Solid wastes will consist of the fiber drumswith empty inner plastic liners which
may contain trace quantities of semduramicin sodium, other paper packaging from
raw materials, floor sweepings, dust collector rejectsand some dry mill material
from equipment cleaning. Production of the premix will not generate hazardous
wastes as defined by the Federal Regulations 40CFR261.4, nor will it generate
hazardous waste as defined by the Missouri Hazardous Waste Management Law
Title 16 Chapter 260. The solid wastes will be disposed by landfill under the
Missouri Solid Waste Rules1OCSR80, or by incineration.
The Lee鈥檚 Summit site makes available to its employees the appropriatedetailed
Material Safety Data Sheets(MSDS) essentially similar to OSHA form 20. The
MSDS sheets for semduramicin sodium and the premix will each have the same
content as the appended copies (Appendix a-1),though they will be in a slightly
different format.
The attached statement (Appendix a-4) certifies compliance with all Federal,
State, and local emissions requirements.
C. Introduction of substances asa result of use:
Use of semduramicin sodium as an anticoccidial agent for broilers would result in
introduction of semduramicin sodium into the environment through excretion by
broilers which have consumed medicated feed and subsequent application of the
broiler manure to soil as fertilizer.
1. Concentration of Semduramicinin Broiler Excreta
The concentration of semduramicin sodium in unaged manure from broilers which
consumed feed containing 25 ppm of semduramicin has been determined to
average 1.6 ppm (Appendix c-1). Most of the ingested drug (about 93%) is
broken down by broilers into many polar metabolites. All but one of the
metabolites were inactive in a test for biological activity that is highly sensitive to
ionophores (Appendix c-1). The sole active metabolite.was present in very low
concentration, only about 1.3 times that of unchanged semduramicin sodium. The
concentration of this metabolitein fresh excreta is therefore about 2.1 ppm.
1.3 x 1.6 ppm = 2.1 ppm
It isrelated in structure to semduramicin, and has molecular weight of the same
order of magnitude. Although it is the only metabolite producing a biological
response, the present calculationassumes it to be as active as semduramicin
sodium, and its concentration has been added to that of semduramicin sodium to
give a combined concentration equivalent to 3.7 ppm of semduramicin sodium in
broiler excreta.
1.6 ppm (unchanged drug)+ 2.1 ppm (metabolite) = 3.7 ppm in excreta
2. Potential Concentration of SemduramicininSoil
Poultry manure is commonly used as a fertilizer. Use of manure containing
semduramicin sodium as fertilizer would result in introduction of the drug into the
soil. The resulting initial concentration of drug in soil can be estimated from the
concentration of drug in manure and the rate of application of manure to soil.
The recommended rate of application for poultry manure ranges up to 7.5 short
tons (6.8 metric tons) per acre, with incorporation into the top six inches in soil
(Reference 3) The top six inches of soil in one acre weigh about 909,000 kg. Use
of broiler manure containing 3.7 ppm semduramicin sodium (Section 6.C.1) would
result in initial concentration in soil of up to only about 0.03 ppm.
3.7 mgkg x 6 800 kg/acre = 25,160 mg/acre
25,160 mg/acre t- 9.09 x 105 kg/acre = 0.0276 mgkg or about 0.03 ppm
The calculations above assume no adjustment for dilutionof manure with bedding
materials (litter) and assume that semduramicin will remain stable in the manure
until the latter is applied to soil. In reality, the concentration of semduramicin in
manure is diluted by bedding materials and is likely to degrade gradually in this
litter and manure mixture. The projected concentrations therefore probably
represent exaggerated estimates.
Amount of Semduramicin Introduced intotheEnvironment
3.
It is possible to estimate the amount of semduramicin sodium that will be
introduced into the environment by a commercial broilerraising facility which uses
semduramicin sodium as its anticoccidial agent. A large commercial broiler
operation can consist of four to five broiler houses containing 80,000-100,000
birds in total. There are usually 6 production cycles per year. Broiler chickens are
raised to an average weight of about 4.2 pounds (Reference 1). It takes a
modern broiler about 42 days to reach this weight (Reference2). During this time,
it excretes about0.054 kg of waste per day (Reference 3), or total of 2.268 kg.
a
0.054 kglday x 42 days = 2.268 kg
A facility containing 100,000 broilers produces 226,800 kg of manure during each
growout cycle:
2.268 kghroiler x 100,000 broilers = 226,800 kg
If the facility has 6 growout cycles per year,it produces about 1,400 metric tons of
manure per year:
226,800 kg/cycle x 6 cycles = 1,360,800 kg
This amount of manure would contain about5.0 kg of semduramicin sodium:
1,360,800 kg x 3.7 mgkg = 5,034,960 mg= 5.035 kg
When applied at the rate of 7.5 short tons (6.8 metric tons) per acre, 1,400 tons of
manure and 5.0 kg of semduramicin sodium would be distributed over about 200
acres:
1,360,800 kg manure 6,800 kglacre = 200 acres
f-
7.FATEOFEMITTEDSUBSTANCESINTHEENVIRONMENT:
&
r
A.
Senfduramicin sodium is a high molecular weight (895), high melting (m.p.
17OoC), ionic solid and would be expected to be non-volatile. In agreement with
this expectation, data where obtained which confirm the vapor pressure must
that
be less than lo-* torr at 20掳C (Appendix c-2). Therefore, semduramicin sodium
would not be expected to partition into the atmosphere under the conditions of
production or use.
Terrestrial
Ecosystems
6.
As discussed above under 6.C.2, low concentrations (about 0.03 ppm) of
semduramicin sodium would be introduced into soil as a result of use of the
the
product. This initial concentration of semduramicin would decline by aerobic
biodegradation as demonstrated in a laboratory test (Appendix c-7).
Semduramicin was observed to degrade to C02 in 3 soils of varying
characteristics that were acquired from differentgeographical regions within the
US. Fifty percent biodegradation was observed inapproximately 94 and 42 days,
respectively, for Ohio and Iowa soils, and 40% degradation for North Dakotasoil
in approximately 94 days. The estimated (vs. experimentally measured) time to
50% biodegradation for Ohio, Iowa, and North Dakota soils was 79, 42, and 104
days, respectively.
Although the kinetics of semduramicin in soils cannot be predicted from the
studies conducted and are likely to be complex (Reference 4), first order kinetics
will be used to illustrate how semduramicin might be eliminated from soils. First
order kinetics have been found applicable describing degradation of a variety
for
of chemicals present at very low (e.g. ppm) concentrations (Reference 4) and
describe a significant portion of the multiphase kinetics of semduramicin
degradation observedin the soil biodegradation study (e.g. clay soil).
dagr&&nx Q.7 S d
1 C- l
4 -an
100
BO
10
I
70
1
*
E
I
I
I
I
I
I
1
n
I
BO
70
0 20 40 50 10
30
10 SO 100
(days)
Tima
Page 18
The concentration of drug in soil at any defined time after its applicationto soil
with manure can bedetermined by the following equation, assuming that theinitial
drug concentration in soil and depletion half life are known:
. -'
Cat time t = Coe
If C equals the concentration semduramicin in soilsimmediately following the
, of
application of manure containing the drug(0.03 ppm, Section 6C3), k equals the
depletion rate constant and e is the natural logarithm, then C equals the
concentration ofsemduramicin in the soil at time t, i.e. one year later when the
soil
is refertilized. Employing the estimated 50% biodegradation rates for 3 soilsof
42, 79 and 104 days, yearly fertilization soils with poultry manure containing
of
semduramicin would not lead to accumulation of increasing concentrations of drug
in soils.
Depletion rates(k)are established from the estimated 50% biodegradation values
listed above:
t1/2 (Days to 50% Biodeqradation)
Soil type k (Days-'
0.01 7
Silty clay loam (Iowa)
42
0.0088
Clay loam (Ohio) 79
0.0067
Clay (North Dakota) 104
The concentration (C) of semduramicin at 365 days (t):
Soil Type C (ppm) at 365 days
Silty loam
clay
(Iowa) 6.1 x 10-5
Clay loam
1.2
(Ohio) X 103
2.6 x 10"
Clay (North Dakota)
As shown, one year after depositing 0.03 ppm semduramicin in soil with manure,
only 61 ppt to 1.2 ppb would remain at the time the soil was refertilized. There
would be no significant increase in concentration of semduramicin above the 0.03
ppm level over time; therefore, the projected maximum environmental
concentration of semduramicin in soils would remain 0.03 ppm.
C. Aquatic Ecosystems
Although semduramicin sodium is appreciably soluble in water (solubility 1,900
ppm), with an acid dissociation constant of 5.39 (Appendix c-2), soil
sorption/desorption experiments indicate that it would largely remain boundto soil
rather than partition into aquatic systems (Appendixc-4).
A maximum potential concentration of semduramicin in surface water can be
estimated using a 40 acre (16.2 hectares) watershed with a 2.5 acre pond
(average depth, 2.5 ft) as the surface water receiving runoff. As has been
demonstrated, the highest potential concentration of semduramicin in soil resulting
fromwse is 0.03 ppm. The 16.2 hectare watershed would contain, at most, 1.091
kg of semduramicinsodium (0.03 mg/kg x 9.09 x 1O5 kg/acre x 2.47 acredhectare
X 16.2 hectares = 1.091 kg). The pond would contain 7.71 x l o 6 liters of water
(6.25 acre ft x 43,560 #/acre ft x 28.32 liters/ft3). Assuming a worst case scenerio
that all the drug runs off thewater shed, the maximum potential concentration of
only 0.14 mg/L or 0.14 ppm (1.091 X
semduramicin sodium in the pond would be
lo6 mg + 7.71 X lo6 L).
Actually, the amount in run off would decline as the runoff traversed
uncontaminated soil and semduramicinsodium was absorbed or deposited onto
its surface. The resulting concentration in the pond would not persist, as
semduramicin sodium decomposes in aqueous solution, particularly in acidic
waters (Appendix c-5), and decomposition is accelerated exposure to sunlight
by
(Appendix c-6).
The logarithm of the octanol-water partition coefficient (log Kow) of semduramicin
sodium is 2.21 to 2.58, depending on pH (Appendix c-3). By comparison,
compounds that are known to bioaccumulate, suchas DDT (log K , 6.19), have
O
log Kow valuesof 4 or greater (Reference 5). Thus the value for semduramicin
sodium suggests that significant accumulation is not predicted. Testing has
demonstrated that semduramicin sodium does not persist and accumulate in living
beings, but is readily converted to more polar metabolites and excreted. Rapid
and extensive metabolic degradation has been demonstrated in chickens, dogs,
and rats (Appendix c-1).
Page 20
8. ENVIRONMENTAL EFFECTS OF RELEASEDSUBSTANCES:
A. Terrestrial Species
As discussed above under 6C, the projected maximum concentration of
semduramicin in soil is 0.03 ppm. This maximum concentration could only occur
when fresh poultry manure had just been mixedinto the soil, i.e. no degradation
had taken place in manureor soil. The greatest possible potential for adverse
effects is assessed below by comparing projected maximum concentration in
the
soil to the results of exposure studies with terrestrial organisms.
Semduramicin sodium was tested in the laboratory for its ability to inhibit the
growth of six different species of soil microorganisms (Appendix c-8). The
minimum inhibitory concentration (MIC) in each case was found to be above 100
ppm, which is more than 3,300 timesthe projected maximum concentration in soil.
When graded concentrations of semduramicin sodium were tested on six species
of crop seeds in the laboratory (Appendix c-9),the highest concentration which
did not affect seed germination or root elongation and the ratio of this
concentration to the projected maximum concentration in soil were found to be as
follows: corn, 17 ppm, 567 times; cucumber, 34 ppm, 1,133 times; pinto beans,
6.3 ppm, 21 0 times; rye, 13 ppm, 433 times; wheat, 6.3 ppm, 210 times. The
lowest concentration of semduramicin that affected soybean germination was 1
ppm in the preliminary test. No effects were observed at concentrations ranging
between 0.36 and 2.2 ppm in the definitive test. A no observable effect
concentration (NOEC) was not established for soybeans.
The lowest concentrations that had any detectable adverse effect on the growth of
seedlings of the same six crop species during a 21 -day observation period
(Appendix c-10) and the ratiosof these concentrations to the projected maximum
concentration in soil were as follows: corn, 4.2 ppm, 140 times; cucumber 2.2
ppm, 73 times; pinto beans, 2.2 ppm, 73 times; soybean, 0.77 ppm, 25 times;
NOECs were established for all parameters in ryegrass and wheat except for root
weight (NOECs 5 0.31 and 0.77 ppm respectively). These values are 10-26 times
the projected maximum concentration in soil.
The above results indicate that use of semduramicin sodium asdirected would not
be expected to have any significant adverse effects on terrestrial organisms in the
environment.
Aquatic Species
B.
The potential exposure of aquatic organisms to semduramicin sodium is expected
to be intermittent, since it depends on rain runoff from soil fertilized with broiler
excreta containing semduramicin sodium; and short-lived,since the concentration
of semduramicin in water would decline due to hydrolysis and
photodecomposition. The greatest possible potential for adverse effects is
assessed below by comparing the projectedmaximum concentration in surface
waters of 0.14 ppm (Section 7C) tothe results of acute exposure studies with
aquatic organisms.
In rainbow trout (Appendix c-14), the concentration of semduramicin sodium
which causes 50% mortality after 96 hours of exposure (the 96-hour L )
C, was
found to be 32 ppm, which is approximately 230 times the projected maximum
concentration. The highest concentration which did not produce any observable
adverse effects was 11 ppm, or about 80 times the projected maximum
concentration.
In bluegill (Appendix c-13), the corresponding numbers are 38 ppmfor the 96-
hour L,,
C which is 270 times the projected maximum concentration, and 13 ppm
for the highest concentration which did not produce any observable adverse
effects, or 93 times the projected maximum concentration.
In the water flea, Daphnia magna (Appendix c-12), the concentration which
caused immobilization of 50% of daphnids exposed for 48 hours (the 48-hour
EC5,) was found to be 38 ppm, approximately 270 times the projected maximum
concentration. .The highest concentration which did not produce any observable
adverse effects was 19 ppm, 136 times theprojected maximum.
In a freshwater algae (Appendixc-1l ) , the lowest concentration which produceda
significant reduction in growth rate and maximum cell densitywas 19 ppm, 136
times the projectedmaximum concentration, and the highest concentration which
had no significant effect on growth rate or culture density was 10 ppm,or 71 times
the projectedmaximum concentration.
Based on these data, the contemplated use semduramicin sodium is highly
of
unlikely to result in any significant adverse effects on aquaticorganisms in the
environment.
USE OF RESOURCES AND
9. ENERGY
Manufacturing semduramicin sodium bulk and premix will require amounts of resources
and energy similarto those used to produce and formulate fermentation-derived
any
additive for animal feeds, such as other ionophores. Disposal of wastes generated
or
from production will not require use of unusual amounts of energy natural resources.
No effects are anticipated uponendangered or threatened species nor upon properties
listed inor eligible for listing in the National Registerof Historic Places.
10. MITIGATION
MEASURES
The proposed action would not beexpected to have any substantial adverse effect on
human health or the environment. The label for semduramicin sodium premix will
instruct users to wear protective clothing and a dustmask when mixing medicated feed
and to wash hands thoroughly afterwards. Other than these precautions listed on the
label, no mitigation measures are necessary for semduramicin sodium.
11. ALTERNATIVES TO THE PROPOSED ACTION
The proposed action would not expected to have any substantial adverse effect on
be
human health or the environment. Therefore, alternatives to the proposed action do
not need to be considered.
12. LIST OF PREPARERS
The following are all members of the staff of Pfizer Central Research, Pfizer Inc.,
.
Groton, Connecticut:
Daniel P. Brannegan, M.A.
Manager of Environmental Health and Safety
M.A. in Organic Chemistry
8 years experience in laboratory studies; 8 years experience in present position.
Larry R. Chappel, Ph.D.
Manager of Environmental Safety
Animal Health Product Development
20 years experience m R&D on animal health drugs.
Roderick 8. Douqherty, D.V.M.
Manager of Regulatory Affairs, Animal Health Product Development
6 years veterinary practice experience; 11 years experience animal health R&D.
Michael J. Keyes, B.S.
Assistant Director, Fermentation Process R&D
B.S. in Chemical Engineering
of
19 years of experience in the experimental and commercial production
pharmaceuticals and food additives.
Richard C. Koch, Ph.D.
Director of Project Liaison, Animal Health Product Development
Ph.D. in Organic Chemistry
30 years experience in humanand animal health drugs and agricultural chemicals.
Martin M. Lynch, M.A., M.B.A.
Manager, Department of Drug Metabolism
M.A. in Analytical Chemistry
M.B.A. in Management
20 years of experience in studies of the fate of drugs in mammals.
Jon L. Schaeffer, D.V.M, Ph.D.
Senior Regulatory AffairsScientist
Animal Health Product Development
3 years veterinary practice and3 years experience in R&D on animal health drugs.
Jeffrey A. Richards, Ph.D.
Manager, Analytical R&D
Ph.D. in Analytical Chemistry
14 years of experience in analytical chemistry.
The following individual isa member of the production staffof Pfizer鈥檚 U.S. Animal
Health Operations at Lee鈥檚 Summit, MO.:
Richard H. Bartel, B.S., P.E.
B.S. in Chemical
Environmental Affairs Supervisor, Lee鈥檚 Summit plant.
Engineering; 30 years of experience in chemical manufacturing and environmental
engineering.
13. CERTIFICATION
The undersigned official certifies that the information presented in Environmental
this
Assessment is true, accurate and complete to the best of his knowledge.
Larry R. Cvappel, Ph.b. Date
Manager, Environmental Safety
Animal Health Product Development
Pfizer Central Research
Pfizer Inc.
Page 25
-_ 鈥? .
14.
REFERENCES
Industry. May 1987. USDA figures in for 1986 broiler sales.
1. Broiler
2. Arbof Acres.1988.BroilerFeedingandManagementGuide.ArborAcres,
Glastonbury, CT, pp. 11 2.
-1
3. of
Food and Drug Administration, 1986. Finding of No Significant Impact
Selenium Supplementation of Animal Feeds. FAP 2201, Food and Drug
Administration, Washington, DC,p. 5 .
4. Alexander, M. and K.M. Scow. 1989. Kinetics of biodegradation in soil pp. 243-
269 in Reactions and Movement of Organic Chemicals in Soils. Sawhney, B. L.
and K. Brown, Eds. Soil Science of America Inc. Special Publication Number 22,
American Society of Agronomy.
5. FoodandDrugAdministration.1987.EnvironmentalAssessmentTechnical
Assistance Handbook. Technical Assistance Document 3.02. Food and Drug
Administration, Washington, D.C.
..
.
Appendix a-1
Material Safety Data Sheets
- --
--a. *.a
**- A..IU---A-
.-u..
-"... u..,-."..
-...I..... I
EXPERIMENTAL SUBSTANCE EasternPointRoad
Groton, Connecticut 06340
MATERIAL SAFETY DATA SHEET Emergency Telephone: (203)441-4100
MSDS # 0109
J u n e , 1992
.
(Superceedes April 1989)
SEMDURAMICIN SODIUM
Ionophore1
[UK-61,689-2;
SECTION I: PHYSICAL DATA
Appearance:
White solid
Melting Point: 169"-170掳C
Molecular Formula: C45H75016Na
895
MolecularWeight:
Chemical Family: PolyetherCarboxylic Acid (ionophore),
Solubility:
Soluble
common solvents;
in organic sparingly in
soluble water
and hexane.
Description:Semduramicin
sodium is animal
an health
drug for use an
as
anticoccidial agent in poultry. The materialis orally activeand all
forms of ingestion or inhalation must be avoided. Semduramicin
sodium is pure, undiluted material.
SECTION 11: FIRE AND EXPLOSlON HAZARD
The dust explosibility rating has not been determined. As with any organic solid,
precautions to minimize dust generation are required. Process equipment for the
handling of dry Semduramicin sodium should be provided with proper explosion relief
devices.
Semduramicin sodium does not present any unusual or significant fire hazards. If the
material becomes involved in a fire, the latter may be suppressed with appropriate
extinguishing medium, including water.
ZECTION 111: HEALTH H A Z A R D INFORMATION_
Semduramicin sodium is an orally active animal health drug In toxicology studies, the
no observed effect level was 0.3 mgkg in dogs(1 year) and 1.0 mg/kg in rats (3 months).
Semduramicin sodiumhad no effect upon reproduction or fertility of rats in a
3-generation toxicity study. There was noevidence of teratogenicity in appropriate
animal studies, and no evidence of mutagenic potential in a standard batteryof tests for
genetic toxicity. Carcinogenicity testing is underway, but related compounds which have
been tested are carcinogenic.
not
Semduramicin sodium has been tested for skin and eye irritation it is not an irritant
and
to intactor abraded rabbit skin, and is not ocular irritant to unrinsed rabbit eyes.
an
Semduramicin sodium, like similar ionophore antibiotics,causes muscle damage in dogs
(2 mg/kg),detectable by serum creatinine phosphokinase (CPK)elevations and
eventually by affected stante and gait. A dose of 0.5 mgkg in dogs produces retinal
lesions after 6 months. No significant cardiovascular changes indogs were observed
administration up to 3 mg/kg.
after oral
D
- lNFORMAT105
Ingestion: Intheevent of ingestion of Semduramicinsodium(solid or liquid
solutions), medical attention should summoned immediately.
be
Inhalation: Personnel who have inhaled Semduramicin sodium should be removed to
fresh air andobserved by medical personnel.
Skin Contact: Skin contacted solids or solutions of Semduramicin sodiumshould be
with
washedthoroughlywithwater.Contam.inatedclothingshould be
-removed. If any effects are observed, medical attention should be sought.
In case of eye contact, immediately wash with water for 15 minutes and
seek medical attention.
FECTION V: REACTNZTY DATA
Semduramicin sodium is stable bulk form for 12 weeks at temperatures up to 50掳C. It
in
is unstable in acidic and strongly alkaline
conditions.
SECTION VI: SPILL OR LEAKPROCEDURE
Spills of Semduramicin sodium should be collected (scooped) into an appropriate
recovery
container.Personnel involved in clean-up of spills, particularly solids, must wear
respiratory protection,gloves and eye protection.
SECTION VII: PRECAUTZON ZNFORMATZON
When handling Semduramicin sodiumavoid contact with skin, eyes or mucous
membranes. Avoid inhaling dust. Wear a dust mask, safety glasses and gloves when
handling the undiluted material. Wear gloves and eye protection when handling the
material in a fume hood. All animal health drugs and/or medicated feeds may produce
undesirablephysiologicaleffectstotheworker, if handledimproperly.Itis
recommended that good manufacturing practices be observed at all times and that
further precautions be followed to minimize exposure to the active drug. These include
minimizing the generation of dusts, avoiding contact with the skin, eyes, and mucous
membranes,andprovidingadequateventilation.Personalprotectiveequipment
including gloves and safety glasses should be worn when handling Semduramicin
equipment, such as a dust mask, use of a fume hood,
sodium and additional protective or
may be appropriate for certain handling activities.
D. P. Brannegan
issued by:
Page 2 of 2
CEN'I'KAL KKYKAK-Ckl Central Research Division
EXPERIMENTAL SUBSTANCE Eastern Point Road
Groton, Connecticut 06340
MATERIAL SAFETY DATA SHEET Emergency Telephone: (203)441-4100
MSDS # 0135
June, 1992
(Superceedw April 1989)
SEMDURAMICIN SODIUM 5% PREMIX
Ionophore1
[UK-61,689-2;
SECTION 1:P~HzZCAL
DATA
Apuearance: Finely
ground
meal
Composition: Semduramicin Sodium 5 % Premixcontainssemduramicin
sodium, sodium carbonate and mineral oil in a soybean mill run
.base.
Description: Thepremixisintended for use as an anticoccidial agentinpoultry
after suitable dilution into
poultry feed.
Chemical Familv: Semduramicin sodium is a polyether carboxylic acid ionophore.
400 g Semduramicin Sodium 5% Premixper 1,000 kg of feed,
TvDical Use Level:
providing a final semduramicin level of 20 ppm.
SEC_TIO_NII:XPLOSZON HAZARD
The dust explosivity rating has not been measured. As with any organic solid,
precautions to minimize dust generation arerequired. Mineral oil has been included as
a premix component to minimize dusting.
Semduramicin Sodium 5% Premix does not present any unusual or significant fire
hazards. If the material becomes involved in a fire, it maybe suppressed with
appropriate extinguisher media, including water.
SECTION 111: HEALTH HAZARD INFORMATION
Semduramicin sodiumis an orally active animal health drug. toxicology studies, the
In
no observed effect level was 0.3 mg/kg in dogs year) and1.0 m&g in rats (3 months).
(1
Semduramicin sodium had no effect upon reproduction or fertility of r a t s i n a
3-generation toxicity study. There was no evidence of teratogenicity in appropriate
animal studies, andno evidence of mutagenic potentialin a standard battery of tests for
genetic toxicity. Carcinogenicity testing is underway, but related compounds which
have been tested not carcinogenic.
are
Semduramicin sodiumhas been tested for skin and irritation andit is not a n irritant
eye
to intact or abraded rabbit skin, i not an ocular irritant to unrinsed rabbit eyes.
and s
muscle damage in dogs
Semduramicin sodium, like similar ionophore antibiotics, causes
(2 mg/kg), detectable by serum creatinine phosphokinase (CPK) elevations and
eventually by affected stance and gait. A dose of 0.5 mg/kg in dogs produces retinal
lesions after 6 months. No signiticant cardiovascular changes in dogs were observed
after oral administration up 3 m&g.
to
Page 1 of 2
Page 30
SECTION IV: FIRST AID INFORMATION
of ingestion of Semduramicin Sodium 5% Premix, medical
Inaestion:Intheevent
attention shouldbe summoned immediately.
who have inhaled Semduramicin Sodium 5% Premix should be
Inhalation: Personnel
removed to fresh air and observed by medical personnel.
contacted with solids or solutions of semduramicin sodium should be
Skin Contact: Skin
washedthoroughlywithwater.Contaminatedclothingshould be
&moved. If any effects are observed, medical attention should be sought.
In case of eye contact, immediately wash with water for 15 minutes and
seek medical attention.
SECTION V: REACTIVITY DATA
Semduramicin Sodium 5% Premix is stable inbulk form for 12 weeks at temperatures up
to 50掳C and at least 2 years at room temperature. Semduramicin sodium is unstable in
acidic and strongly alkaline conditions.
SECTION VI: SP
Spills of Semduramicin Sodium 5% Premix should be collected(scooped) into an
appropriate recovery container. Personnel involved in clean-up of spills, particularly
solids, must wear respiratoryprotection, gloves and eyeprotection.
SECTION VU: PRECAUTIONINFORMATION
When handling Semduramicin Sodium 5% Premix avoid contact with skin, eyes or
mucous membranes. Avoid inhaling dust. Wear a dust mask, safety glasses and gloves
when handling the undiluted material. Wear gloves and eye protection when handling
the material in a fume hood. All animal health drugs andlor medicated feeds may
produce undesirable physiological effects to the worker, if handled improperly. It is
recommended that good manufacturing practices be observed at all times and that
further precautions be followed to minimize exposure to the active drug. These include
minimizing the generation of dusts, avoiding contact with the skin, eyes, and mucous
membranes,andprovidingadequateventilation.Personalprotectiveequipment
including gloves and safety glasses should be worn when handling Semduramicin
Sodium 5% Premix and additional protective equipment, such as a dust mask,or use of a
fume hood, may be appropriate for certain handling activities.
D. P. Brannegan
issued by:
Page 2 of 2
__
Page 31
.
Appendix a-2
NPDES Permit Explanation
NPDES PERMIT EXTENSION
The paragraph that follows explains the administrative extension by the Connecticut DEP of
the NPDES pewnit that was to expire in 1985 and provides the name of the person at the
DEP who is familiar with the situation. A copy of the NPDES permit is found on pages 8066-
8079 (NADA 140-940).
The plant's NPDES permit was issued by Connecticut Department of Environmental
the
Protection (CT DEP), the delegated agent of the United States Environmental Protection
Agency, on May 20, 1980 with a life of five years or until May 19, 1985. Pfizer submitted a
timely permit renewal application at least 180 days prior to the
permit expirationdate. Under
the conditions set forthin 40 CFR 124 and 125 the NPDES permits in place at the time of
expiration continueto be in effect during the permit renewal process, therefore, the permit
issued in 1980 remains in effect inthe Groton plant. This situation is not unusual. There is a
significant backlog of pending permits within Connecticut and throughout the U.S. Over the
years there have been a number of meetings with the CT DEP and the USEPA regarding
this permit. The plant environnent manager contacts the CT DEP on a periodic basis to
remind the agency of the pending permit application. According to the agency, the pending
permit is under active review, and willbe issued in the near future.
Joseph Holmes of the Water Management Bureau, Departmentof Environmental Protection,
Hartford, Connecticut is familiar with the situation.
Page 33
.
Appendix a-3
Certification of Compliance - Bulk Manufacturing Sites
February 23,1993
.
TO WHOM IT MAY CONCERN:
This is to certify that, to the bestof our knowledge, Pfizer Inc's
manufacturing plant at Groton, Connecticut is in compliance
with a l applicable state,
l federal, and local emissions
requirements and is expected to remain in compliance when
semduramicin sodium bulk is produced at the site.
Robert T. Pfisterer
PlantEnvironmental Director
Food Science Group
I
I DATE: April 9, 1993
.
our knouledge, Pfizer鈥檚 manufacturing
to certify that i t s best
This i s oh of
plant s t Taketoyo, Aichi Prefectuie, Japan is in m p l i a m with all appt icable
national and l o c a l emissions requirments and i s expected to remain In compl tancu!
when semdufamicln sodim i s produd nt t e a i b .
h
H. Kikuchi
Vice President, knufacturing
.
Pfizet Phamawticals tnc.
Plant
Nagoya
Page 36
. -. .
Appendix a-4
Certification of Compliance - Premix Manufacturing Site
. April 5, 1993
TO WHOM IT MAY CONCERN:
This is to certify that, to the best of our knowledge, Pfizer Inc's manufacturing
plant at Lee's Summit, Missouri is in comptiance with all appllcable federal,
state and local emissions' requirements and is expected to remain in
compliance when the Semduramian Type A Medicated Article is produced.
Richard H. Bartel
Manager, Environmental Compliance
North American Animal Health Division
.
Appendix b
Data Summary Charts
APPENDIX b
DATA SUMMARY CHARTS
PHYSICAL-CHEMICALAND ENVIRONMENTAL FATE DATA
Generic Name: Semduramicin sodium
Structural Formula:
Molecular Formula: C45 H75 016 Na
Molecular Weight: 895
Solubility in water: pH Solubility
(mg/ml)
1.7
6
1.9
7
1.9
9
N-Octanol-Water Partition Coefficient: pH Lg ,
o K,
6 2.58
. 7' 2.37
9 2.21
Vapor Pressure: Non-volatile
Dissociation Constant: pKa = 5.39 in 1:1 acetone:water
Ultraviolet-Visible Absorption Spectrum: No detectable absorption at 290 to 800 nm and pH
5,7 or 9.
Melting Temperature: 1 7OoC.
lid
Absorption to Soil: Soil Type lloC
Mississippi Silty Clay Loam 1800 25.7
1400 10.6
Arkansas Silty Loam
4.65 150
Iowa Sandy Loam
6
- - -
Hydrolysis: pH: 5 -
7 8 9
77.1 115 89.936.111.1
Half-life (days):
- -
7
Photodegradation: pH: 6 - 9
11.32Half-life (days):
8.76 7.1
Estimated Half-life (Days)
Biodegradation in Soil:
Soil
Type At 25 ppm
Iowa Silty
Loam
42
Clay
Clay Ohio 79 Loam
Clay North
Dakota 104
ACUTE AND SUBACUTE TOXICITYSTUDIES
. TERRESTRIAL ORGANISMS
ENDPOINT
ORGANISM
- Minimum InhibitoryConcentration (MIC)
Soil Microbes
145
Clostridium n o w i
170
Bacillus stearothermophilus
170
Flavobacterium meninqosepticum
125
Nostoc
145
Trichoderma viride
240
Penicillum italicum
NOEC for Seed Germinationand Root Elongation (ppm)
Crop Seeds -
17
Corn
34
Cucumber
6.3
Pinto Bean
Not determined
Soybean
13
Rye
6.3
Wheat
LOEC For Survival, Root Weight, Shoot Weight, and Shoot Length
Crop Seedlings
(PPm)
4.2
Corn
2.2
Cucumber
2.2
Pinto Bean
0.77
Soybean
Ryegrass 0.31
0.77
Wheat
ACUTE AND SUBACUTE STUDIES
.
AQUATIC ORGANISMS
ENDPOINT
ORGANISM
MIC = 19 ppm
Freshwater algae
48-hour EC50 = 38 ppm
Daphnia
96-hour LC50 = 38 ppm
Bluegin
96-hour LCso = 32 ppm
Rainbow trout
.
Appendix c-1
Excretion of Semduramicin and its Metabolites by Broilers
OF SEMDURAMICIN AND ITS METABOLITES BY
Report Summary: EXCRETION
BROILERS
.
Study Numbers: 1515N-60-87-003, 18,027-97 and 17499-97
Test Material: Excreta from broilers medicated with radiolabeled semduramicin
Studies were conducted to assess the levels, mass balance and identity of products
excreted by poultry dosed with carbon-14 labeled semduramicin sodium.
Concentrations of Semduramlcin In Poultry Excreta.
A.
Thirty-seven day old broilers consumed feed containing 24 ppm C-14 radiolabeled
of
semduramicin sodium for 7 days. On days 4 through 7, excreta from 15 males and 14
females were collected and pooledby sex and date. Triplicate samples of each collection
were assayed for total radioactivity by combustion and scintillation counting.
Samples of the day 6 collection from males and of the day 7 collection from females were
extracted with solvents. The extracts were fractionated by high performance liquid
chromatography (HPLC), and the amount of radioactivity in each sample was measured.
The fractions obtainedfrom the excreta of male broilerswere also examined by the most
sensitive known test for a biological activity characteristic of polyether antibiotics, an in vitro
anticoccidial assay.
The balance between ingestion and excretion of radiolabeled material was calculated for
these two collections of excreta from the measured concentration of drug in feed (f),
measured feed intake (i), standard ratio of excreta to feed intake (r), and measured
concentration of total radioactivity in the excreta (e). This made it possible to calculate the
percentage of the ingested dose represented by each of the mostabundant HPLC fractions.
e = the determined concentration of total residues (radioactivity) in excreta.
r = the standard ratio of excreta to feed intake = 0.054 kg excreta per day for
consumption of 0.064 kg feedlday, (Reference2, this summary)
i = the measured feed intake.
f = the determined concentration of semduramicin in feed.
In the dataprovided:
e = 27 mg/kg semduramicin equivalents, the actual measured concentration of radioactivity
in excreta from female broilers on day 7 (Study: 1515N-60-87-003);
r= 0.054/0.064, a literature value (Reference 2, this summary);
i= 0.142 kg of feed (mean measured value of feed consumed by 14 female broilers on
day 7 of study 1515N-60-87-003),and
24 mg/kg, the actual measured concentration for semduramicin (corrected for 97%
f=
recovery) in feed (Study: 1515N-60-87-003).
Accordingly, inserting these values in the above equation shows 95% of the dose
ingested was found in excreta of female broilers during the seventh dayof medication.
[(27) x (0.054/0.064) x (0.142) x 1001
= 95%
[(24) x (0.1 42)]
A similar calculation formale broilers revealed 105%of the ingested dosewas excreted on
day 6 of the study by male broilers. In this case, the measured concentration in excreta (e)
on day 6 for male birds is 29.8 mg/kg (Study: 1515N-60-87-003), and (i) measured feed
the
intake for 15 male broilers is 0.165 kg, (Study: 1515N-60-87-003). Thus the percent of dose
ingested =
[(29.8) x (0.054/0.64) x (0.1 65)x 100
= 105%
[(24) x (0.1 65)]
Thus an excellent mass balance the dose ingested was calculated for
for male and female
broilers in the semduramicin study.
Separately, small samplesof the most abundant metabolites were isolatedfrom the bile of
medicated broilers for structure identification by mass spectral and nuclear magnetic
resonance analysis.
Summary of Results:
The following levels of radioactive material (in ppm) were measuredin the eight separate
collections of excreta:
24-Hour Collection Females
Males Mean
Day 4 19.1 29.8 24.5
19.4 16.6 22.2
Day 5
6 26.6 23.3 29.8
Day
1.
87 27.0-
22.9
Day 7
23.4 Mean 21.5 25.1
The HPLC profile of extracts from excreta is illustrated in the accompanying figure. The most
prominent HPLC peaks, in order of increasing polarity, were as follows:
of Ingested Dose
oo
/
I
HPLC Peak Females Males
Semduramicin 3.7 7.8
4.0 4.8
C
E 5.9 7.8
10.7 11.5
F
Figure 1. HPLC-LSC profile obtained ffom pooled extract derived from the 24-hour collection
and last day of 14 female broilers fed carbon-14 labeled senlduramicin sodium in feed at 25
ppm for 7 days. Experiment: 1515N-60-87-003.
F
Scrndurnmicin
Sodium
E
I-JPLC Retention Time
The abundance of unchanged semduramicin shown above translates intoconcentrations of 2.1
ppm and 1.1 ppm in the excreta of female and male broilers, respectively. These results in 6-
week old broilersagree with the level of 1.6 ppm of unchanged semduramicin measured by a
.
specific assay in the excreta of 2-week old medicated broilers (Report 17499-97).
The remainder of the radiolabeled material in excreta consisted of an array of polar metabolites.
Peak F was the only metabolite that exceeded 10% of the ingested dose. It and peak E were
shown to represent 0-desmethyl metabolitesof semduramicin. Neither E nor F was active in
the sensitive in vitro assay; this is consistent with the greatly reduced biological activity in
several tests, including the in vitro anticoccidial assay, that results from 0-demethylation of the,
polyether ionophore monensin (Reference 1, this summary). Peak C, representing about 4-5%
of the ingested dose, was the only component of the extracts other than unchanged
semduramicin that was active in the in vitro assay. Its structure could not be established with
certainty, but probably corresponds to ring-opening of the terminal cyclic hemiketal, followed by
reduction of the resulting ketone. A similar metabolite of monensin has been described
(Reference 3, this summary) and was reported to be about half as active as monensin in a
microbiological assay.
B. Mass Balance In Semduramicln Excretion Studies in Broilers
A mass balance of radiolabeled material ingested and excreted by medicated broilers is
calculated below for both excreta collections from experiment 1515N-60-87-003that were used
to obtain a chromatographic profile of excreted metabolites.
1) Female Broilers
The percentage of the excreted dose represented by various peaks in the HPLC was
determined in excreta collected during the last day of dosing from 14female broilers which
for 7 days consumed feed medicated with radiolabeled semduramicin.
of
Determination of the actual amount semduramicin inqested and excreted
Females housed in two separate pens consumed feed medicated with radiolabeled
semduramicin for 7 days. During the seventh day, they consumed 0.93 kg of feed in one
pen and 1.06 kg in the other, or an average of 0.995 kg per pen. Each pen contained 7
broilers, so on average each broiler consumed 0.142 kg feed:
of
0.995 kg/pen+. 7 broilers/ pen = 0.142 kghroiler
The intended level of semduramicin in feed was 25 ppm; the actual measured level was 23 .
ppm. The mean recovery in the assay procedure is 97%. When the measured level of 23
ppm iscorrected for 97% recovery the assay, a level of 24 ppm in feed obtained:
in is
23 ppm f- 0.97 = 24 ppm
Therefore, the broilers in these pens ingested an average of 3.41 mg of semduramicin on
day 7:
. 0.142 kghroiler x 24 mg/kg = 3.41 mghroiler
Total Amount of Druq and Metabolites Excreted
The total amount of waste excreted was estimated from a formula which related feed
consumed to waste excreted. During growout, a broiler consumes an average of 0.064kg
feed/day and excretes 0.054 kg of raw waste/day (Reference 2, this summary). Applying
these relative proportions of consumed feed and excreted waste to thesemduramicin study
leads to an estimate of 0.120 kg of raw waste excreted on day 7:
0.142 kg feedlbroiler x 0.054 kg excreta/0.064 kg feed = 0.120 kg excreta/broiler
The measured concentration of radioactivity in excreta collected from female broilers on day
7 was 27.0 ppm of semduramicin equivalents (Study: 1515N-60-87-003). Therefore, the
total amount of radiolabeled material excreted by each broiler onday 7 was 3.24 mg of
semduramicin equivalents:
0.120 kghroiler x 27 mg/kg = 3.24 mghroiler
Mass Balance of RadiolabeledMaterial
Of the 3.41 mg ingested by each broiler on day 7, 3.24 mg (95%) can be accounted for by
radioactivity measured in excreta.
2) Male Broilers
Radioactivity and in vitro anticoccidial activity were determined for HPLC fractions obtained
from material excreted by 15 male broilers which consumed medicated feed for 6 days.
Only two fractions were found to possess anticoccidial activity: unchanged semduramicin
sodium and a slightly more polar material. The relative amounts of this more polar material
and unchanged drug were 6.4% and 4.9%, respectively, a ratio of 1.3:l .O. Even though it
was present in a larger amount, the polar metabolite was less potent i n vitro than
semduramicin sodium.
Feed consumption on day 6 (Study: 1515N-60-87-003):
Pen 1 M: 1.14 kg + 7 broilers = 0.1 63 kglbroiler
Pen 2 M: 1.33 kg + 8 broilers = 0.1 66 kglbroiler
Average: 0.1 65 kgbroiler
Drug level infeed: 24 mg/kg
Amount of drug ingested on day 6:
24 mg/kg x 0.165 kghroiler = 3.96 mg/broiler
Amount of excreta produced on day 6:
0.165 kg/broiler x 0.054 kg/0.064 kg = 0.139 kghroiler
Measured concihtration of radioactivity in excreta collected from male broilers on day 6 (Study
1515N-60-87-003):
29.8 mglkg
Amount of radiolabeled material in day 6 excreta:
29.8 mg/kg x 0.139 kghroiler = 4.14 mg/broiler
Mass balance: 4.1 4 mg + 3.96 mg x 100 = 105%
3) Conclusion: An excellent mass balance can be calculated for excreta that were used to
investigate the abundance and polarityof metabolites.
Identification of residues in poultry excreta and comparative metabolism with the
C.
dog and rat.
Comparative radiotracer metabolism studies were conducted with carbon-1 4 labeled
semduramicin sodium in poultry and laboratoryspecies to elucidate its biotransformation among
species, and to provide data supporting the use of this anticoccidial compound in broiler
chickens.
These metabolism studies complement earlierradiotracer studies that defined the target tissue,
and the quantitative assessmentand depletion of total and marker residues in edible tissues
and excreta of broilers that were fed and withdrawn from carbon-14 labeled semduramicin
sodium under projected use conditions.
In comparative metabolite identification studies, liver, bile and excreta were obtained from
poultry fed 14C-semduramicin sodium in feed at projected use level of 25 ppm for 7 to 11 days.
The rat and dog were dosed by aqueous gavage with 14C-semduramicin sodium at 1
mg/kg/day SID x 5, and samples of liver recoverd fromeach species at sacrifice.
As defined by HPLC, liver of poultry sacrified six hours after withdrawal, is comprised mainlyof
unchanged semduramicin sodium (-45%) and an array of more polar, low-level ( ~ 0 . 1 ppm)
metabolites. None of these metabolites represent more than 10 percent of total radioactivity.
HPLC peaks corresponding to these metabolites aredetected in the liver of the rat and the dog
receiving aqueous oral gavagesof the drug, tentatively confirming exposure of the toxicology
species to the metabolites of semduramicin sodium in the use animal target tissue. Several of
these metabolites are also found inbile and excreta of the chicken at levels sufficient for
identification. When isolated from chicken bile two metabolites were tentatively identified by
FAB mass spectrometry as the A-ring 0-desmthyl, and the G-ring 0-desmethyl compounds. A
third and lower abundant metabolitewas tentatively identified by FAB MS as a ring-opened
product of semduramicin. The mass spectral assignments of the desmethyl compounds were
confirmed by proton NMR.
REFERENCES
Donoho, A.L. 1984. J. An.Sci.58:1528-1539.
1.
Food and Drug Administration, 1986. Finding of No Significant Impact of Selenium
2.
Supplementation of Animal Feeds. FAP 2201. Food and Drug Administration,
Washington, DC, p. 5.
M. Delort, G. Jeminet and G.Dauphin. 1990. J. Antib. 43:1189-1191.
3. Vaufrey, F.,A.
.
Appendix c-2
Physical-chemical Propertiesof Semduramicin Sodium
Report Summary: PHYSICO-CHEMICAL PROPERTIES OF SEMDURAMICIN SODIUM
Solubllity in Aqueous Buffers. The solubility ofsemduramicin sodium was determined by
stirring 50 mi v6lumes of pH 6,7, and 9 acetate or ammonium aqueous buffers maintained at
25 2 1掳C containing an excess (210 mg)of crystalline semduramicin sodium. Samples (4 ml)
were removed periodically, filtered, and assayed by HPLCuntil a constant solubility value was
reached. The pH values remained constant over the course of the experiment. There were
three to five replicates at each pH. Solubility results are summarized in Table 1.
It was observed that the solubility of semduramicin sodium is essentially constant over thepH
range six to nine. There was no evidence of aggregation or supersaturation. The solubility of
semduramicin sodium was reduced upon addition ofsodium ions (common ioneffect) and
enhanced upon addition of calcium or magnesium ions.
Solubility results were not obtained at pH 5 due to instability of semduramicin at low pH over a
long period of time; however, below pH 5 the solubility of semduramicin sodium falls due to
off
the intrinsic solubility (about 0.06 mg/mL)of the free acid form (pKa is 4.2).
Dissociation Constant. The dissociation constant(pKa) of semduramicin was determined by
potentiometric titration, in quintupicate, of approximately 6.25 mM solutions [200 mg/40mi] of
semduramicin sodium dissolved in 1:1 acetonewater using 0.5N hydrochloric acid as titrant at
23 2 2掳C. The theoretical nei!tral equivalent was obtained. ThepKa value was computed from
the standard relationship, pH=pKa at the half titrated point. An apparent pKa value 5.39 2
of
0.01 was obtained. From this result, the pKa in aqueous solution was estimated as 4.2. This
value is reasonable, considering semduramicin as a substituted acetic acid. An example
titration curve is provided in Figure 1.
Ultravlolet-Visible Absorption Spectrum. The absorption spectra of 0.1 mg/mL solution of
semdurarnicin sodium in 1:l methanol:pH bufferwere determined in triplicate at5 , 7 and 9.
There were no detectable absorption bands (withan extinction coefficient exceeding 25
Umole-cm) over the wavelength rangeof 290 to 800 nm. Absence of absorption is expected,
based on the structure of semduramicin, whichdoes not contain any common chromophoric
groups such as double bonds, conjugatedK systems, etc.
Melting Temperature. A capillary tube containing semduramicin sodium and anothertube
containing a melting point reference standardwere placed into the heating well a melting
of
point apparatus. The temperature was raised at aconstant rate (l"C/min) and the
temperatures were noted at which changes wereobserved in either material. The
determination were carried out in quadruplicate for the sample and in triplicate for the standard.
The average melting temperature for semduramicinsodium was 170掳C; the replicates gave a
range of 168 to 170掳C. The average melting point for the standard was 173"C, which fell within
the manufacturer's specification (ThomasStandard E, 172.5-17323掳C).Since the melting point
of semduramicin is 17OoC,no change of state is expected for the compound duringits
transport, storage, use or disposal.
Thermogravlmetric Analysis. A sample of semduramicin sodium washeated (30"C/min)
under nitrogen in a commercial, Perkin-Elmer TGA instrument, whichcontinuously and
accurately monitors the weight of the sample. The sample was run in triplicate. The samples
exhibited a small weight loss corresponding to water contentto a temperatureof approximately
150掳C. Beginntng at about 162掳C there was a rapid loss of weight caused bythermal
degradation of semduramicin to volatile decomposition products. The TGAplot is shown in
Figure 2.
These results indicate that semduramicin sodium has very low vapor pressure and is non-
volatile, as expected for a salt of high molecular weight. Additional thermo ravimetric data
were obtained which confirm that the vapor pressure must be less than10-3 torr at 20掳C. One
?
gram samples of semduramicin sodium and pyrene (for which the vapor pressure has been
reported as 7x1V7 torr at 20掳C) were examined for weightloss at a severechallenge
condition of 100掳C for 24 hours undervacuum. The semduramicin sodium sample did not lose
any significant weightbeyond solvated water, but the pyrene sample was nearly completely
volatilized.
Table 1. Solubility Results of Semduramlcln Sodium
Sarhpling
Time Solubility (mq
semduramicin/mlf
pH=6 pH=9
pH=7
(hours)
0.5 1.32 1.69 1.83
1 1.29 1.73 1.83*
3
1.86. 1.70 1.38
6 1.40 1.69 1.84.
24 1.44 1.93: 1.75.
1 .924 1.97
96
120 1.96
1 .954
144
168 1.95.
1.77*
193
216 1.67:
240 1.68
1.9 1.7 1.9
Solubility
Values averaged to calculate the solubility
Figure 1, Potentlometric titration plot for semduramlcin sodium.
.
1
Figure 2. Therrnogravimetric plot of sernduramicin sodium
Appendix c-3
The Octanol-water Partition Coefficient of Semduramicin Sodium
Report Summary: THEOCTANOL-WATERPARTITIONCOEFFICIENTOF
SEMDURAMICIN SODIUM
.
Study Number: 2438-1287-61 14-705
Two-phase solvent system
Test System:
Summary of Experimental Design: Solutions of radiolabelled semduramicin sodium were
prepared at 6 ~ 1 0and 7 ~ 1 0molar concentrations inpH 6, 7, and 9 aqueous buffers. Each
-~ -~
combination of pH and concentration was prepared in triplicate. (Solutions in pH 5 buffer were
not studied becausethey were known to be unstable.) A 40 ml volume of each solution was
shaken gently for two hours at 25 1掳C in a centrifuge tube with 2 ml of n-octanol. It had been
determined in a preliminary experiment that this length of time was sufficient to attain
equilibrium.
The phases were then separatedby centrifugation and the amount of radiolabel present in each
phase was determined by liquid scintillation counting of aliquots. The radiocount for each
aliquot was divided by the volume of the aliquot, and the resulting radiocount per unit volume of
the phase was divided by the specific radioactivity of the test material to obtain the final
concentration of semduramicin sodium in the phase.
The partition coefficient ( O) for semduramicin sodium in each system was calculated by
K!
dividing the final concentratlon of semduramicin sodium in the octanol phase by its final
concentration in the aqueous buffer phase. The partition coefficient was converted to its
logarithm, log Kow.
The radiometric mass balance was checked by multiplying the radiocount per unitvolume of
the volume of the phase, summing the resulting total radiocount per phase for
each phase by
the two phases of each system, and dividing the totalby the amount of radioactivity originally
added to the system in the buffer solution. The result was expressed as a percentage.
Summary of Results: The table below lists the mean values of the final concentration in the
octanol phase, final concentration in the aqueous phase, partition coefficient, logarithm of
partition coefficient, and percent radiolabel recovered, for each combination of pH and initial
concentration in the aqueous buffer. Semduramicin demonstrated aslight preference for the n-
octanol phase at all three pH鈥檚. Based on the low Kow of less than 3, significant accumulation
of semduramicin in aquatic organismsis not prediged.
Concentration (mg/ml)
- %Recovery
kq Kow
Kow
pH
initial Water
Octanol
6 6.3~1 3.3~10-~
0-4 0.010 2.47k0.01
291.60k2.27 96.37k0.51
6 54x10-3 0.097 19x10-4 2.70k0.01
499.49k10.22 98.87k1.64
7 6.3~1 0.009 5 . 410-5
0-4 ~ 2.24k0.02
172.38k9.83 97.18k1.75
7 54x10-3 0.092 2.9~10-~ 326.53+9.78 2.51+0.01 97.54k2.18
9 6.3~1 0.009 7.6~1 0-5 2.08k0.01
0-4 119.15k3.48 98.0351.14
54x10-3 40x10-4
0.089 2.35k0.01
9 222.24k7.69 95.84k1.35
Page 60
.
Appendix c-4
Soil Sorption and Desorption of Serndurarnicin Sodium
Report Summary: SOIL SORPTION AND DESORPTION OF SEMDURAMICIN SODIUM
Study Number: 2438-6115
Test System: Three types of soil in contact with aqueous solutions.
Summary of Experimental Design: The same general procedure was used to conduct a
screening test, a soil kineticstest, and an isotherm determination. All tests were conducted in
triplicate. An Iowa Sandy Loam, an Arkansas Silty Loam, and a Mississippi Silty Clay Loam
were used inthe isotherm determination. The characteristics of these soils are shown in Table
1.
To study sorption, samples of each soil were shaken in capped centrifugetubes with solutions
of radiolabelled semduramicin sodium in 0.01 M aqueous calcium chloride. The ratio of solution
to soil was 5:l in the screening test and 5:l or 20:l in the soil kinetics and isotherm tests. For
every combination of soil type and initial concentration, the concentration remaining in the
aqueous phase (Ce) was determined by radioassay, andthe amount sorbed onto soil (x) was
calculated from the differencebetween the initial andfinal concentration in the aqueous phases.
To study desorption, soilsamples containing sorbed semdurarnicin sodium were equilibrated
twice in succession with fresh 0.01 M aqueous calcium chloride, and the concentrations in the
aqueous phases were again determined by radioassay. In the screening test,a separate set of
sorption and desorption experiments was carried out with deionized water as the aqueous
vehicle.
Calculations: The logarithm of the experimentally determined equilibrium concentration, log Ce,
was plotted against log (x/m) for each soil, where x/m is the concentration in the soil. The
points on the graph were fitted to a logarithmic transformation of the Freundlich isotherm
equation:
where Kd is the Freundlich sorption coefficient and / n , an empirical constant, is the slope of
l
the graph. Log Kd was read off the graph as the intercept. The antilog, Kd, was then
calculated and converted to K , the sorption constant adjusted for the organic carbon content
O
of the soil, according to the equation:
KO, = (Kd x 100)1?/~organic carbon
For the two soils that were studied at a ratio of solution to soil of 20:l (Arkansas and
Mississippi), the percent of the initially addedsernduramicin sodium that would be sorbed from
aqueous solution onto each of the soils at a solution ratio of 5:l was calculated from the Kd
values determined for sorption the isotherm test:
in
+ 5 ) )x 100
% sorbed = [Q/(Q
Similarly, the percent of the semduramicin sodium sorbed onto soil that would be desorbed
when the soil is exposed to fresh 0.01 M calcium chloride solution, at aso1ution:soil ratio of 5:1,
was calculated from the Q values determined for desorption:
. % desorbed = [5(Kd + 5)]X 100
Summary of Results: The results of the screening test showed that calcium chloride did not
significantly interferewith the sorption or desorption of semduramicin as compared to distilled
water. Semduramicin readily sorbed ( ~ 2 5 %and did not readily desorb(>75% of the fraction
)
sorbed), indicating that an advanced test should be conducted. Equilibrium was attained at
approximately 72 hours for all three soil types.
The results of the isotherm test confirmed that sorption of semduramicin sodium in soil is
the
moderately strong though reversible (Tables 2,3, and 4). As shown below, the value of Kd, the
Freundlich sorption coefficient, in these three soils ranged from 4.65 to 25.7 for sorption and
from 2.41 to 16.2 for desorption. The corresponding ranges ofKO, were 150 to 1,750 and 79 to
1,100, respectively. Compounds having a KO, value of 1,000 or larger are considered relatively
immobile in soil and have a low potential for leaching into the water table or into runoff water.
Thus, semduramicin sodium would be expected to be relatively immobile intwo of the three
soils (MS STCYLM and AR SYLM) and to be only moderately mobile even in a soil that is
predominantly sand (IA SDLM).
Desorption Sorption
Kd
Kd
Soil Type K,
O 1In
1In
1750 0.949 1100
25.7 16.2 1.016
MS STCYLM
1400 0.843 0.798
AR STLM 10.6 6.35 840
150 0.824 2.41 79
4.65 0.765
IA SDLM
It was calculated that a so1ution:soil ratio of 5:l from 48 to 84% of the initially added
sernduramicin sodium will be sorbedonto these soilsfrom the aqueous solution, and 24 - 67%
will be desorbed upon subsequent exposureof the soils to freshcalcium chloride solution.
SOIL TYPE SORBED DESORBED
Kdes
KS '/o
Oo
/
24 16.2
84 25.7
MS STCYLM
68 10.6 6.35
AR STLM 44
48 4.65
2.41 67
IA SDLM
Table 1. Characterization of the Soils Used in the Sorption/Desorption Coefficient
Determination
. Mississippi
Arkansas
Iowa
Source
Silty Loam Silty Clay Loam
Sandy Loam
Texture
72.0
16.0
YoSand 53.2
58.0 50.0
37.6
Silt
o/
o
28.O
26.0
YoClay 9.2
YoOrganic 1.3 2.5
5.2
Matter
6.2 7.7
5.6
PH
Cation
6.0 12.2
12.5
Exchange
Capacity
(meq/lOOg)
Field
o/
o
24.97
16.9
Moisture 23.9
Capacity
Table 2. Isotherm Test: Concentrations of Semduramicin in Soil and in Solution
Mean of three values and standard deviation
. Soil type/concentration .
Mean
Measured
Initial
MISSISSIPPI SILW CLAY LOAM
IOWA SANDY LOAM ARKANSAS SILTY LOAM
Concn.
Aqueous,uglmlSoil,ug/g Aqueous,
ug/ml Soil, ug/g
Aqueous,ug/ml Soil, ug/g
SORPTION STUDY
5.62
Mean
107.9 4.21 59.13 6.76 20.45
9.7
-__a
0.1 2
SD 0.59 0.26
0.21 5.23
2.66
Mean
52.45 2.00 24.55 3.40 9.83
4.6
___ a
__-a 0.14 2.71
0.07
SD 0.33
16.31
1.24 1.13 23.31
Mean 1.48
5.28
2.3
2.55 0.05
0.05 0.90
SD 0.1 3
0.23
Mean 0.44
0.37 0.93 6.82 0.59 2.46 11.32
_-_ --_a _--a
a ---a
SD 0.20 0.01
DESORPTION STUDY
Mean 3.1 62.14 6.91 17.60
54.27 2.75
9.7
_--a
_-_
a
SD 0.1 7
0.03 0.22 7.49
Mean 1.70
1.22 2.71 4.74
21.97 1.57
4.6
_--a
-_-
a 1 2.1
0.04
SD 0.05 0.75
0.71 10.17
Mean 0.75 3.91
2.1 7 0.67
2.3
1.35
0.01
SD 0.90
0.05 0.03 0.06
5.46 Mean
0.93 0.31 2.380.31
0.23 1.05
--_a _-_
--_a a
0.66 0.03 0.94
SD
%ne or two samples lost due to broken centrifuge tubes, hence standard deviation not applicable.
Linear regression analysis of the Sorption Data usingtheFreundlich Isotherm
Table 3.
[log,, (x/m) = log,, (Kd) + l / n log,, (C,)] UK 61,689-2 with three soil types.
for
.
Soil Type
9.7 2.03 0.62 1.31 0.75 0.83 1.77
4.6
0.99 0.42 0.30 1.39
1.72 0.53
0.05 2.3 0.09 1.37
1.21 0.17 0.72
'
0.93 0.39 -0.36 1.05
0.83 -0.23
-0.43
0.988
Correlation: 0.989 0.998
0.843
Slope (1In): 0.949 0.824
Kd): 1.026
(Log
Int. 1.41 0.667
1.186
n 1.05 1.214
10.6 4.65
25.7
Kd
Yo Organic
0.76
1.47 Carbon' 3.06
1400 150
K, 1800
Yo Organic Carbon = o/o Organic Matterll.7.
..
Linear Regression analysis of the Desorption Data using the Freundlich Isotherm
Table 4. ,
[loglo (x/m) = log10 (Kd) + l / n loglo (Ce)] for UK 61,689-2 with three soil types.
. Soil Type
0.33
0.44 1.73
9.7 1.25 0.50 0.84
0.09
0.20 1.34 0.68 0.23
4.6 0.43
-0.17
-0.15 1.01 0.59 -0.1 2
2.3 0.34
0.38 -0.640.93 0.74 -0.51. -0.48 0.02
Correlation: 0.891
0.986 0.963
1.01 6 0.798
Slope (1In): 0.765
1.803 0.383
.1.253 1.307
6.35 2.41
Kd .16.2
Yod Organic
1.47Carbon' 0.76 3.06
'.
K79 1100 840
O,
Y Organic Carbon = Yo Organic MatterIl.7
o
+
.
Appendix c-5
Hydrolysis of Semduramicin Sodium
Report Summary: HYDROLYSIS OF SEMDURAMICIN SODIUM
Study Number: 2438-0189-6142-715
- Solutions in aqueous buffers
Test Systems:
Summary of Experimental Design:
Sterile solutions containing 10 ppm of semduramicin sodium in-pH5, 6, 7, 8 and 9 aqueous
buffers were prepared and three replicates of each solution were placed into glass
volumetric flasks. The flasks were fully covered with aluminum foil to shield them from light
and placed in a shaker water bath. In a preliminary experiment, this bath was held at 49.8 2
0.4OC; in the definitive experiment, it was maintained at a constant temperature of 25.0
0.2OC. An aliquot of each replicate at each pH was analyzed by a high pressure liquid
chromatography assay specific for semduramicin sodium at the initiation of the experiment
and then at periodic intervals. The logarithms of the observed concentrations at each pH
were plotted as a function of time and correlated with time by linear regression analysis
according to the equation for first-order reaction kinetics:
log C = - (W2.30)t + log CO
Where C is the concentration at time t, Co is the initial concentration, and k is the rate
constant. The half-life (t1/2) of sernduramicin sodium was thencalculated from the equation:
t1/2 = In 2 k
In addition, at initiation. and near termination of the definitive experiment each solution was
tested for microbiological activity against Baci-lfus stearothermophifus by the Kirby-Bauer
Method. Zones of inhibitation that surrounded discs containing either semduramicin sodium
in buffered solution or penicillin G were compared following incubation of plates at 65" for 2
hours and 45 minutes.
Summary of Results:
The following rate constants and half-lives were calculated from the preliminary experiment
conducted at 50掳C:
-')
pH Rate Constant (days Half-life (days)
2.1 1
0.328
4.94
0.140
10.9
0.0637
21 .o
0.0330
11.3
0.0615
The corresponding results from the definitive test at 25掳C are as follows:
-'
pH Rate Constant (days Half-life
(days)
)
11.1
6.23 x
5-
1.93 x 1o-2 36.1
6
89.9
7.85 x
7
6.02 x 10-3 115.0
8
1.oo x 1o-2 77.1
9
Mean differences in zone sizes between semduramicin sodium and the penicillin G standard
for each pH at the initiation and conclusion of the definitive test are as follows:
(mm) 4.4 6.1 6.5 7.5 7.0
Difference at initiation
-0.4 4.3 3.1 6.2 6.0
Difference on day 23 (mm)
The hydrolysis study supports an observation that UK-61,689-2 is hydrolyzed rapidly at pH 5
and pH 6. The microbial inhibition dataindicate that the inhibitory activity of UK-61,689-2
was lower than the inhibitory effect of penicillin at pH 5 toward the target microbe. At the
or
other pHs tested, the inhibitory effect of UK-61,689-2 was greater than equal to the
inhibitory effect of penicillin toward the target organism. While the data provide anindication
that semduramicin reacts under the conditions of the test, a more extensive study would be
required to determine the extent of degradation.
.
Appendix c-6
Photodegradation of Semduramicin Sodium in Aqueous Solution
Report Summary: PHOTODEGRADATIONOF SEMDURAMICIN IN AQUEOUS SOLUTION
Study Number: 2438-0488-6125-720
Test System: Irradiation of Aqueous Solutions with Simulated Sunlight.
Summary of Experimental Design:
Solutions containing 10 ppm of semduramicin sodium in pH 6, 7, and 9 buffers were prepared.
Samples of each buffered solution were used to fill four sets of 13 X 100 mm quartz glass
tubes. Three of the sets were saturated with air, and one of these was shielded from light by
covering the tubeswith aluminum foil. The fourth set was saturated with nitrogen and covered
with foil. All the tubes were placed in aninstrument that simulates natural sunlight by means of
a Xenon arc lamp and appropriate filters. A fifth set filled with a solution containing an
and 1.5 x M pyridine, was
actinometer (reference material), M p-nitroacetophenone
also placed in the instrument. Irradiation was started, the temperature was maintained at 34"C,
and a sample from each set was analyzed by HPLC for semduramicin sodium or p-
nitroacetophenone at initiation and at periodic intervals thereafter. At initiation and termination,
a sample of each set containing semduramicin sodium was also assayed microbiologically. For
each pH, the ratio of the concentration of semduramicin, C, to the initial concentration, Co was
plotted versus time, t, according to the equation for first order decomposition.
In C/Co=-kt
The slope of the line, k, was obtained from the graph and used tocalculate the half-life, t 1/2,
according to the relation:
t 112 = In 2/k
Summary of Results:
The concentration of semduramicin sodium in the irradiated samples declined fairly rapidly,
more so at lower than at higher pH:
Measured Concentration (mg/L) of UK-61689-2and p-nitroacetophenone (PNAP).
1.64
9.94 10.0
0 9.30
7.82 1.48
6.1 4 7.81
21
1.41
7.65 8.1 2
38 6.39
1.34
4.42 6.03 6.1 6
48
1.31
5.72
3.63 6.59
66
4.77 1.24
5.69
4.59
90
1.10
2.69 3.78 5.58
136
5.1 7 1.15
3.40
166 3.03
2.37 3.56 3.54 1.08
187
Values for UK-61,689-2 represent a mean of three replicates on day and amean of two
0
replicates for all subsequent sampling intervals. Values for PNAP are for a single unaerated
replicate.
The following rate constants and half-liveswere determined; thehalf-livesdetermined under
continuous irradiation were multiplied by two to obtain the half-livesin days with 12-hour
exposure to light:
pH Rate Constant (lhours) 12-hour exposure/day)
Half-Life(days)
Half-Life
(Days,
7.10
3.55
x 10-3
8.14
6
8.76
7 6.59 X 10-3 4.38
1 1.32
5.66
5.10
9 X 10-3
PNAP 11.3
2.55 X 10-3 22.6
The concentration in the aerated samples kept in the dark also declined, but much more slowly
than in the irradiated samples. The concentration in the nitrogenated sampleskept in the dark
.
declined even more slowly. The reference material decomposed at the expected rate.
The microbiological activity of the irradiated solutions of semduramicin sodium was markedly
lower at termination than at initiation for all three pH's, indicating that activity against
the
indicator organism was lost as semduramicin sodium decomposed. In a disc assay, ateach
pH, the initial solution produced a larger zone of inhibition than a fixed concentration of a
reference standard at the same pH. The decline in the activity of the irradiatedsolutions
manifested itself as a reduced diameterof their zones of inhibition and consequently, a smaller
difference from the reference standard.
- -
-
6 9
pH 7
Difference at initiation
4.9 5.1 4.6
(mm)
(mm) 187 after
Difference
hours. 0.6
0 0.5
Semduramicin sodium was found to undergo photodegradation in aqueous solution even
though if does not show detectable absorption in the UVhisible spectrum. This appears to be a
case of photo-induced degradation, a type of indirect photolysis in which a reactive chemical
species formed by the action of light reacts chemically with a compound, which does not itself
absorb light, so as to decompose it (Choudry, G.G. and G.R.B. Webster. 1985. Protocol
guidelines for the investigations of photochemical fate of pesticides in water, air, and soils. I.
Introduction. Residue Reviews 96:80-81). While there is evidence that oxidant species such as
singlet oxygen have been reported in a variety of natural waters, there is no direct evidence that
these oxidatively degrade organic pollutentsin natural waters. Due to the lack of absorption of
semduramicin, it was not possible to calculate a quantum yield.
.
Appendix c-7
Biodegradation of Semduramicin Sodium in Soil
Report
Summary: AEROBICBIODEGRADATION OFSEMDURAMICINSODIUM IN
SOIL
.
Number:
Study SC910074
4C semduramicin sodium admixed with
soils at 25 ppm.
Test System:
Summary of Experimental Desiqn:
Characteristics of3 soils employed in the studyare as follows:
Field
Soil Identification Cation Exchange
Organic
Moisture Texture ( / )
?.
Capacity (meq/l OOg) Matter (Yo) pH Capacity (/)
(Location) ". Sand Silt Clay
Loam Clay 2.6 9.3 24.93 5.7 24 48 28
(West Jefferson,OH)
28.6 Clay 5 -6
41.80 6.4 22 38 40
(Casselton, ND)
25.8LoamClay
Silty 3.9 31.83 7.3 10 56 34
(Iowa)
Three treatments were employed: 1) 14C semduramicinat a final concentration of 25 ppm in
soil (5.5 x l o 6 DPM activity), 2) glucose (a combination of 14C and unlabeled) at a final
concentrations of 10 mg C/50 g soil (6.03 x 1O6 DPM activity), 3) untreated control. Each
treatment was evaluated in triplicate for each of the3 soils. A series of 27 incubation flasks,
each containing 50 g of soil, were arranged in a system modified from Marinucci and Bartha
(Apparatus for monitoring the mineralization of volatile 14C-labelled compounds. Appl.
Environ. Microbiol. 38: 1020-1 022) for trapping14C02 and where appropriate, organic
volatiles. Flasks were incubated in the dark at 22k3"C. The amount of radiolabeled carbon
dioxide in the traps was measured periodically by liquid scintillation counting. All treatments
were monitored for 66 days; two of the soils (Ohio and North Dakota) were monitored an
additional 28 days (94 days total)because C 0 2 production in the semduramicin treatment
had not plateaued by day 66.
Material balance was computed for the glucose and semduramicin treatments at the end of
the incubation period. Ohio and North Dakota soils containing semduramicinwere extracted
with organic solvents followed by base and acid hydrolysis to recover as much radioactivity
from the soils as possible. Methylene chloride and ethylacetate extracts were analyzed by
high pressure liquid chromatography (HPLC) and thin layer chromatography (TLC) to
determine the relative proportions of unchanged drug and metabolites.
Summary of Results:
The semduramicin treatment demonstrated significant degradation to C 0 2 with 50%
biodegradation in approximately 94 and 42 days,respectively, for Ohio and Iowa soils, and
40% degradation for North Dakota soil in approximately 94 days.
The glucose treatment demonstratedrapid degradation to C 0 2 in all three soils with 50%
biodegradation achieved in approximately 7, 28, and 14 days, respectively, for Ohio, North
Dakota and Iowa soils.
For semduramicin, the estimated (vs experimentally measured) time to 50% biodegradation
for Ohio, Iowa, and NorthDakota soils was 79,42 and 104 days,respectively:
to Days 50%
oo Dearaded
/
Type
Soil 14
'Day Day Day
66 94 Biodegradation
1.oo ----
Loam Clay 51.95 79
----
1.76 Clay 104 40.30
----
63.38 1.41 Loam Clay Silty 42
The untreated control demonstrated noC 0 2 evolution in any of the soils. No appreciable
quantities of organic volatiles (-0.2%) were trapped during the test period from any
treatment.
At the termination of the experiment, material balance achieved for the glucose treatment
was 92% (66.5% degraded, 25.3% bound to soil), 93% (58.5% degraded, 34.7% bound to
bound to soil), respectively, for Ohio, North Dakota,
soil), and 94% (61.6% degraded, 31 -7%
and Iowa soils. For the semduramicin treatment, it was 95% (52.0% degraded, 10.8
extracted by methylene chloride and 32.1% bound to soil); 98% (40.3% degraded, 19.3%
extracted by methylene chloride and38.0% bound to soil), and 97% (63.5% degraded, 4.9%
extracted by methylene chloride and 28.5% bound to soil), respectively, for the same three
soils as follows:
Clay Loam Silty Clay Loam
Clay
(West Jefferson,OH) (Casselton, ND) (Iowa)
1% glucose (66 days)
(94 days) (94 days)
61.61
66.46 58.55
degraded
o/
o
0.14 0.36
Methylene chloride extract YO 0.13
31.70
25.30 34.70
Bound to soil (Yo)
94M.66
92kl 93k1.95
Mass balance
4C semduramicin
51.95 40.30 63.38
o degraded
/
o
19.3 10.8 4.9
Methylene chloride extract o/o
28.5
38.0 32.1
Bound to soil ("Io)
98k1.02 95k1.95 97M.54
Mass balance .
The Ohio and NorthDakota soils inthe semduramicin treatment were monitored 94 days for
C 0 2 evolution. At trial termination, these soils contained 48 and 6O%, respectively, of the
total applied radioactivity. Mutlisolvent extractions, followed by base and acid hydrolysis
released 22% and 35% total applied radioactivity from the two respective soils. Methylene
chloride extractions of the two soils contained an average of 11 and 19% of total applied
radioactivity. Ethyl acetate extractions of the same soils contained an average of 6 and 12%
total appliea radioactivity. Both extractions wereanalyzed by HPLC to determine the relative
proportion of unchanged drug and metabolites. Methylene chloride extracts contained three
major components in Ohio clay loam and 3 to 5 components in North Dakota clay soil, with
the largest of the major components being semduramicin. No single component accounted
for more than 0.3% of the total applied radioactivity more than 0.05 ppm in either soil.
or
Ethyl acetate extracts contained 5 components each in Ohio and North Dakota soils. No
single component accountedfor more than 0.05% of the total applied radioactivity or more
than 0.01 1 ppm in either soil.
.
Appendix c-8
Effect of Semduramicin Sodium on Soil Microbes.
OF SEMDURAMICIN SODIUM ON SOIL MICROBES
ReDort Summary: EFFECT
Study Number:
2438-01 89-6143-795
Test Species: Soil-dwelling microbes
Summary of Experimental Desiqn:
The lowest concentrationsof semduramicin sodium that will inhibit the growth of pure cultures
of representative soil bacteria,ascomycetes, fungi, and blue-green algae were determined by
the agar plate dilution technique. The following organismswere used:
Clostridium novyi, a free-living nitrogen-fixing bacterium
Nostoc, a blue-green alga
Bacillus stearothermophilus and Flavobacterium meninxosepticum, soil bacteria
Trichoderma viride, an ascomycete
Penicillium italicum, a mold
Each of the above organisms was maintained inpure culture under conditions appropriate for
the species. A preliminary range-finding study was conducted at widely spaced concentrations,
approximately 1,000, 100, 10 and 1 ppm. The results were used to select a series of four
closely spaced concentrations.
In the definitive test, semduramicin sodium was introducedinto molten agarat concentrations of
0,100, 125, 145, 167 and 186 ppm, except that concentrations of 0,186, 205, 233, 240, and
256 ppm were used for Penicillium italicum. All concentrations were prepared in triplicate. Each
agar preparationwas then poured into a Petri dish, allowed to cool and solidify, inoculated with
one of the organisms, and incubated at an appropriate temperature. When colony growth was
well developed on the plates which did not contain any drug, the plates containing
semduramicin sodium were examined visually for microbial growth. All three replicates were
evaluated at each concentration, and if microbial growth was found to have been prevented in
one or more replicate, that concentration was recorded as inhibitory (I); if growth (more than a
single colony) was observed in all three replicates, the result was recorded as "growth" (G).
Summary of Results:
The recorded observations are shown the following table:
in
. Blank
Species125
100mg/L
mg/L
mg/L
Control
186
167
145
Control
G G
G G
I I I
Clostridium novyi
G
G G G G
I I
Bacillus stearothermophilus
G G G G G
I I
Flavobacterium meningosepticum
G I G G
I I I
Nostoc
G G G
G
I I I
Trichoderma viride
Blank
Solvent
186mg/L 205 mg/L 233 mg/L 240 mg/L 256 mg/L Control Control
Species
G G G G G
I I
Penicillium italicum
These observations indicate that the lowest concentration which inhibits growth, the minimum inhibitory
concentration (MIC), is:
145
Clostridium novyi
167
Bacillus stearorhermophilus
167
Flavobacterium meningosepticum
125
Nostoc
145
Trichoderma viride
240
Penicillium italicwn
.
Appendix c-9
Effect of Semduramicin Sodium on Seed Germinationand Root Elongation
Report Summary: EFFECT OF SEMDURAMICIN SODIUM ON SEED GERMINATION AND
ROOT ELONGATION
.
Study Number:
2438-1287-61 17-600
Test Species: species
Six of plant seeds
Summary of Experimental Design:
Seeds of the following species were used:
Monocotyledons:Loliumperenne L. - rye
Triticum aestivum L. - wheat
-mays L. - corn
Zea
L. - cucumber
Dicotyledons:
Cucumis
sativus
Glycine max L. - soybean
Phaseolus vulgaris L. - pinto bean
After soaking in distilled water, the seeds were placed on two sheets of filter paper saturated
with test solution and containedin a glass Petri dish, 50 seeds of a given species to a dish.
Preliminary tests were conducted with each species using nominal concentrations of
semduramicin sodium of 0.96, 9.6, 96 and 960 pprn. Based on the results of the preliminary
test, the following measured concentrationswere evaluated. The drug was dissolved in distilled
water and added to six replicate dishes:
Corn: 0,17,34,56 and 86 ppm
0,17,34,56 and 86 ppm
Cucumber:
Pinto bean: 0.6.3, 13, 16 and 31 ppm
0,4.0,6.3, 13, and 31 ppm
Rye:
0,0.36, 0.61,0.93, and 2.2 ppm
Soybean (germination):
0,17,34,56 and 86 ppm
Soybean (elongation):
0,4.0, 16, and 31 ppm
6.3,
Wheat:
The dishes were incubated at 3OoC and 100掳/o humidity for 3 to 4 days. Every day during the
test, the number of seeds thathad germinated was determined, and the seeds wereexamined
for any morphological abnormalities such as discoloration, swelling or lesions. Upon
termination of the exposure, the radicle (primary root) lengths of ten impartially selected,
germinated seeds from each dish were measured.
Summary of Results:
For soybean, test concentrations between 0.36 and 2.2 ppm had no effect upon germination;
however, because germination was reduced statistically in the preliminary test at 1 ppm, the
lowest concentration evaluated, a NOEC was not established. In the preliminary test, root
elongation was reduced statistically at 960 and 96 ppm but not at9.6 or 0.96 ppm. In the
definitive test, all concentrationstested (86-17 ppm) reduced root length statistically and a
NOEC was not established.
For other species tested, the highest concentrations at which no adverse effects on
germination, radicle length or appearance which were observed were as follows: for corn, 17
ppm; cucumber, 34 ppm; pinto bean, 6.3 ppm; rye, 13 ppm; and wheat, 6.3 ppm. Higher
concentrations inhibited germination and/orroot elongation.
.
No adverse effects were observed in any species on appearance such as discoloration,
swelling or lesions. For all species except soybean, the lowest concentration at which seed
germination or root elongation were inhibited (the LOEC) and the highest concentration that
had no statistically significant effect as the exposed seeds as compared to controls (the NOEC)
are summarized below:
Elongation Root Germination
LOEC~ NOEC~ LOEC~
NOEC~
Species
34 17b 34 56
Corn
86 56 34 56
Cucumber
13 6.3b 6.3
Pinto Bean 13
31
31 Rye 13
31 16 6.3 16
Wheat
SoybeanC
aNOEC and LOEC shown are measured concentrations (ppm)
bLowest concentration tested.
CNOEC and LOEC not established.
The following two tables show theeffects of various concentrations of semduramicin on
germination andon radicle length(root elongation):
Mean Number of Germinated Seeds and Percent Germination.of Plant Seeds Exposed
to Semduramicin Sodium in the Definitive Test.
Germination
Plant
Germination Data
Species Time (days)
Concentration Corn
(ppm): 4 86 56 34 17
0
48+1
46+2 42+2a40+4a
39+4a
No. germinated seeds:
95+2
93+4 84+478+8
81
+7
o germination:
o
/
Concentration
(ppm):
Cucumber
4 86 56 34 17
0
50+0 50+1 48+2
49+1 42+3a
No. germinated seeds:
100+0
99+1 97+3
98+2 85+5
oo germination:
/.
Concent:ation
(ppm): 3 Pinto 31 16 13 6.3
0
Bean No. germinated
43+3
seeds: 43+4 27+4a
27+4a
37+3a
. .
germination:
86+7
86+6 7 3 454+8
54+8
o/
o
.Concentration
(ppm): 3 Ryegrass 4.0 6.3 13 31
0
.
No. germinated seeds: 46+1 47+1
47+2 46+2 32+7a
germination: 9 1 94+3
94+3 9255 64+15
o/
o
..
2.2
0.93
0.610.36
0
Concentration
(ppm):
3
Soybean
22+10
17+6
25+3 33+9 29+5
No. germinated seeds:
44+2035+13 50+5 65+19 57+10
germination:
o/
o
4.0 6.3 . 16 31
0
Concentration Wheat
(ppm): 3
No. germinated seeds: 48+1 48+1 47+2 46+1 41+5a
germination: 96+1 96+2 9 4 4 93+2 82+11
o/
o
astatistically different (Pr0.05) from mean for the control.
Radicle Lengths (mm) of Six Plant Species After Exposure to Semduramicin in the Definitive
Test.
.
Measured Test Concentration (ppm)*
Specles Control 56 17 34 86
Corn (12) 29 (3) 28 26 (5) 18 (6)a 16 (4)a
8 (2)a
Cucumber 22 (6) 15 (3) (2) 17
12 (3)a
Control 6.3 13 16 31
Bean
Pinto 31 (4) 29 (3) 19 (7)a 14 (3)a 13 (3)a
Control 17 56 86
34
Soybean 17 (8)
25 (3)a 10 (2)a 9 (2)a 6 (l)a
Control 4 .O
16 6.3 31
0)
Wheat 43 (2)
47 (5) (1 40 35 (6)a (3)a33
Values shown are means for six replicates per test concentration with standard deviation in
parentheses.
a Treatment data were significantly different (P20.05) from the control data.
.
Appendix c-10
Effect of Semduramicin Sodium on Seedling Growth
EFFECT OF SEMDURAMICIN SODIUM ON SEEDLING GROWTH
Report Summary:
Study
Number: 2438-0288-6116-620
-
Test Species: Six species of plant seedlings
Summary of Experimental Design:
Seedlings of the following species were used:
Loliumperenne L. - rye
Monocotyledons:
Triticum aestivum L. - wheat
Zea mays L. - corn
sativus L. - cucumber
Dicotyledons: Cucumis
Glycine max L. - soybean
Phaseolus vulgaris L. - pinto bean
Groups of five sprouted seedlings grown on-site from seed were transplanted into washed silica
sand held in plastic containers and housed in an environmentalchamber, where favorable
conditions for growth were maintained. A nutrient solution containing semduramicin sodium, or
nutrient solution devoid of drug, was added daily to each group of seedlings. Preliminary tests
were conducted with each speciesusing nominal concentrations of semduramicin sodium of
1.O,10, 100, and 1000 ppm. Based on the results of the preliminary test, the following
measured concentrations of semduramicin sodium were tested.
Corn: 0,0.77,2.2,4.2, and 7.0 ppm
Cucumber: 0, 0.23,0.31, 0.77, and 2.2 ppm
Pinto bean: 0,0.77,2.2,4.2, and 7.0 ppm
0, 0.31 0.77,2.2, and 4.2 ppm
Rye:
0, 0.23,0.31, 0.77, and 2.2 ppm
Soybean:
Wheat: 0,0.77,2.2,4.2 and 7.0 ppm
Each treatment was replicated 5 times. Seedling shoot lengths were measured on days1, 3, 5,
7, 14, and 21. At test termination, dry weights of the shoots and roots were measured
separately and any abnormal appearance was noted. The percent elongation of the shoots
was calculated from the equation:
% elongation = (length of treated tissue-length of control) (x100) t- (length of control).
Summary of Definitive Test Results:
Corn: The highest concentration at which no significant effects on plant survival, root weight,
shoot weight, shoot length, or appearance werenoted was 2.2 ppm. At 4.2 ppm, the number of
surviving seedlings was reduced and at 7.0 ppm, the average root weight was lower than in the
seedlings that were not exposed to semduramicin sodium. Shoot weight and shootlength were
not affected by the highest concentration tested, 7.0ppm.
Cucumber: The highest concentration at which no significant effects were observed was 0.77
ppm. At 2.2 ppm, both shoot weight and shoot length were reduced. Survival and root weight
were not affected by the highest level tested, 2.2 ppm.
Pinto Bean: The highest concentration at which no significant effects were observed was 0.77
ppm. At 2.2 ppm, root weight was reduced, and at 4.2 ppm, there were reductions in survival,
shoot length and shoot weight.
Ryegrass: The lowest concentration at which aneffect on root weight was still observed was
0.31 ppm. Survival, shoot length and shoot weight were reduced at2.2 ppm.
Soybean: The highest concentration at which no significant effects were observed was 0.31
ppm. At 0.77 ppm, shoot length was reduced. Survival, root weight and shoot weight were not
affected by the highest concentrationtested, 2.2 ppm.
Wheat: The concentration at which an effect was still observed on root weight was 0.77 ppm.
At 2.2 ppm, shoot weight and length were reduced and so was survival at 7.0 ppm. Survival
was not affected at 2.2 ppm and shoot length andweight were not affectedby 0.77 ppm.
The next three pages contain thefollowing information:
- The lowest concentration that had a statistically significant effect on a particular parameter
(the LOEC) and the highest concentration that had no statistically significant effect on the
exposed plants as compared tothe controls (the NOEC);
The mean shoot length of the seedlings after various periods of exposure;
-
- A summary of percent mortality, mean shoot and root weight, and observed abnormalities
in exposed seedlings.
LOEC and NOEC Values forAll Species of Plants Exposed toSemduramicin.
LOECa
. NOECa
Species
Corn
4.2 2.2
Mortality
7.0b 4.2
Root Weight
>7.Ob 7 .Ob
Shoot Weight
>7.Ob 7.0b
Shoot Length
Cucumber
>2.2b 2.2b
Mortality
>2.2b 2.2b
Root Weight
2.2b 0.77
Shoot Weight
2.2b 0.77
Shoot Length
Pinto Bean
4.2 2.2
Mortality
2.2 0.77
Root Weight
4.2 2.2
Shoot Weight
4.2 2.2
Shoot Length
Ryenlass
2.2 0.77
Mortality
~0.31~
0.31
Root Weight
2.2 0.77
Shoot Weight
2.2
Shoot Length 0.77
Soybean
>2.2b 2.2b
Mortality
>2.2b
Root Weight 2.2b
>2.2b
Shoot Weight 2.2b
0.77
Shoot Length 0.31
Wheat
4.2 2.2
Mortality
0.77c ~0.77~
Root Weight
2.2 0.77c
Shoot Weight
2.2 0.77c
Shoot Length
aBased on test concentrations (mg/kg) measured to 0-hour.
prior
bThe highest measured concentration UK-61,689-2 tested.
of
CThelowest measured concentrationof UK-61,689-2 tested.
Mean Shoot Lengths of Seedlings Exposed to Semduramicin
cm) Length Plant
DaylShoot Concen-
Species tration
3 1 (PPW 21 14 5 7
28.6d.8
19.0k2.1 23.9k3.0
11.5+2 43.8k5.1
Corn 38.5d.8
0
as) 25.2k6.3
15.6k3.2 20.9k4.6
9.6kl.2 34.0d.3 38.9k9.8
26.8k2.0
16.3k1.1 21.3k1.1
0.77 9.8k0.8 36.94.3 41.6k5.0
23.323.1
12.7k0.7 1 7.4k1.1
2.2 8.6k0.8 34.3d.2 39.2k2.6
14.2k2.0
10.3k1.7
9.3k0.7
4.2 10.0+0.9 34.3k0.2 35.9k1.6
15.1k7.3
11.3k5.0
9.3k1.5 9.8k1.6 27.2k4.0
7.0 26.0k5.7
5.1k0.4
4.4k0.4 4.9k0.5
4.1k0.5 6.4k0.5 10.2k0.6
Cucumber 0
5.4k0.6
4.5k0.6 4.8k0.6
4.1k0.5 6.2k0.5 9.4k0.6
OF)
5.6k0.2
4.8k0.3 5.2k0.3 10.3k0.9
0.23 4.5k0.4 6.6k0.4
5.2k0.5 5.7k0.5
0.31 4.3k0.4 4.750.5 6.5k0.3 10.1k0.6
4.7k0.5 5.1k0.5
0.77 4.3k0.5 lO.Ok1.2
4.0k0.5 5.7k0.6
4.7k0.5 4.9k0.6
4.3k0.4 4.4k0.5 7.2k0.4
2.2 5.5+0.5a
17.0k1.1 23.9k1.9 31.5k3.3 55.6k5.8 62.7k5.9
Pinto 11.5k0.8
0
21.1k2.7 27.0k3.3
16.6k1.2 65.3k8.8
Bean 11.9k0.8 58.3a.5
O(S)
25.1k1.4
19.4k1.4
0.77 15.0k0.9 53.9d.7 39.0k5.1
9.6k1.4
18.8k1.8 23.6k2.3 49.4k6.7
2.2 10.3k1.8 15.6k1.9 53.6k5.3
18.44.6
14.9k2.4
4.2 31.2k10.8
9.2k1.6 11.6k1.6 36.0k8.8
15.7k2.5
12.7k2.1
11.1k1.6 35.8k12.3
7.0 9.4k1.6 29.24.3
9.2k1.3 11.6k1.5 21.1k1.5 29.7k2.5
7.8k0.9
Ryegrass 5.3k0.7
0
11.3k0.7
8.0k0.5
ow 6.7k0.6 20.8k1.6 27.3k1.6
4.3k0.5
10.1k1.7
6.920.6 19.1k2.1 26.8k1.1
5.6k0.6
0.31 3.5k0.5
9.Ok1.1
6.4k0.9 17.2k1.1 25.2+1 .O
0.77 5.4k0.8
3.3k0.5
7.6k1.7
5.9k1.1 6.6k1.2 12.3d.O 19.9+3.0a
2.2 4.4k0.8
----- -----
5.6k0.8 5.8+1 .O
4.2 4.4k0.8
4.3k0.7
17.2k0.4 22.0k0.7
12.3k0.5 41.7k1.6 65.0k4.7
Soybean 8.3k0.8
0
22.8k1.3
18.2k1.1 41.0k1.9 57.3k4.1
9.4k0.8 13.1k1.6
O(S)
18.9k0.7 24.0k0.9 43.4k2.2 59.1k3.5
9.7k0.8 13.6k0.4
0.23
22.6k1.5
16.8k2.1 40.2k2.7 57.1k2.2
8.7k1.5 12.0k1.8
0.31
17.6k2.6 23.54.3 51 .0+4.2a
0.77 12.3k2.0 38.4g.2
8.4k0.9
15.5k1.6 21.2k1.3 34.6k1.6 48.9+4.4a
2.2 7.7k1.2 10.9k1.7
21.1 k1.7
15.7k1.5 27.922.6 32.8k1.8
12.5k0.6
Wheat 9.Ok1.1
0
22.1~2.8
14.1k1.7 17.1 k1.2 31.4k2.0 37.0k1.4
11.1k1.8
ow
21.5k0.7
16.4k0.5 29.952.1 34.9k1.7
13.7k1.6
0.77 10.2k1.4
14.6k1.4 18.4k2.6 30.5+2.3a
25.4k2.0
8.7k0.8 11.5k0.8
2.2
9.9k1.6 10.7k1.3 22.1+1.9a
14.7k2.4
9.3k1.7
4.2 8.3k1.2
10.3k2.1
9.5k1.9 12.3k3.4 19.1+4Sa
8.7k1.6 8.9k1.4
7.0
aMean is significantly different(P20.05) from the control.
O(S) is a solvent control.
Percent Survival, Mean Shoot and
Root Weight, and Observed Abnormalities of Seedlings Exposed
to
Semduramicin
Mean Dry WeigM(mg)
Concen- Percent
Plant
- Observed
Shoot
Abnormalities
tration Mortality Root
Species
369.1 51 137.7k30.9
00.0
20.0
Corn 0
102.1k44.8
376.7k195.6
24.0
O(S)
.o
11 1.0521
208.1k67.0
0.77 8.0
100.1+15.7
165.8k48.6
28.0
3.2
75.7k29.0
wilted
plants
468.3k14.3
4.2 92.0
77.4d0.5 wilted
plants
130.4+78.1a
7.0 88.0
368.4540.0
174.6k106.9
Cucumber 0 0.0
278.7248.3
133.9k62.1
0.0
OF)
303.6536.1
124.9k62.1
0.23 0.0
319.9k40.7
97.2d3.0
0.3 1 0.0
31 1.6k29.2
97.293.0
0.77 0.0
192.0+28.6aDriedout,reducedsize
74.1k51.4
2.2 0.0
530.8k38.8
901.9k603.7
4.0
Pinto 0
ow 449.3k59.8
953.1k473.6
4.0
Bean
404.0k36.2
552.82285.3
0.77 0.0
384.2k52.8
321 .3+92.0a
2.2 16.0
1 62.3+88.4a yellow colored leaves,
364.0+151.1a
4.2 44.0
216.1+78.gabrowntippedleaves,wilted
275.1+110.2a
44.0
7.0
29.3G.7
32.9k6.2
Ryegrass 0.0
0
20.6k2.1
20.5d.4
0.0
O(S)
19.8k2.4
7.7+3.1a
0.31 0.0
17.1 yellow
k2.6 colored leaves,
8.1+4.3a
0.77 4.0
1
2.7+2.gabrowntipped leaves,
5.5+1 .Oa
2.2 40.0
--- --- plants
wilted
4.2 100.0
360.3k49.8
141.2k49.2
Soybean 0.0
0
248.3k21.9
82.844.1
4.0
O(S)
237.7k28.9
83.1Q3.5
0.23 0.0
278.7k14.3
None
74.0k14.5
0.3 1 0.0
236.0k35.3
77.7k19.0
0.77 4.0
230.9k27.2
54.351 3.9
2.2 0.0
58.8k7.3
199.4k68.3
Wheat 0 0.0
68.9k11.2
223.7555.6
0.0
O(S)
60.8k7.6
74.1+1 2.4a
0.77 0.0
43.0+3.1a
70.2+24.6a
2.2 4.0
24.2+3.4a
54.5+11 .5a
4.2 28.O
7.0+6.7awilted
1 6/9
39.6+6.5a
64.0
7.0
aMean is significantly different (P20.05) control.
from
O(S) is a solvent control.
Appendix c-1 1
Effect of Semduramicin Sodium on Algae
THE EFFECT OF SEMDURAMICIN SODIUM ON FRESHWATER ALGAE
Report Summary:
Study Number: 2438-1287-6113-550
-
Test Species: capricornutum, a freshwatergreen alga
Selenasrrum
Summary of Experimental Design:
The test was conducted in 125 ml flasks, each containing50 ml of Algal Assay Procedure
(AAP) medium. The following average measured concentrations of semduramicin sodium were
tested in triplicate: 150 70, 39, 19, 10, and 0 (negative control) ppm. Each flask was
inoculated with about 1O4 algal cells per ml and placedon a gyrotory shaking table in an
environmental chamber. Light and temperature favorable to algal growth were maintained. At
24 hours and at each subsequent 48 hour interval, triplicate cell counts were conducted on
each flask using a hemocytometer and a compound microscope. The test was continued until
day 13 when cell density in all flasks increasedby less than5% per day.
Test endpoints were 1) cell density and2) growth rate (p)
Cell density = Number of Cells + (Number of Microscopic Fields x Field Volume)
1)
Field Volume = Volume of hemocytometer grid (0.1 x 0.1 x 0.01 cm)
growth rate (p)was calculated using the formula:
2)
where In = natural logarithm, X1 and X2 are cell density measured at times t l and t2 and p is
expressed in units of days - I . The maximum growth rate (p max) for each culture vessel is the
highest value for p calculated for any 24 hour interval during the test.
From the observed values for maximum culturedensity and the calculated values for maximum
growth rate, the highest test concentration thatcaused no significant growth inhibition or
stimulation (No Observed Effect Limit, NOEL) and the lowest test concentration that caused
significant inhibition (Minimum Inhibitory Concentration, MIC) were determined using one-way
analysis of variance (Sokal and Rohl 1981)and Dunnett's Procedure (Dunnett 1955,1964).
Summary of Results:
The concentration of semduramicin in AAP media was determined by HPLCanalysis withpost-
column derivatization and found on day 13 to average 104% of the level found on day 0. As
shown in Table I, growth of Selenustrum capricornutum was completely inhibited by concentration
at at above 39 mg/L semduramicin. For culture density and for max, Dunnett's Test indicated
p
that the Minimum Inhibitory Concentration (MIC) of semduramicin was 19 mg/L and the No
Observed Effect Limit (NOEL) was 10 mg/L.
Table I. CellDensityandGrowthRate of AlgaeExposed to SemduramicinSodium
Observation Time (hours)
Control
4.00 36.39 241.72 266.56 643.61 744.17 626.1 1 744.17
1.30
cell density
0.75 8.82 38.82 32.26 30.10 21.03 42.45 21.03
S.D. 0.00
1.334 1.100 0.942 0.051 0.448 0.076 0.089 1.363
Growth rate
0.228 0.178 0.134 0.022 0.054 0.027 0.049
S.D. 0.185
10 mglL
2.86 26.58 224.81 289.44 530.28 576.67 549.17 590.28
1.30
cell density
0.19 1.34 47.96 18.33 84.60 62.07 50.26 48.29
S.D. 0.00
0.945-0.025
1.115 1.136
1.049
0.134
0.302
0.046
Growth rate
0.046
0.100
0.79
0.090
0.105
0.026
0.055
0.024
S.D.
19 mglL
2.39 11.28 65.97 149.16 393.06 413.33 390.56 430.28
1.30
cell density
0.19 2.00 45.64 85.80 96.77 47.35 60.12 59.99
S.D. 0.00
0.727 0.772 0.780 0.433 0.555 0.035 0.031 0.91 1
Growth rate
0.099 0.127 0.322 0.076 0.260 0.076 0.058 0.1 29
S.D.
39 mglL
2.50 0.94 0.31 2.50
1.30 0.00
cell density 0.64 0.00 0.00
0.10
0.22 0.61 0.53 0.00
0.00 0.00
S.D. 0.00 0.22
Growth rate 0.781-0.187 -0.487
-0.362 0.781
0.108 0.563 0.089 0.108
S.D.
70 mgIL
1.61
1.30 1.63
cell density 0.00
0.00
0.44 0.00
0.00 0.00
0.32 0.05 0.00 0.29
0.00
0.00 0.00
0.00
S.D. 0.00
-0.639 Growth rate 0.241
0.096 0.251
S.D.
150 mglL
1.36 0.81 1.52
1.30 0.00
cell density 0.00 0.00 0.00 0.00
0.54 0.34
S.D. 0.35
0.00 0.00
0.00 0.00
0.00
0.00
0.080 -0.264 2 -0.01
Growth rate
0.501
0.201
S.D.
0.363
.
Appendix c-12
Acute Toxicity Study with Semduramicin Sodiumin Daphnia
Report Summary: ACUTE TOXICITY STUDY WITH SEMDURAMICIN SODIUM IN DAPHNIDS
Study Number: 2438-1287-6113-110
Test Species: Water Flea (Daphnia magna)
Summary of Experimental Design:
The test was conducted in 250 ml glass beakers, each containing 200 ml of test solution. The
following measured concentrations of semduramicin sodiumwere present in four beakers each:
48, 31, 19, 11, 6.2, and 0 (negative control) ppm. Daphnids which were no more than 24 hours
old were obtained from a laboratory culture and distributed impartially, five a beaker. Test
to
solution temperatures, oxygen content and pH were monitored and when necessary, adjusted.
At 0, 24, and 48 hours after initiation,the number of immobilized daphnids in each beaker and
any other signsof toxicity were recorded.
Summary of Results: The test concentrations remained stable during the experiment. A mean
of 85% of the daphnids exposed to 48 ppm and 20%of those exposed to 31ppm were found to
be immobile, and hence presumed dead, after 48 hours (see Table). From the test data, the
concentration of semduramicin sodium which causes immobilization in 50% of exposed
daphnids (the EC50) was estimated by standard statisticalprocedures to be as follows:
=5O 95% Confidence Interval
24-hour 42 38-49
48-hour 38 31-48
The no-observed effect concentration(NOEC) through 48 hours was 19 ma/L. The NOEC is
the highest concentration the test material that has-no statistically significantadverse effect
of
on the exposed organisms as compared to the controls (15). One out of 20 daphnids (5%) was
immobilized at 19 ppm and at 11 ppm; also, one daphnid was caught on particulate matter at 11
ppm. However, any apparent difference between a test group and the controlswould have to
be at least 15% to be statistically significant. Furthermore, although immobilization and
entrapment on particulate matter did not occur in the control group in thisparticular test, they
for
are observed randomly at a low incidence in control groups and are not unusual daphnids.
Therefore, the observations at 19 and 11 ppm are not considered drug-related. No other
physical or behavioral abnormalities, such as flared carapace, w e e observed.
Concentrations Tested, Corresponding Cumulative Percent of Immobilized Organisms and
Observations During the 48-hour Static Exposure of Daphnids (Daphnia magna) to UK 61,689-2
(N=20).
. Cumulative Number of Immobilized Organismsa
Mean
Measured
Concentration 24-hour 48-hour
A B D B
(mg/L) C Mean A C D Mean
19
11
6.2
aCumulative percent mortalities arelisted with the corresponding number of dead
organisms in parentheses.
of the surviving daphnids was lethargic caught on particulate matter.
and
of the surviving daphnids was caught on particulate matter.
of the surviving daphnids was lethargic.
eTwo of the surviving daphnids were lethargic.
.
Appendix c-13
Acute Toxicity Study with Semduramicin Sodium in Bluegill
ReportSummary: ACUTETOXICITYSTUDYWITHSEMDURAMICINSODIUMIN
BLUEGILL
.
Study Number: 2438-0188-6113-100
Bluegill (Lepornis rnacrochirus)
Test Species:
Summarv of Experimental Design:
Groups of ten bluegill wereselected impartially froma population which had been maintained in
a common holding tank and ranged from 0.21 to 1.56 g in weight. Each group was placed in 14
liters of test solution contained in an 18.9 liter glass aquarium. Duplicate groups were exposed
for 96 hours to mean measured concentrations of 100, 62, 37, 22, 13, 7.6, and 0 (negative
control) ppm of semduramicin sodium. Test solution temperatures, oxygen content, and pH
were monitored and when necessary, adjusted. At 0, 24, 48, 72 and 96 hours after initiation,
mortalities were recorded; dead fish were removed, and surviving fish were observed for signs
of toxicity.
Summary of Results: The test concentrations remained stable throughout the experiment.
Mortalities were observed attest concentrations of 22 ppm and above (see Table). From the
test data, the concentration of semduramicin sodium in the test solution which causes 50%
mortality in exposed bluegill (the LC50) was calculated by standard statistical methods to be as
follows:
95% Confidence Interval
G O
31 - 51
40 48-hour
31 - 47
38 72-hour
31 - 47
38 96-hour
No mortalities and no physical or behavioral abnormalities,such as lethargy, loss of equilibrium,
or darkened pigmentation were observed in bluegillexposed to 13 or 7.6 ppm.
Concentrations Tested, Corresponding Cumulative Percent Mortalities and Observations Made
During the 96-hour Static Exposure of Bluegill ( k p o m i s macrochirus) to UK 61,689-2 (N=20).
.
Mean
Measured
Cumulative Mortality
Concentration
(mg/L) 24-hour 48-hour 72-hour 96-hour
100 45(9)b 75( 15) 85( 7) 1
85( 1 7)d
62 60(60( 2)
15(3)
1 1 60( 2) 2)
1
50( 10)
50( 10)
45(9)b 50( 0)
37 1
'
50(10) 50(10)
22 50( 10)
45(9)b
13 O(0) O(0)
O(0) O(0)
7.6 O(0)
O(0)
O(0) O(0)
o(o)c
Control 5(1) 5(1) 5(1)
acumulative percent mortalities are listed with the corresponding number of
dead
organisms in parentheses.
of the surviving fish exhibited a complete loss of equilibrium.
of the surviving fish was atthe surface of the test solution.
of the surviving fish exhibited darkened pigmentation.
.
Appendix c-14
Acute Toxicity Study with Semduramicin Sodium in Rainbow Trout
Page 102
-- ~~ ~
Report Summary: ACUTETOXICITYSTUDYWITHSEMDURAMICINSODIUMIN
RAINBOW TROUT
Study Number: 2438-0188-6113-1
03
.
Test Species:
Rainbow
trout (Onchorhynchus mykiss)
Summary of Experimental Design:
Groups of ten trout were selected impartially from a populationwhich had been maintained in a
common holding tank and ranged from 0.39 g to 1.58 g in weight. Each group was placed in 14
liters of test solution contained in an 18.9 liter glass aquarium. Duplicate groups were exposed
for 96 hours to mean measured concentrations 50, 30, 18, 11, 6.4 and 0 (negative control)
of
ppm of semduramicin sodium. Test solution temperatures, oxygen content, and pH were
monitored and when necessary, adjusted. At 0, 24, 48, 72 and 96 hours after initiation,
mortalities wererecorded;dead fish were removed, and surviving fish were observed for signs
of toxicity.
Summary of Results: The test concentrations remained stablethroughout the experiment. A
significant incidence of mortality was observed at test concentrations of 30 and 50 ppm;
surviving fish exhibitedloss of equilibrium, lethargy, and/or darkened pigmentation (see Table).
From the test data, the concentration of semduramicin sodium in the test solution which causes
50% mortality in exposed rainbow trout (the LC50) was calculated by standard statistical
methods to be as follows:
95% Confidence Interval
4 0
---
24-hour
43
30-50
48-hour 39
18-50
72-hour 33
18-50
96-hour 32
The no-observed effect concentration (NOEC) through96 hours was 11 ppm. The NOEC is the
highest concentrationof the test material that has no statistically significant adverse effect on
the exposed organisms as compared to controls (15 ) . At 18 ppm, there was no mortality, but
several fish exhibited darkened pigmentation. One out of 20 trout (5%) exposed to 11 ppm died
and one exposed to 6.5 ppm also showed abnormal pigmentation. However, the difference
between a test group and the controls would have to be at least 15% to be statistically
significant. Furthermore, although no deaths or abnormal pigmentation occurred in the control
group in this particular study, they occur randomly at a low incidence in controls (up to 10% is
considered acceptable) and are not unusual for trout. Therefore, the observations at 11 and
6.4 are not considered drug-related. No other physical or behavioral abnormalities, such as
loss of equilibrium or lethargy, were observedat 11 or 6.4 ppm.
Concentrations Tested, Corresponding Cumulative Percent Mortalities and Observations Made
During the 96-hour Static Exposure of Rainbow Trout (Onchorhynchus mykiss) to UK 61,689-2
(N=20).
.
Mean
Measured
Cumulative Mortality (%)a
Concentration
~~~~
96-hour
72-hour48-hour24-hour (mg/L)
aCumulative percent mortalities are listed with the corresponding numberof dead
organisms in parentheses.
bAll of the surviving fish exhibited darkened pigmentation.
鈥楽everal of the surviving fish exhibited darkened pigmentation.
dTwo of the surviving fish exhibited a complete loss equilibrium.
call of the surviving fish were lethargic.
fOne of the surviving fish exhibited a complete loss of equilibrium.
gone of the surviving fish exhibited darkened pigmentation.
of the surviving fish exhibited a partial loss of equilibrium.
鈥楢ll of the surviving fish exhibited a partial loss of equilibrium.
.
Appendix c-15
Acute Dermal and Ocular Irritation Studies with Semduramicin Sodium in Rabbits
ReportSummary: ACUTE DERMAL AND OCULAR IRRITATION STUDIES WITH
SEMDURAMICIN SODIUM IN RABBITS
.
Study Number: 88-564-09
Albino rabbit (New Zealand White)
Test Species:
Summary of Experimental Desiqn:
Dermal Irritation: One male and two female rabbits were used. Their bodyweights ranged
1)
from 3.35 to 3.66 kg. A dose of 0.5 gram of semduramicin sodium was applied to one
intact andone abraded site on the back of each rabbit and was held in continuouscontact
with the skin under an occlusive patch for 24 hours. Each test site measured
approximately two inches square. During the dosing procedure, both the compound and
the skin were thoroughly wetted with distilled water until an aqueous paste of the
compound was formed. The total dose of 1 gram applied to each animal was equivalent to
a dose of 273 to 299 mg/kg of semduramicin sodium. All rabbits were observed for 7 days
after dosing.
Ocular Irritation: One male and two female rabbits were used. Their bodyweights ranged
2)
from 3.66 to 3.97 kg. A dose of 21.5 mg semduramicin sodium, equivalent to the 0.1 ml
volume of solid specified in procedure, was introduced into the conjunctivalsac of the
the
left eye. The treated eye of each rabbit was not rinsed after dosing. The animals were
observed for 7 days. On the day of dosing (day 0), the eyes were evaluated with minimal
manipulation and without the use of fluorescein.
Skin reactions and ocular changes were evaluated visually according to the standard Draize
scoring system, in which a score of zero denotes no effect and higher scores denote
increasingly severe reactions. A Primary Irritation Score for skin was calculated as the sum of
the mean erythema scores at 24 and 72 hours, divided by 4.
Summary of Results:
Only mild dermal irritation was observed. Following a 24-hour exposure to the compound,
1)
the skin at all intact sites appeared normal, and no gross tissue changes were observed at
any of these sites during the remainderof the 7-day observation period.
2)
At 24 hours, well-defined (value of erythema, but no edema, was apparent at eachof
the abraded sites. The erythema was confined primarily to an area approximately 2 mm to
either side of the abrasion lines. Also, at 24 hours, the skin along the abrasion marks was
slightly separated and appeared reddish or golden-brown. By 48 hours post dose, dry,
crusty (scab-like) tissue was apparent along the abrasions.
The erythema at each of the abraded sites subsided completely within 4or 5 days of
dosing. Sloughing on the dry tissue along the abrasions occurred, but at the time of
sacrifice on day 7, there was still some scab-like tissue along the lines of abrasion at each
site. (Erythema and subsequent scab formation along and confined to the abrasion lines
are not uncommon or unexpected findings following abrasionof rabbit skin.)
Condition of skin Time after
application Mean value of score
(hours) Erythema Edema
. all observations
Intact 0 0
2 .oo
Abraded 24 0
0
48 2.00
0
72 1.67
0
96 0.67
120-168 0 0
The Primary Irritation Score calculated from the data is 0.92. (The maximum possible score
would be 8).
All rabbits were alert and active throughout test period, and there were no obvious clinical
the
signs of toxicity. However;each animal exhibited decreased food consumptionon two or three
days at various times during the study andweighed slightly less at the time of sacrifice than it
did prior to dosing.
These results indicate that semduramicin sodium is nota primary skin irritant.
2) Immediately after dosing, each rabbit held itseye closed for only a few seconds, and within
one minute, two animals were briefly rubbing the treated eye; however, none of the rabbits
exhibited signs of obvious pain or discomfort. Within 1 to 5 hours of dosing, slight
reddening of the conjunctivae, slight chemosis, and/or slight discharge were apparent. In
addition, circumcorneal injection was noted in each animal, and a small, localized areaof
iritis was observedin one of the three rabbits. By 24 hours after dosing and throughout
the remainder of the study, the treated eye of each rabbit appeared normal.
The mean values obtained from the individual scores are summarizedbelow.
Scores After Application
Time Mean
(Hours) Cornea Iris Conjunctivae Total
1 0 0
2.00 2.00
3.33
1.67
0
3 5.00.
3.33
5 1.67 5.00
0
0 0
24-1 68 0 0
80 110 20
Maximum possible score
10
All rabbits were asymptomatic throughout the 7-day test period, and they gained weight.
The results of this test indicate that semduramicin sodium is not an ocularirritant.
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