Document xewoY6EjKagym75qRGm6qB2y
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REPORT
DETERMINATION OF THE PARTITION COEFFICIENT (N-OCTANOL/WATER) OF T-5874
BY HIGH PERFORMANCE L I Q U I D CHROMATOGRAPHY (HPLC)
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NOTOX P r o j e c t 121275 NOTOX substance 38187
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NOTOX P r o j e c t 121275
STATEMENT OF GLP COMPLIANCE
NOTOX B . V . , 's-Hertogenbosch, The Netherlands
The study d e s c r i b e d i n t h i s r e p o r t was conducted i n compliance w i t h t h e most recent e d i t i o n o f : The OECD P r i n c i p l e s o f Good Laboratory P r a c t i c e which are essentially i n conformity with: The U n i t e d S t a t e s Food and Drug A d m i n i s t r a t i o n . T i t l e 21 Code o f Federal Regul a t i ons P a r t 5 8 . The U n i t e d S t a t e s Environmental P r o t e c t i o n Agency (FIFRA). T i t l e 40 Code o f Federal R e g u l a t i o n s P a r t 160. The U n i t e d S t a t e s Environmental P r o t e c t i o n Agency (TSCA). T i t l e 40 Code o f Federal Regulations P a r t 792.
Study Director:
Drs. R . de Vries
. . ./. . ..... . ... * . . . . . .
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QUALITY ASSURANCE STATEMENT NOTOX B.V., 's-Hertogenbosch, The Netherlands.
Study procedures were s u b j e c t t o p e r i o d i c inspections and general non study specific: processes were also inspected a t p e r i o d i c i n t e r v a l s .
T h i s r e p o r t was a u d i t e d by t h e NOTOX Q u a l i t y Assurance U n i t and t h e methods and r e s u l t s a c c u r a t e l y r e f l e c t the raw data.
I DATES OF QAU INSPECTIONS/ AUDITS
REPORTING DATES
25 March 1994 28 March 1994 2 1 A p r i l 1994
25 March 1994 28 March 1994 21 A p r i l 1994
Q u a l i t y Assurance Manager
C.J. M i t c h e l l B.Sc.
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T-5874 REPORT APPROVAL STUDY DIRECTOR:
MANAGEMENT:
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NOTOX P r o j e c t 121275
Drs. R. de V r i e s
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J.A.M.W. van H e l v o i r t Section head Physic0 Chemistry
09 ....................... ':\
lob I'99'c
Date:
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PREFACE Sponsor
Study Monitor Testing Facility
Study Director Study plan
TEST SUBSTANCE IdentSfi c a t i o n ---. Description Batch Purity Storage conditions S t a b i 1it y under s t o r a g e condi tions Expiry date
3M Belgium - Chemical EBC
Canadastraat 11 B-2070 ZWIJNDRECHT Bel g i um M r . R.H. Cox NOTOX B.V. Hambakenwetering 3 5231 DO 's-Hertogenbosch The Netherlands Drs. R . de Vries S t a r t : 30 March 1994 Completed: 07 A p r i l 1994
T-5874 Cream s o l i d 2334 100%
A t room temperature i n the dark
Stable January 01, 1996
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PURPOSE AND PRINCIPLE
The purpose o f t h e s t u d y was t o determine t h e p a r t i t i o n c o e f f i c i e n t of t h e t e s t substance between n-octanol and water.
The p a r t i t i o n c o e f f i c i e n t (n-octanol/water) (Pow) i s defined as the r a t i o o f the equilibrium concentrations i n a two phase system consisting o f n-octanol and water.
The r e t e n t i o n t i m e o f a substance i n a reversed-phase High Performance l i q u i d Chromatography (HPLC) chromatographic system i s r e l a t e d t o i t s partition coefficient (n-octanol/water). The HPLC method i s u s e f u l f o r l o g Po, v a l u e s between 0 and 6 . Using t h e HPLC method, i m p u r i t i e s a r e o f minor importance. The HPLC method must a l s o be a p p l i e d i f t h e t e s t substance i s a m i x t u r e o f compounds b u t i s n o t applicable t o strong acids and bases, metal complexes, substances which react with the eluent or surface-active agents.
GUIDELINES
The study procedure d e s c r i b e d i n t h i s r e p o r t was based on t h e f o l l o w i n g guidelines:
Organization f o r Economic Co-operation and Development (OECD), OECD Guideline f o r Testing of Chemicals, guideline no. 117: " P a r t i t i o n c o e f f i c i e n t (n-octanol/water) High Performance L i q u i d Chromatography (HPLC) method" (adopted March 30, 1989).
European Economic Community (EEC), EEC-Directive 92/69 EEC, Annex V , P a r t A , Methods f o r the determination o f physico-chemical p r o p e r t i e s , A.8: " P a r t i t i o n c o e f f i c i e n t " . EEC Pub1i c a t i o n no. L383, December 1992.
ARCHIVING
NOTOX B . V . w i l l a r c h i v e t h e f o l l o w i n g d a t a f o r a t l e a s t 10 years: p r o t o c o l , r e p o r t , t e s t substance r e f e r e n c e sample and raw data.
TEST SYSTEM AND RATIONALE
A High Performance L i q u i d Chromatograph with a spectrophotometric detector. The s t a t i o n a r y phase was bonded s i l i c a C18. The m o b i l e phase was 75/25 ( v / v ) a c e t o n i t r i l e l w a t e r . Both HPLC i n s t r u m e n t a t i o n and c o n d i t i o n s a r e described i n the section "method o f chemical analysis".
The t e s t system was recognized by the i n t e r n a t i o n a l g u i d e l i n e s (OECD, EEC).
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REFERENCE SUBSTANCES
S i x chemicals f o r which l o g Pow has been reported were used t o c a l i b r a t e t h e e l u t i o n t.ime i n u n i t s o f l o g Pow. The m i x t u r e o f r e f e r e n c e substances consisted of:
Reference substance
- benzylalcohol (p.a., Merck) - toluene (p.a., Merck) - 1,4-dichlorobenzene (z.s., Merck) - f l u o r a n t h e n e (98%, GC, A l d r i c h Chemie) - dibenzyl (99%, GC, A l d r i c h Chemie) . - 2,4-DDT (99%, HPLC, Riedel de Haen, Seelze,
FRG)
l o g Po, values according t o t h e OECD guideline.
1.1 2.7 3.4 4.7 4.8 6.2
PERFORMANCE OF THE TEST
Preparation o f the solutions
S o l u t i o n s o f the reference substances (except f o r ethylmethylketone) were prepared i n methanol (HPLC-grade, Labscan L i m i t e d Co., Dublin, I r e l a n d ) a t a c o n c e n t r a t i o n o f 1.5-2 g / l . A m i x t u r e o f t h e r e f e r e n c e sirbstances was
prepared by adding 125 u l o f each s o l u t i o n t o a v o l u m e t r i c f l a s k .
Subsequently, t h i s f l a s k was made up with m o b i l e phase t o a f i n a l volume o f 25.0 m l . For the determination o f to ( r e t e n t i o n time o f the unretarded component), a
s o l u t i o n o f formamide (p.a., Merck, Darmstadt, FRG) i n methanol was prepared a t a c o n c e n t r a t i o n o f 1 . 7 g / l and t h e r e a f t e r d i l u t e d 10 times w i t h mobile
phase. A s t o c k s o l u t i o n o f T-5874 was prepared by d i s s o l v i n g 165 mg t e s t substance i n 50.0 m l a c e t o n i t r i l e (3 minutes s o n i c a t i o n ) . The s t o c k s o l u t i o n was d i l u t e d 10 times with mobile phase, r e s u l t i n g i n the t e s t s o l u t i o n .
Performance 0.f t h e test
The s o l u t i o n s were i n j e c t e d i n the f o l l o w i n g sequence: the m i x t u r e o f reference substances, mobile phase, the formamide solution, the t e s t s o l u t i o n ( i n duplicate), mobile phase, the mixture o f reference substances and the formamide solution.
Temperature o f measurement
The temperature o f t h e mobile phase was recorded s e v e r a l times d u r i n g the measurements.
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METHOD OF CHEMICAL ANALYSIS
The c o n d i t i o n s used f o r t h e High Performance L i q u i d Chromatographic method are described below:
Anal v s i s
Col umfl Mobile phase
F1ow Detect! on I n j e c t i o n volume
LiChrospher 100 RP-18; 125 x 4 (I.D.)
d = 5 (Merck, Darmstadt, FRG) 7!/25 ( v / v ) a c e t o n i t r i l e (HPLC-grade, L i m i t e d Co., D u b l i n , I r e l a n d ) / M i l l i - Q ( M i l l i p o r e Corp., Bedford, MA, USA) 1 ml/min UV a t A= 210 nm 10 u1
mm;
Labscan water
Instrumentation
HPLC Pump Autosampl er Detector
Integrator
S e r i e s 410 LC ( P e r k i n Elmer, Norwalk, CT, USA) ISS-200 ( P e r k i n E l m e r ) SpectroMonitor 3100 (LDC A n a l y t i c a l , R i v i e r a Beach, FL, USA) SP 4290 (Spectra Physics, San Jose, CA, USA)
DATA HANDLING -_-.
Using High Performance L i q u i d Chromatography, e s p e c i a l l y l a r g e l o g Pow values can be accurately determined,from the chromatographic retention data. To t h i s end, t h e c a p a c i t y f a c t o r ( k ) was used, s i n c e i t i s p r o p o r t i o n a l t o the p a r t i t i o n c o e f f i c i e n t . The c a p a c i t y f a c t o r was c a l c u l a t e d from t h e r e t e n t i o n o f the substance concerned ( t r )and t h e unretarded component (to):
k' = (tr - to)/to
From t h e r e s y l t s o f t h e r e f e r e n c e substances, a p l o t of l o g Pow (x-value) versus 109 k (y-value) was constructed, u s i n g l i n e a r r e g r e s s i o n a n a l y s i s . The l o g k v a l u e o f each component o f t h e t e s t substance was compared with t h e l o g k ' values o f t h e reference substances w i t h known l o g Pow values.
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RESULTS
Representative HPLC chromatograms o f t h e t e s t s o l u t i o n , the blank-.(mobile phase), the mixture o f reference substances and the formamide s o l u t i o n are shown i n F i g u r e s 1 t o 4.
I n the HPLC chromatograms o f T-5874, u s i n g d e t e c t i o n a t 210 nm, one l a r g e peak a t 4 . 1 minutes and a small peak a t 7.7 minutes were observed. Between 1 . 6 and 4 minutes, s e v e r a l s m a l l peaks were observed. I t was assumed t h a t the l a r g e peak derived from t h e major component o f t e s t substance whereas the small(er) peaks derived from impurities.
The towas determined t o be 0.90 minutes as a mean value o f b o t h measurements ( i . e 0.89 and 0.90 minutes).
The mean values o f t h e r e t e n t i o n times, k ' values, l o g k ' values, l o g Pow and Pow v a l u e s t a r e summarized i n Table 1. The p l o t o f l o g Pow (x-value) versus l o g k (y-value) from the reference substances i s shown i n Figure 5.
Table 1 Results o f the t e s t .
Substance
trl
k'
l o g k ' l o g Po,
pow
benzyl alcohol t o 1uene 1,4-dichlorobenzene fluoranthene d ibenzyl
2,4-DDT
t e s t substance peak 1 peak 2 (major comp.) several small peaks
1.14 2.24 2.89 5.67 4.07
9.12
0.274 1.503 2.229 5.330 3.547
9.190
-0.563 0.177 0.348 0.727 0.550 0.963
1.1 2.7 3.4 4.7 4.8 6.2
7.70 4.05 1.6 I 4
7.598
0.881
5.6
3.525
0.547
4.4
1.791 3.47 -0.1010.542.214.4
1 1
Note
Mean v a l u e o f t h e r e t e n t i o n t i m e s o f b o t h chromatograms.
I n t e r p o l a t e d f r o m t h e r e g r e s s i o n l i n e : y = 0 . 2 8 8 ~- 0.730
: The c a l c u l a t i o n s were p e r f o r m e d u s i n g n o t r o u n d e d v a l u e s .
(r=0.972, n=6)
39.8 ~ 1 0 4 2. 51x104 1.5Ox1O2 I 2. 51x104
The temperature o f t h e m o b i l e phase was 20.2-20.6OC d u r i n g the measurements.
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HPLC chromatogram o f t h e t e s t s o l u t i o n o f 330 mg/l
T-5874 i n mobile phase.
Column Mobile phase F1ow Detection I n j e c t i o n volume
LiChrospher 100 RP-18; 125x4 (I.D.) m m ;
75/25 (v/v) acetonitrile/Milli-Q water 1 ml/min UV, a t X = 210 nm 10 ~1
dp=5
Note: The peaks between 0 and 1.6 m i n u t e s were also observed i n t h e chromatograms o f t h e b l a n k (see F i g u r e 2 ) . T h e r e f o r e i t was assumed t h a t t h e s e peaks d i d n o t d e r i v e
from the test substance.
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7 .
:,-:.
T 17
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t.=-!.-.,-*> -5..4
F i g u r e 2 HPL.C chromatogram o f a blank (mobile phase).
Column Mobi 1e phase F1OW 0et:ecti on
I n j e c t i o n volume
LiChrospher 100 RP-18; 125x4 ( 1 . 0 . ) mm; 75/25 ( v / v ) a c e t o n i t r i l e / M i l l i - Q water 1 ml/min
U V , a t X = 210 nm
10 l.11
dp=5 Um
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HPLC chromatogram o f t h e m i x t u r e o f reference substances i n mobile phase.
Column Mobile phase F1ow Detection I n j e c t i o n volume
LiChrospher 100 RP-18; 125x4 (I.D.) mm;
75/25 ( v / v ) acetonitrile/Milli-Q water 1 ml/min UV, a t X = 210 nm 10 111
dp=5 urn
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F i g u r e 4 HPLC chromatogram o f a formamide s o l u t i o n of 0.2 g / l i n mobile phase.
Column Mobile phase
F1 ow
Detection I n j e c t i o n volume
LiChrospher 100 RP-18; 125x4 (I.D.) mm;
75/25 (v/v) a c e t o n i t r i l e / M i l l i - Q water 1 ml/min UV, a t X = 210 nm
10 I.I~
dp=5 urn
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13/04/94T R J
Pow
1.000
0.800 0.600
0.400
0.200 0.000
0.0 1.0 2.0 3.0 4.0 5.0 6.0 log Pow
F i g u r e 5 P l o t o f l o g Po, (x-value) versus l o g k ' (y-value) from t h e
reference substances.
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