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REPORT
DETERMINATION OF THE PARTITION COEFFICIENT (N-OCTANOL/WATER) OF T-5874
BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
NOTOX Project 121275 NOTOX substance 38187
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RECEIVED
NO y ~ 9 :994
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TOa i^ w lO V
T-5874
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STATEMENT OF GLP COMPLIANCE
NOTOX B.V., 's-Hertogenbosch, The Netherlands
The study described in this report was conducted in compliance with the most recent edition of: The OECD Principles of Good Laboratory Practice which are essentially in conformity with: The United States Food and Drug Administration. Title 21 Code of Federal Regulations Part 58. The United States Environmental Protection Agency (FIFRA). Title 40 Code of Federal Regulations Part 160. The United States Environmental Protection Agency (TSCA). Title 40 Code of Federal Regulations Part 792.
Study Director:
Drs. R. de Vries Date : '\
\
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QUALITY ASSURANCE STATEMENT NOTOX B.V., 's-Hertogenbosch, The Netherlands.
Study procedures were subject to periodic inspections and general non study specific processes were also inspected at periodic intervals.
This report was audited by the NOTOX Quality Assurance Unit and the
methods and results accurately reflect the raw data.
.
DATES OF QAU INSPECTIONS/ AUDITS
REPORTING DATES
25 March 1994 28 March 1994 21 April 1994
25 March 1994 28 March 1994 21 April 1994
Quality Assurance Manager
C.J. Mitchell B.Sc.
Date:
%
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T-5874 REPORT APPROVAL
STUDY DIRECTOR:
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Drs. R. de Vries
MANAGEMENT:
J.A.M.W. van Helvoirt Section head Physico Chemistry
Date:
Iot> I `0 0 4 *
C)r. Ilona C. Enninga Technical Director
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T-5874
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PREFACE Sponsor
Study Monitor Testing Facility
Study Director Study plan
TEST SUBSTANCE
Identification Description Batch Purity Storage conditions Stability under storage conditions Expiry date
3M Belgium - Chemical EBC Canadastraat 11 B-2070 ZWIJNDRECHT Belgium Mr. R.H. Cox NOTOX B.V. Hambakenwetering 3 5231 DD 's-Hertogenbosch The Netherlands Ors. R. de Vries Start: 30 March 1994 Completed: 07 April 1994
T-5874 Cream solid 2334 100% At room temperature in the dark
Stable January 01, 1996
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PURPOSE AND PRINCIPLE
The purpose of the study was to determine the partition coefficient of the test substance between n-octanoi and water.
The partition coefficient (n-octanoi/water) (P0w) defined as the ratio of the equilibrium concentrations in a two phase system consisting of n-octanol and water.
The retention time of a substance in a reversed-phase High Performance liquid Chromatography (HPLC) chromatographic system is related to its partition coefficient (n-octanol/water). The HPLC method is useful for log Pow values between 0 and 6. Using the HPLC method, impurities are of minor importance. The HPLC method must also be applied if the test substance is a mixture of compounds but is not applicable to strong acids and bases, metal complexes, substances which react with the eluent or surface-active agents.
GUIDELINES
The study procedure described in this report was based on the following guidelines:
Organization for Economic Co-operation and Development (OECD), OECD Guideline for Testing of Chemicals, guideline no. 117: "Partition coefficient (n-octanol/water) High Performance Liquid Chromatography (HPLC) method" (adopted March 30, 1989).
European Economic Community (EEC), EEC-Directive 92/69 EEC, Annex V, Part A, Methods for the determination of physico-chemical properties, A.8: "Partition coefficient". EEC Publication no. L383, December 1992.
ARCHIVING
NOTOX B.V. will archive the following data for at least 10 years: protocol, report, test substance reference sample and raw data.
TEST SYSTEM AND RATIONALE
A High Performance Liquid Chromatograph with a spectrophotometric detector. The stationary phase was bonded silica C^g. The mobile phase was 75/25 (v/v) acetonitrile/water. Both HPLC instrumentation and conditions are described in the section "method of chemical analysis".
The test system was recognized by the international guidelines (OECD, EEC).
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REFERENCE SUBSTANCES
Six chemicals for which log Pow has been reported were used to calibrate
the elution time in units of log Pow. The mixture of reference substances consisted of:
Reference substance
- benzylalcohol (p.a., Merck) - toluene (p.a., Merck) - 1,4-dichlorobenzene (z.s., Merck) - fluoranthene (98%, GC, Aldrich Chemie) - dibenzyl (99%, GC, Aldrich Chemie) . - 2,4-DDT (99%, HPLC, Riedel de Haen, Seelze, FRG)
1 log PQW values according to the OECD guideline.
log Pq *1
1.1 2.7 3.4 4.7 4.8 6.2
PERFORMANCE OF THE TEST
Preparation of the solutions
Solutions of the reference substances (except for ethylmethylketone) were prepared in methanol (HPLC-grade, Labscan Limited Co., Dublin, Ireland) at a concentration of 1.5-2 g/1. A mixture of the reference substances was prepared by adding 125 yl of each solution to a volumetric flask. Subsequently, this flask was made up with mobile phase to a final volume of 25.0 ml. For the determination of t0 (retention time of the unretarded component), a solution of formamide (p.a., Merck, Darmstadt, FRG) in methanol was prepared at a concentration of 1.7 g/1 and thereafter diluted 10 times with mobile phase. A stock solution of T-5874 was prepared by dissolving 165 mg test substance in 50.0 ml acetonitrile (3 minutes sonication). The stock solution was diluted 10 times with mobile phase, resulting in the test solution.
Performance of the test
The solutions were injected in the following sequence: the mixture of reference substances, mobile phase, the formamide solution, the test solution (in duplicate), mobile phase, the mixture of reference substances and the formamide solution.
Temperature of measurement
The temperature of the mobile phase was recorded several times during the measurements.
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METHOD OF CHEMICAL ANALYSIS
The conditions used for the High Performance Liquid Chromatographic method are described below:
Analysis
Column
Mobile phase
Flow Detection Injection volume
LiChrospher 100 RP-18; 125 x 4 (I.D.) mm;
dg= 5 ym (Merck, Darmstadt, FRG)
75/25 (v/v) acetonitrile (HPLC-grade, Labscan
Limited Co., Dublin, Ireland)/Milli-Q water
(Millipore Corp., Bedford, MA, USA)
1 ml/min
UV at X= 210 nm
10 yl
'
Instrumentation
HPLC Pump Autosampler Detector
Integrator
Series 410 LC (Perkin Elmer, Norwalk, CT, USA) ISS-200 (Perkin Elmer) SpectroMonitor 3100 (LDC Analytical, Riviera
Beach, FL, USA) SP 4290 (Spectra Physics, San Jose, CA, USA)
DATA HANDLING
Using High Performance Liquid Chromatography, especially large log Pow values can be accurately determined^from the chromatographic retention data. To this end, the capacity factor (k') was used, since it is proportional to the partition coefficient. The capacity factor was calculated from the retention of the substance concerned (tr) and the unretarded component (t0):
k = (tr - t0)/t0
From the results of the reference substances, a plot of log Pow (x-value) versus log k ' (y-value) was constructed, using linear regression analysis. The log k value of each component of the test substance was compared with the log k ' values of the reference substances with known log Pow values.
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RESULTS
Representative HPLC chromatograms of the test solution, the blank-(mobile phase), the mixture of reference substances and the formamide solution are shown in Figures 1 to 4.
In the HPLC chromatograms of T-5874, using detection at 210 nm, one large peak at 4.1 minutes and a small peak at 7.7 minutes were observed. Between 1.6 and 4 minutes, several small peaks were observed. It was assumed that the large peak derived from the major component of test substance whereas the small(er) peaks derived from impurities.
The t0 was determined to be 0.90 minutes as a mean value of both measurements (i.e 0.89 and 0.90 minutes).
The mean values of the retention times, k ' values, log k ' values, log Pow and Pow values?are summarized in Table 1. The plot of log Pow (x-value) versus log k ' (y-value) from the reference substances is shown in Figure 5.
Table 1 Results of the test.
Substance
tr1 k'
log k ' log Pow
pow
benzylalcohol toluene 1,4-dichlorobenzene fluoranthene dibenzyl 2,4-DDT
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
test substance peak 1 peak 2 (major comp.) several small peaks
7.70 4.05 1.6 / 4
7.598
0.881
5.6 2
3.525
0.547
4.4 2
0.79/ 3.47 -0.10/0.54 2.2/4.4 2
1 Mean value of the retention times of both chromatograms. ' Interpolated from the regression line: y = 0.288x - 0.730 (r=0.972, n=6) Note : The calculations were performed using not rounded values.
39.8 xlO4 2.51X104 1.50X102 / 2.51X104
The temperature of the mobile phase was 20.2-20.6C during the measurements.
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T-5874 CHANNEL H
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INJECT 87-84-94 89:44:55 STOk'EB TU BIN # S3
38136/121286
FILE 1.
METHOB
FINALVST : KRH
PEAK#
areas:
1-3*
0. 495 2. 459
3 1, 083
4"
18.792 1. 288
6 2. 538
?=1; 9
H. 468 2. 476
i, 977
18 0. 636
11 1. 864
12 2. 06 4
13 1. 718
14 11.232
15 10. 435
16 38. 458
1? 2. 014
I 0 !HL
h:'0.
37-84-94 88s 44:5b 8. RUN 10 INDEX
PT
0. 15 0. ?5 0. 34 0. 53 0. 79 1- Pi 1- <9 1. 6 1, 71 1. 91 ~.r -32. 62 7. 19 3. 51 3. 67 4. 05 7. 69
AREA BC
1399 02 6951 8? 3061 02 53 l 9 88 364fl 85 7175 85 132? 8? 70100 02 3588 02 1797 03 5268 92 5842 03 4857 02 31750 02 29495 92 108707 93 5694 01
Iyvyb1
CH= "H" NS- 1. BIN 53
Figure 1 HPLC chromatogram of the test solution of 330 mg/1 T-5874 in mobile phase.
Column Mobile phase Flow Detection
Injection volume
LiChrospher 100 RP-18; 125x4 (I.D.) mm; dp=5 um 75/25 (v/v) acetoni tri 1e/Mi 11i-Q water 1 mi/min
UV, at X = 210 nm 10 yl
Note: The peaks between 0 and 1.6 minutes were also observed in the chromatograms of the blank (see Figure 2). Therefore it was assumed that these peaks did not derive from the test substance.
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T-5874
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CHANNEL H
INJECT 87-04-94 89:94:84 STORED TO BIN 4 56
DATA SAVED TO BIN # 5b
30 196/121286
FILE 1.
METHOD
ANALYST : KRH
IAK#
AREfi/I
1 1. 543
L
3
10--.5p1ST5--
4. ~...icrm 3 .cJr
7 7 y K 6. 901
. i' 2. 589
8 11.576
TOTAL
100.
87-84-94 09:34:84 8. RUN 12 INDEX 12
RT
0. 15 8. 25 8. 24 8, 57 8. 67 8. 99 1. 18 :1- 61
AREA BC
Ff7'7 82 5976 82 21 98 8? 21 449 87 4415 87: 2922 82 1476 8 -i 6579 81
5637:4
CH= "A" P5= 1. BIN 56
Figure 2 HPL.C chromatogram of a blank (mobile phase).
Column
Mobile phase Flow Detection Injection volume
LiChrospher 100 RP-18; 125x4 (I.D.) mm; dp=5 ym 75/25 (v/v) acetonitrile/Milli-Q water
1 ml/min UV, at X = 210 nm 10 yl
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T-5874 L-HHHHEL fi
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I N J E C T 0 7 - 0 4 - 9 4 0 9 : 5 0 : 1 7 sT HE Eli TO BIN #
lifilfi SfiVEB TO BIN # 57
3819 6,-"'121286
F IL E
1.
METHOD
0.
07-04-94 09:58:17
R U N 14
INDEX 14
fiN R L V S T : KRH
PEAK#
1
w ?
4
r;
6
r
8 9 10 11 12 13 14 15
to tal
fiR E fi*;
0. 023 0. 145 Ci. 06 0. 879 01. 073 0. 139 8. 977 0. 118 11.317 0. O H 10. 567 15. 524 38. 584 0. 816 18. 878
1 0101.
RT
8 . 14 0 . 25 0. 34 0. 53 -8 , 79 0 . 96 1 . 14 1 . 61 2 . 24 2 . 61 2. 89 4* HH 5. s 6 98 9. 13
H R F f i ec
1131 02 7079 02 291 7 08 42751 08 3542 05 6771 06 476774 03 5460 01 550640 0-1
611 02 514-177 03 755374 01 1877339 01
15361 01 645770 01
4865613
CH= ufi" PS= 1. BIN -57
Figure 3 HPLC chromatogram of the mixture of reference substances in mobile phase.
Column Mobile phase Flow
Detection Injection volume
LiChrospher 10 RP-18; 125x4 (I.D.) mm; dp=5 ym 75/25 (v/v) acetonitrile/Milli-Q water 1 ml/min
UV, at X = 210 nm 10 yl
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CHANNEL
INJECT 0 7 - 0 4 - 9 4 07:1:7:44 STORED T O BIN # 49
DATA
3 8 1 9 6 / 1 2 1 4 8 fc.
FILE 1,
METHOD
ANALYST: KRH
PEAK#
ARERX
1 2
S4T s
~7
o Q 10 11
0. 35 1. 189 8. 79 18. 44 1. 884 54.548
6. 246 8. 12 3. 094 8. 424 20, 915
TOTAL
100.
07-04-94 07:37:44
0. RUN 6 INDEX 6
RT
8. 16 0.
-. 55 0. 78 0, 89 1- 11 1. 47 1. 87 7, 9 3. 65
AREA BC 2624 82 8926 07 5930 02 78363 88 14143 86 4 0 9 4 37 08 * .`79 05
999 85 73224 02
3185 83 1,56999 01 750599
CH= "A" PS= 1. BIN 49
Figure 4 HPLC chromatogram of a formamide solution of 0.2 g/1 in mobile phase.
Column
Mobile phase Flow Detection Injection volume
LiChrospher 100 RP-18; 125x4 (I.D.) mm; dp=5 ym 75/25 (v/v) acetonitrile/Mi11i-Q water 1 ml/min UV, at X = 210 nm
10 yl
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Figure 5 Plot of log Pow (x-value) versus log k ' (y-value) from the reference substances.
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