Document Z4MdBy4GJnB1JqwpB9NEMNY68
N OTOX
Sof ty a Environm*ntal Restarch LV.
REPORT
EVALUATION OF THE MUTAGENIC ACTIVITY OF T-5874
IN THE AMES SALMONELLA/MICROSOME TEST (WITH INDEPENDENT REPEAT)
NOTOX Project 115932 NOTOX Substance 38187
page 1 of 23 -
RECI-=IVED
NOV 9 1994
T(:)AiGULOGY Hambakenwetering 3
P.O. Box 3476, S203 DL 's@Hertogenbosch. The NeO*fk*ml@@ Tel.:073 - 41 9S 75 Fax: 073 - 41 85 43
REPORT
EVALUATION OF THE MUTAGENIC ACTIVITY OF
T-5874
IN THE AMES SALMONELLA/MICROSOME
TEST
(WITH INDEPENDENT REPEAT)
NOTOX Project 115932 NOTOX Substance 38187
page 1 of 23 -
REC-,F-IVED
,NO@i 9 1994
TOA,@.,uLOGY
T-5874 STATEMENTOF GLP COMPLIANCE
NOTOX Project 115932
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.
With the exception that the stability of the test substance in the vehicle was unknown.
Study Director
Ing. E.J. van de Waart Date:
page 2
T-5874 QUALITY ASSURANCE STATEMENT
NOTOX Project 115932
NOTOX B.V., Is-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
24-01-1994 26-01-1994 15-03-1994
REPORTING DATES
24-01-1994 26-01-1994 15-03-1994
Quality Assurance Manager
C.J. Mitchell B.Sc.
Date: -)-o page 3 -
T-5874 REPORT APPROVAL STUDY DIRECTOR:
MANAGEMENT:
NOTOX Project 115932
Ing. E.J. van de Waart
.......................... Date:CI64@V*
Dr. I.C. Enninga Technical Director
.....................
Date: :Zo ot+ I
Li.
page 4
T-5874
PREFACE Sponsor
Study Monitor Testing Facility
Study Director Technical Coordinator Study Plan
NOTOX Project 115932
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
Ing. E.J. van de Waart
G. van Oort
Start Completed
January 25, 1994 February 10, 1994
TEST SUBSTANCE
Identification Description Batch Purity Specific Gravity Instructionsfor test substance storage Stability under storage conditions Expiry date Stable for at least 4 hours in vehicle
T-5874 Cream solid 2334 100% 1.7
At room temperature in the dark
Stable January 01, 1996
Water
no
Dimethylsulphoxide: not indicated
VEHICLE
The test substance was dissolved in dimethylsulphoxideof spectroscopic quality (Merck). Test substance concentrations were prepared directly prior to use.
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T-5874
NOTOX Project 115932
GUIDELINES
The study procedures described in this report were based on the following guidelines:
. Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Guideline no. 471: "Genetic Toxicology: Salmonella typhimurium Reverse Mutation Assay", (adopted May 26, 1983).
- European Economic Community (EEC), Directive 92/69/EEC. Annex V of the EEC Directive 67/548/EEC, Part B: Methods for the Determination of Toxicity; B.14: "Other Effects-Mutagenicity: Salmonella typhimurium Reverse Mutation Assay". EEC Publication no. L383 (adopted December, 1992).
ARCHIVING
NOTOX B.V. will archive the following data for at least 10 years: protocol, report, test article referencesample and raw data.
OBJECTIVE
Purpose of the study
The objective of this study was to evaluate the test substance for its ability to induce reverse mutations in a gene of histidine-requiring Salmonella typhimurium bacterial strains to produce histidine-independent strains of these micro-organisms.The assay was conducted in the absence and in the presence of a metabolic system (S9-mix).
Justificationfor selectionof the test system
The Ames test has been shown to be a rapid and inexpensive indicator for the mutagenic activity of a wide range of chemical compounds. The bacterial strains employed were capable of detecting both frameshift and base-pair substitution mutagens.
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T-5874
NOTOX Project 115932
MATERIALS AND METHODS
TEST SYSTEM
Test System
Salmonella typhimurium bacteria
Rationale
Recognized by the international guidelines as the recommended test system (e.g. EPA, OECD, EEC).
Source
Dr. Bruce N. Ames, University of California at Berkeley, U.S.A. (TA1535, TA1537 and TA98; 1987 and TA100; 1993).
The characteristicsof the individualstrains were as follows:
Strain
Histidine mutation
Mutation type
TA1537
hisC3076
Frameshift
TA98
hisD3052/R-factor*
Frameshift
TA1535
hisG46
Base-pair substitutions
TAIOO
hisG46/R-factor*
Base-pair substitutions
*: R-factor = plasmid pKMI01 (increases error-prone DNA repair)
Each tester strain contained the following additional mutations:
rfa : deep rough (defective lipopolysaccharidecellcoat)
gal : mutation in the galactose metabolism chl : mutation in nitrate reductase bio : defective biotin synthesis uvrb: loss of the excisionrepairsystem (deletionof the ultraviolet-
repair B gene)
Stock cultures of the four strains were stored in liquid nitrogen (-1960C).Strains were regularly checked to confirm their histidinerequirement,crystal violet sensitivity,ampicillin resistance(TA98 and .TA100),UV-sensitivity and the number of spontaneous revertants.
CELL CULTURE
Preparation of bacterial cultures
Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxcid no. 2) and incubated in a shaking bath (370C, 150 spm), until the cultures reached an O.D. of 0.4 at 700 nm (109 cells/ml). Freshly grown cultures of each strain were used for a test.
- page 7 -
T-5874
NOTOX Project 115932
Agar plates
Top agar Environmental conditions
Agar plates (o 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Oxoid, code L28) in Vogel-Bonner Medium E (*), 10 g glucose, 0.5 mg biotin and 0.6 mg histidine. (*) Vogel, H.J. and Bonner, D.M., 1956, Acetylornithinaseof Escherichia coli: partial purification and some properties. J. Biol. Chem., 218, 97-106.
Vogel-Bonner Medium E containing 0.6% (w/v) purified agar was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 1200C.
All incubations were carried out in the dark at 370C. The temperature was monitored during the experiment.
REFERENCE SUBSTANCES
Negative control: The vehicle of the test article.
Positive controls:
Without Strain TA1535 TA1537
TA98 TA100
metabolic activation (-S9-mix): Chemical sodium azide (SA) (Fluka) 9-aminoacridine (9AC) (Janssen Chimica) daunomycine (DM) (Sigma) methylmethanesulfonate (MMS) (Merck)
Concentration/plate 1 pg
60 pg
4 jig 650 jig
Solvent Saline Saline
Saline DMSD
With metabolic activation (+S9-mix):
Strain TA1535,
Chemical
TA1537 2-aminoanthracene (2AA)
TA98,
TA100
2-aminoanthracene (2AA)
(Sigma)
Concentration/plate 5 Ug 0.5 ug
Solvent DMSO DMSO
Solvents for reference substances Saline = physiological saline (Medital Pharma Ned. B.V.) DMSO = dimethylsulphoxide of spectroscopic quality (Merck).
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T-5874
NOTOX Project 115932
METABOLIC ACTIVATION SYSTEM
Preparation of S9-homogenate:
Rat liver microsomal enzymes were routinely prepared from adult male Wistar or Sprague Dawley rats, which were obtained from BRL, Switzerland.
The animals were housed at NOTOX in a special room under standard laboratory conditions, as described in the SOP'S. The rats were injected intraperitoneallywith a solution (20% w/v) of Aroclor 1254 (500 mg/kg body weight) in corn oil. Five days later, they were killed by decapitation; (they were denied access to food for at least 12 hours preceding sacrifice). The livers of the rats were removed aseptically, and washed in cold (OOC) sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na2-EDTA. Subsequently the livers were minced in a blender and homogenized in 3 volumes of phosphate buffer with a Potter homogenizer. The homogenate was centrifuged for 15 min at 9000 g. The supernatant (S9) was transferred into sterile ampules, which were stored in liquid nitrogen (-1960C).
Preparation of S9-mix:
S9-mix was prepared immediately before use and kept on ice during the test. S9-mix contained per 10 ml: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 ml aqua bidest; 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml 0.08 M MgCl2 solution; 1 ml 0.33 M KC1 solution, and 0.5 ml S9. The above solutionswere mixed and filter (0.22 um)-sterilized(apart from the S9fraction,which was added after filter-sterilizationof the S9-mix components).
EXPERIMENTAL PROCEDURE
Selection of dose levels
Selection of an adequate range of doses was based on a preliminary test with strain TA100, both with and without S9-mix. Eight concentrations were tested in triplicate(this preliminarytest was reportedas a part of the first experiment of the mutagenesis assay). The highest concentration of test article used in the subsequent mutagenesis assay was 5 mg/plate.
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T-5874
NOTOX Project 115932
Direct plate incorporationassay
Five different doses (with approximatelyhalf-log steps) of the test substance were tested in triplicate in each strain. The test substance was tested both with and without S9-mix in each strain, in two independent experiments. Top agar.in top agar tubes was melted and heated to 450C. The following solutions were successively added to 3 ml top agar: 0.1 ml of a fresh bacterialculture (109 cells/ml) of one of the tester strains,0.1 ml of a dilution of the test substance in DMSO, and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidificationof the top agar, the plates were turned and incubated in the dark at 370C for 48 h. After this period revertant (histidineindependent) colonies were counted.
Ames, B.N., McCann, J. and Yamasaki, E., 1975, Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test, Mutation Res., 31, 347-364. Maron, D.M. and Ames, B.N., 1983, ifevisedmethods for the Salmonella mutagenicity test, Mutation Res., 113, 173-215. Erratum, 1983, Mutation Res., 113, 538.
Colony counting
The revertant colonies (histidine independent)were counted automatically with an Artek model 880 colony counter or manually, if less than 40 colonies per plate were present. Plates with sufficienttest article precipitate to interfere with automated colony counting were counted manually.
ACCEPTABILITY OF ASSAY
An Ames test is consideredacceptable if it meets the followingcriteria: a) The negative control data (number of spontaneous revertants per plate)
should reasonably be within the laboratory background historical range for each tester strain. b) The positive control chemicals should produce responses in all tester strains which also reasonably are within the laboratoryhistorical range documented for each positive control substance. Furthermore, the mean plate count should be at least two times the concurrent vehicle control group mean. c) The selected dose range should include a clearly toxic concentration as demonstrated by the preliminary test with strain TA100 or should extend to 5 mg/plate.
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T-5874
NOTOX Project 115932
DATA EVALUATIONAND STATISTICALPROCEDURES
No formal hypothesis testing was done. A test substance is considered negative (not mutagenic) in the Ames test if: a) The total number of revertantsin any tester strain at any concentration
is not greater than two times the solvent control value, with or without metabolic activation. b) The negative response should be reproduciblein at least one independently repeated experiment.
A test substance is considered positive (mutagenic)inthe Ames test if: a) It induces at least a 2-fold, dose related increase in the number of
revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant.If the test substanceshowed in the first test only a positive response at one or two concentrations,the assay was repeated with doses just below and exceeding those showing positive effects in the firsttest. b) The positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluationdecision.
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T-587 4
NOTOX Project 115932
RESULTS
TOXICITY OF THE TEST SUBSTANCE
The survival of the TA100 culture was determined by comparing the number of colonies on the plates containing the test substance with those on the solvent control plates. Both in the absence and presence of S9-mix the survival of strain TA100 was not reduced up to and including test substance concentrations of 5000 pg/plate (Table 1). Based on these data, the test substance was tested up to and including a concentration of 5000 ug/plate in the absence and presence of S9-mix.
THE AMES SALMONELLA/MICROSOME PLATE TEST
Tables 1 and 2 show the results of the Salmonella/microsome plate test with T-5874. All bacterial strains showed negative responses over the entire dose range of the test substance, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments. The negative and strain-specific positive control values were within our laboratory background historical ranges indicating that the test conditions were optimal and that the metabolic activation system functioned properly.
CONCLUSION
Based on the results of this study it is concluded that the test substance is not mutagenic in the Ames Salmonella/microsome assay.
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T-5874
NOTOX Project 115932
TABLE 1 MUTAGENIC RESPONSE OF T-5874 IN THE AMES SALMONELLA/MICROSOME PLATE TEST.
Experiment 1
Dose (ug/plate)
Mean number of revertant (His+) colonies/ 3 replicateplates ( S.D.) with different strains of S.typhimurium
TA1535
TA1537
TA98
TA100
Without S9-mix
3 10 33 100 333 1000 33302
50002
10 + 4
8t 3 8+ 5
10 t 3 10 + 2
8+ 2 7+ 3 9+ 2 8+ 3
8+ 3
24 + 4 26 + 3 23 + 2 22 + 2 22 + 3
124 + 12 129 + 7
146 + 22 138 + 32 149 + 17
138 + 11 146 + 15 144 + 14
Solvent control'
6+ 2
7+ 3
21 + 2
163 + 11
Positive control 274 t 28 577 + 89 643 + 54 1372 + 29
----------------------------------------------------------------------
With S9-mix
3 10 33 100 333 1000 33302
50002
9+ 2 11 + 2
8t 4 5+ 5 7t 3
7+ 3 6+ 4 6+ 3 6+ 3 7+ 1
23 + 4 25 + 2 28 + 3 24 + 5 25 + 4
141 + 8 143 t 4 130 + 10
136 7 147 + 7 140 + 5 150 + 19
147 + 8
Solvent control'
7+ 2
8+ 4
25 + 3
170 + 11
Positive control 218 + 34 665 + 86 253 + 7
601 + 31
----------------------------------------------------------------------
1 0.1 ml DMSO 2 Test-substanceprecipitated slightly on the plates
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T-5874
NOTOX Project 115932
TABLE 2 MUTAGENIC RESPONSE OF T-5874 IN THE AMES SALMONELLA/MICROSOME TEST.
Experiment 2
PLATE
Dose (ug/plate)
Mean number of revertant (His+) colonies/ 3 replicateplates ( S.D.) with different strains of S.typhimurium
TA1535
TA1537
TA98
TA100
Without S9-mix
100 333 1000 33302
50002
7+ 3
10 t 1 8+ 2
21 t 10 17 + 4
8+ 2 8+ 2 6+ 3 61 12 153
30 + 2 28 + 7 29 + 1 29 -+ 3 25 + 5
108 + 18 126 + 3 124 + 10 161 + 6 129 + 6
Solvent control'
13 + 4
4+ 2
28 + 1 123 + 7
Positive control
257 + 13 391 + 45 477 + 69 1292 + 59
----------------------------------------------------------------------
With S9-mix
100 333 1000 33302
50002
11 + 4 6+ 4 7+ 3
11 + 3 5+ 2
5+ 3 5+ 4 7+ 2 6+ 1 7+ 2
34 + 13 30 + 5 36 + 8 40 + 12 31 + 5
120 + 6 118 + 15 133 + 6 114 + 17 143 + 2
Solvent control'
5+ 1
4+ 2
26 + 7 114 + 3
Positive control 174 t 55 216 + 56 339 + 23 649 + 56 ----------------------------------------------------------------------
0.1 ml DMSO 2 Test substance precipitatedslightly on the plates 3 One plate infected with other bacteria
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T-5874
NOTOX Project 115932
APPENDIX I
Evidence of test article precipitate on the plates is recorded by addition of the following precipitation code.
PRECIPITATION CODE
Definition
Characteristics
Slight Precipitate
Moderate Precipitate Heavy Precipitate
Distinguished by noticeable precipitate on the plate. However, the precipitate does not influence automated counting of the plate. Distinguished by a marked amount of precipitate on the plate, requiring the plate to be hand counted. Distinguished by a large amount of precipitate on the plate, making the required hand count difficult.
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NOTOX Project 115932
APPENDIX 2
Individual plate counts; (following pages)
Strain Experiment
TA1535 1
----------------------------------------------------------
WITHOUT S9-MIX
plate
1
2
3
MEAN
SD
Dose(pg/plate)
100 333 1000 33301 50001
11
5 13
6 8 11
12
3
8
13
9
7
9 13
9
10 + 4 8+ 3 8+ 5
10 + 3 10 + 2
Solvent control
8
4
7
6+2
Positive control
296 283 242
274 + 28
----------------------------------------------------------
WITH S9-MIX
plate
1
2
3
MEAN
so
Dose(pg/plate)
100 333 1000 33301 50001
12
8
8
11 13
9
5 7 12
1 10
5
5 5 11
9+2 11 + 2
8+4 5+5 7 +3
Solvent control
9
5
8
7 +2
Positive control
185 217 253
218 + 34
----------------------------------------------------------
Test substance precipitated slightly on the plates
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T-5874
NOTOX Project 115932
Strain Experiment
TA1537
---------------------------------------------------------
WITHOUT S9-MIX
plate
1
2
3
MEAN
SD
Dose(ug/plate)
100 333 1000 33301 50001
6 10
7
4
9
9
10 11
7
9 10
4
6 11
8
8+2 7+3 9+2 8+ 3 8 +3
Solvent control
5 11
6
7+3
Positive control
679 516 535
577 + 89
---------------------------------------------------------
plate
WITH S9-MIX
1
2
3
MEAN
SD
Dose(ug/plate)
100 333 1000 33301 50001
5 5 10
692
5
9
4
3
7
8
7
8
7
7+3 6+4 6+3 6+3 7 ,1
Solvent control
3 11 10
8+4
Positive control
645 760 591
665 + 86
---------------------------------------------------------
Test substance precipitated slightly on the plates
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T-5874
NOTOX Project 115932
Strain
TA98
Experiment
-----------------------------------------------------------
WITHOUT S9-MIX
plate
1
2
3
MEAN
SD
Dose(pg/plate)
100 333 1000 33301 50001
22 29 21 29 26 23 25 23 22 20 22 23 24 19 23
24 + 4 26 + 3 23 + 2 22 + 2 22 + 3
Solvent control
22 19 21
21 + 2
Positive control
696 588 644
643 + 54
----------------------------------------------------------
plate.
WITH S9-MIX
1
2
3
MEAN
SD
Dose(ug/plate)
100 333 1000 33301 50001
19 27 24 26 22 26 31 28 25 23 29 20 27 28 21
23 + 4 25 + 2 28 + 3 24 + 5 25 + 4
Solvent control
28 23 23
25 + 3
Positive control
260 246 254
253 + 7
-----------------------------------------------------------
Test substance precipitated slightly on the plates
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T-5874
NOTOX Project 115932
Strain Experiment
TA100 1
----- ----------------------------------------------------
WITHOUT S9-MIX
plate
1
2
3
MEAN
SD
Dose(ug/plate)
3 10 33 100 333 1000 33301 50001
125 112 136 123 129 136 157 160 120 101 158 154 134 145 168 138 148 127 149 159 130 158 131 144
124 + 12 129 + 7 146 + 22 138 + 32 149 + 17 138 + 11 146 +-15 144 + 14
Solvent control
150 168 170
163 + 11
Positive control
1383 1339 1395
1372 + 29
----------------------------------------------------------
WITH S9-MIX
plate
1
2
3
MEAN
SD
Dose(ug/plate)
3 10 33 100 333 1000 33301 50001
143 133 148 144 146 138 140 128 121 137 142 128 150 139 153 146 138 136 130 151 168 139 155 148
141 + 8 143 + 4 130 + 10 136 +, 7 147 + 7 140 + 5 150 + 19 147 + 8
Solvent control
162 182 166
170 + 11
Positive control
637 586 581
601 + 31
----------------------------------------------------------
Test substance precipitated slightly on the plates
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T-5874
NOTOX Project 115932
Strain Experiment
TA1535 2
----------------------------------------------------------
WITHOUT S9-MIX
plate
123
MEAN
SD
Dose(ug/plate)
100 333 1000 33301 50001
11 6 5 10 9 10
8 7 10 17 33 14 21 16 14
7+3 10 + 1
8+2 21 + 10 17 + 4
Solvent control
13 17 9
13 + 4
Positive control
245 256 270
257 + 13
----------------------------------------------------------
plate
WITH S9-MIX 123
MEAN
SD
Dose(ug/plate)
100 333 1000 33301 50001
7 14 11 3 11 5 4 10 8
8 11 13 45 7
11 + 4 6+4 7+ 3 11 + 3 5+ 2
Solvent control
45 6
5+ 1
Positive control
ill 215 196
174 + 55
----------------------------------------------------------
Test substance precipitatedslightly on the plates
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Strain Experiment
TA1537 2
----------------------------------------------------------
plate
WITHOUT S9-MIX
12 3
MEAN
SD
Dose(pg/plate)
100 333 1000 33301 50001
a 10 6 7 7 10 68 3
56 7 INF 12 15
8+2 8+2 6+3 6+1 12 ; 15
Solvent control
426
4+2
Positive control
441 355 378
391 + 45
----------------------------------------------------------
plate
WITH S9-MIX 12 3
MEAN
so
Dose(pg/plate) '
100 333 1000 33301 50001
7 25 9 25
785 567 488
5+3 5 +4 7 +2 6+1 7+2
Solvent control
462
4+2
Positive control
276 208 165
216 + 56
----------------------------------------------------------
1 Test substance precipitatedslightly on the plates
INF = Plate infected with other bacteria
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ME-In
T-5874
NOTOX Project 115932
Strain Experiment
TA98 2
----------------------------------------------------------
WITHOUT S9-MIX
plate
1
2
3
MEAN
SD
Dose(pg/plate)
100 333 1000 33301 50001
30 31 28 30 34 21 30 29 29 26 32 30 31 24 21
30 + 2 28 + 7 29 + 1 29 + 3 25 + 5
Solvent control
27 29 28
28 + 1
Positive control
437 557 438
477 + 69
----------------------------------------------------------
WITH S9-MIX
plate
123
MEAN
SD
Dose(ug/plate)
100 333 1000 33301 50001
29 25 49 25 35 30 43 38 28 31 36 53 36 27 30
34 + 13 30 + 5 36 + 8 40 + 12 31 + 5
Solvent control
18 30 31
26 + 7
Positive control
314 343 360
339 + 23
----------------------------------------------------------
Test substance precipitated slightly on the plates
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Strain Experiment
TAIOO 2
-----------------------------------------------------------
WITHOUT S9-MIX
plate
1
2
3
MEAN
SD
Dose(pg/plate)
100 333 1000 33301 50001
110 124 89 123 126 128 114 125 134 168 160 156 128 135 123
108 + 18 126 + 3 124 + 10 161 + 6 129 + 6
Solvent control
124 129 115
123 + 7
Positive control
1252 1360 1265
1292 + 59
-----------------------------------------------------------
WITH S9-MIX
plate
1
2
3
Dose(pg/plate)
MEAN
SD
100 333 1000 33301. 50001
118 116 127 104 134 115 133 138 127 100 133 110 141 143 144
120 + 6 118 + 15 133 + 6 114 + 17 143 + 2
Solvent control
114 111 116
114 + 3
Positive control
585 677 685
649 + 56
-----------------------------------------------------------
Test substance precipitated slightly on the plates
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