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EC number: 946-383-6 | CAS number: -
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 12. Jun. 2018 to 21. Jun. 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- OECD Guidelines for the Testing of Chemicals Part 471, adopted 21. Jul. 1997 “Bacterial Reverse Mutation Test“
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- Commission Regulation (EC) No. 440/2008, EU-Method B.13/14 adopted 30. May 2008 “Mutagenicity –Reverse mutation test using bacteria”
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
Test material
- Reference substance name:
- Reaction mass of 2-ethylhexyl (3-isocyanato-4-methylphenyl)carbamate and 2-ethylhexyl (5-isocyanato-2-methylphenyl)carbamate and 2-ethylhexyl (3-isocyanato-2-methylphenyl)carbamate
- EC Number:
- 946-383-6
- Molecular formula:
- C17H24N2O3
- IUPAC Name:
- Reaction mass of 2-ethylhexyl (3-isocyanato-4-methylphenyl)carbamate and 2-ethylhexyl (5-isocyanato-2-methylphenyl)carbamate and 2-ethylhexyl (3-isocyanato-2-methylphenyl)carbamate
- Test material form:
- liquid: viscous
- Details on test material:
- Name: Trixene AS
Batch no.: OP544-153/1801239973
Appearance: clear, light yellow-green, viscous liquid
Composition: Reaction mass of 2-ethylhexyl (3-isocyanato-4-methylphenyl)carbamate and 2-ethylhexyl (5-isocyanato-2-methylphenyl)carbamate and 2-ethylhexyl (3-isocyanato-2-methylphenyl)carbamate
EINECS-No.: 946-383-6
Molecular formula: C17-H24-N2-O3
Purity: "multi-constituent substance made up of a mixture of isomers
Homogeneity: Homogeneous
Vapour pressure: not stated
Expiry date: 01. Sep. 2019
Storage Room: Temperature (20 ± 5°C)
Constituent 1
- Specific details on test material used for the study:
- No further details specified in the study report.
Method
- Target gene:
- Histidene
Species / strain
- Species / strain / cell type:
- S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535
- Details on mammalian cell type (if applicable):
- Origin and Culture
All Salmonella typhimurium strains were obtained from TRINOVA BioChem GmbH (batch: TA97a: 4997D, TA98: 5011D, TA100: 4996D, TA102: 4982D, TA1535: 5012D) and were stored as lyophilizates in the refrigerator at 2-8 °C.
The lyophilizates were used to prepare permanent cultures which were filled into vials and stored at < - 75 °C.
Eight hours before the start of each experiment, an aliquot of a permanent culture per strain to be used was taken from the deep freezer to inoculate a culture vessel containing nutrient broth. After incubation overnight for eight hours at 37 ± 1 °C, the cultures were used in the experiment. During the test, the cultures were stored at room temperature as to prevent changes in the titre. - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 was obtained by Trinova Biochem GmbH, Gießen.
Batch nos.: batch: 3850 for the first experiment and batch: 3913 and 3833 for the second experiment
Specification: produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intraperitoneally. - Test concentrations with justification for top dose:
- The stock solution was used to prepare the geometric series of the concentrations to be tested.
The following nominal concentrations were prepared for the first experiment:
5 μL/plate, 1.5 μL/plate, 0.5 μL/plate, 0.15 μL/plate and 0.05 μL/plate
Based on the non- toxic results of the first experiment, the test item was tested up to con-centrations of 5 μL/plate in the presence and absence of S9-mix in all bacteria strains using the pre-incubation method.
The following nominal concentrations were prepared for the second experiment:
5 μL/plate, 2.5 μL/plate, 1.25 μL/plate, 0.63 μL/plate, 0.31 μL/plate, 0.16 μL/plate, 0.08 μL/plate and 0.04 μL/plate - Vehicle / solvent:
- In a non-GLP pre-test, the solubility of the test item was tested in DMSO.
The liquid test item is sufficiently soluble in DMSO.
Based on the non-GLP pre-test, DMSO was chosen as vehicle, because the test item was sufficiently soluble and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
On the day of the start of the first and the second experiment, a stock solution containing 50 mL/L (nominal concentration) of the test item in DMSO was prepared. The test item solution was not sterile filtrated before use.
The following substances were used as solvent controls:
• DMSO, batch: 246245640 and batch: 187256959, for the positive controls nitro-phenylendiamine, benzo-a-pyrene and 2-amino-anthracene and the test item
• Demineralised water, batch: 20170309 and batch: 20180221 for the positive control sodium azide
Controls
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- benzo(a)pyrene
- other: 4-Nitro-1,2-phenylene Diamine; 2-Amino-Anthracene
- Details on test system and experimental conditions:
- Culture of Bacteria
Eight hours before the start of each experiment, one vial permanent culture of each strain was taken from the deep freezer and an aliquot was put into a culture flask containing nutri-ent broth. After incubation for eight hours at 37 ±1 °C, the cultures were used in the experi-ment. During the test, the cultures were stored at room temperature as to prevent changes in the titre.
Conduct of Experiment
Preparations
Different media and solutions were prepared preliminary (exact production dates are docu-mented in the raw data).
On the day of the test, the bacteria cultures were checked for growth visually. The incubation chambers were heated to 37 ±1 °C. The water bath was turned to 43 ±1 °C. The table sur-face was disinfected.
The S9 mix was freshly prepared and stored at 0 °C.
Experimental Parameters
First Experiment
Concentrations tested: 5 / 1.5 / 0.5 / 0.15 / 0.05 μL/plate
Incubation time: 48 h
Incubation temperature: 37 ±1 °C
Tested strains: TA97a, TA98, TA100, TA102, TA1535
Method: plate incorporation method
Second Experiment
Concentrations tested: 5 / 2.5 / 1.25 / 0.63 / 0.31 / 0.16 / 0.08 / 0.04 μL/plate
Incubation time: 48 h
Incubation temperature: 37 ±1 °C
Tested strains: TA97a, TA98, TA100, TA102, TA1535
Method: pre-incubation method
Description of the Method
General preparation
Per bacteria strain and concentration, three plates with and three plates without metabolic activation (-S9) were used.
For the top agar 100 mL agar basis was melted in a microwave oven, 10 mL of the histidine-biotin-solution 0.5 mM was added, then the mixture was placed in the water bath at 43 ±1 °C.
Plate incorporation method
The following materials were gently vortexed in a test tube and poured onto the selective agar plates:
100 μL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
500 μL S9 mix or phosphate buffer (for test without metabolic activation).
100 μL bacteria suspension
2000 μL overlay agar (top agar)
The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37 ±1 °C.
Pre-incubation method
The following materials were gently vortexed in a test tube and incubated at 37 ±1°C for 20 minutes:
100 μL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
500 μL S9 mix or phosphate buffer (for test without metabolic activation).
100 μL bacteria suspension
After the pre-incubation for 20 minutes, 2000 μL top agar was added and the tube was gently vortexed. The mixture was poured onto the selective agar plate.
The plates were closed and left to solidify for a few minutes, then inverted and placed in the incubator at 37 ±1 °C.
References and Validity
Genotype Confirmation
Genotype confirmation is performed for each batch of lyophilized bacteria before stock cul-ture preparation.
Histidine requirement
Each strain was streaked on a biotin and a histidine-biotin-plate, using a sterilized wire loop. The plates were incubated for 24 hours at 37 ±1 °C.
Ampicillin/Tetracycline-Resistance (pKM 101, pAQ1)
Each strain was streaked on an ampicillin agar plate and on an ampicillin-tetracycline agar plate. TA1535 was used as control strain, since it is not ampicillin resistant. The plates were incubated for 24 hours at 37 ±1 °C.
UV-sensitivity (uvrB)
Each strain was streaked on a plate, and one half of the plate covered with aluminum foil so that one half of each streak was protected against light. The plates for the strain TA97a, TA100 and TA102 were irradiated for 8 seconds, the plates for the strain TA 98 were irradi-ated for 10 seconds and the plates for the strain TA1535 were irradiated for 6 seconds with a germicidal lamp (254 nm, 30W).
Keeping a distance of 33 cm for the strains TA97a, TA102 and TA1535.
Keeping a distance of 66 cm for the following strains: TA98, TA100.
Incubation for 24 hours at 37 ±1 °C followed.
Crystal violet sensitivity (deep rough/rfa)
For each strain, two plates were used. 0.1 mL of bacteria suspension were mixed with 2 mL Top-Agar and poured on nutrient agar. Sterile paper discs ( 9 mm), each soaked with 10 μL of crystal violet solution (0.1%) were placed into the middle of each plate, followed by incubation for 24 hours at 37 ±1°C.
Spontaneous Revertants
Three replicates, with/without S9, for each solvent which was used in the test, incubation for 48 hours at 37 ±1°C.
Determination of Titre
The titre was determined by dilution of the overnight culture using sodium chloride solution and placing 0.1 mL on maximal-soft agar. Incubation for 48 hours at 37 ±1 °C followed. It should give a density of 109 cells/mL (at the least), two replicates with and without metabolic activation.
Toxicity Control
Performed in experiment 1 only analogously to the titre control with the maximum dose of test item on maximal-soft agar, two replicates with and without metabolic activation, incuba-tion for 48 hours at 37 ±1°C.
Sterility Control
Performed analogously to the test with solvent only and S9 (without adding bacteria) on top agar, incubation for 48 hours at 37 ±1°C, four replicates.
Solubility
Plates were checked for precipitation of test item at the end of the incubation by visual in-spection.
Positive Controls
Using diagnostic mutagens, three replicates were prepared. The stock solutions of the substances were diluted to achieve an application volume of 0.1 mL/plate, incubation for 48 hours at 37 ±1°C. - Rationale for test conditions:
- In accordance with test guidelines.
- Evaluation criteria:
- The colonies were counted visually and the numbers were recorded. A validated spread-sheet software (Microsoft Excel®) was used to calculate mean values and standard devia-tions of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination were calculated as well as the increase factor of revertant induction (mean revertants divided by mean spon-taneous revertants) of the test item solutions and the positive controls. Additionally, the ab-solute number of revertants (mean revertants minus less spontaneous revertants) was given.
A substance is considered to be mutagenic, if a reproducible increase with or without meta-bolic activation of revertant colonies per plate exceeding an increase factor of 2 for the bac-teria strains TA97a, TA98, TA100 and TA102 and an increase factor of 3 for the bacteria strain TA1535 compared to vehicle controls in at least one strain can be observed and there is a concentration-related increase.
A substance is not mutagenic if it does not meets these criteria.
If the criteria listed above are not clearly met, the results will be assessed as equivocal and will be discussed. - Statistics:
- Not specified.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium, other: TA97a
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 5, 2.5 and 1.25 μL/plate (decrease in the number of revertants)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 5 and 2.5 μL/plate (decrease in the number of revertants)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 5, 2.5 and 1.25 μL/plate (decrease in the number of revertants)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 5 μL/plate (decrease in the number of revertants)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 5 and 2.5 μL/plate (decrease in the number of revertants)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- First Experiment
Confirmation of the Criteria and Validity
All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and nearly all were within the historical control data ranges (one exception, only).
Solubility and Toxicity
In the first experiment, the test item showed no precipitates on the plates in all tested concentrations.
No signs of toxicity towards the bacteria strains could be observed. The bacterial back-ground lawn was visible and not affected. The number of revertant colonies was not reduced.
Mutagenicity
No increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.
Therefore, the test item is stated as not mutagenic under the conditions of this experiment.
To verify this result, a further experiment was performed with the pre- incubation method.
Second Experiment
Confirmation of the Criteria and Validity
All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. Nearly all determined values for the spontaneous revertants of the neg-ative controls (one exception, only) were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.
Solubility and Toxicity
In the second experiment, the test item showed no precipitates on the plates in all tested concentrations.
Signs of toxicity were observed in the following concentrations:
TA97a: 5, 2.5 and 1.25 μL/plate (decrease in the number of revertants)
TA98: 5 and 2.5 μL/plate (decrease in the number of revertants)
TA100: 5, 2.5 and 1.25 μL/plate (decrease in the number of revertants)
TA102: 5 μL/plate (decrease in the number of revertants)
TA1535: 5 and 2.5 μL/plate (decrease in the number of revertants)
In all concentrations the bacterial background lawn was visible and not affected.
Mutagenicity
No increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.
Therefore, the test item is stated as not mutagenic under the conditions of this experiment.
Mutagenicity of Test Item
The test item Trixene AS showed no increase in the number of revertants in all bacteria strains in both experiments.
Nearly all negative (one exception, only) and nearly all strain-specific positive control (one exception, only) values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Based on the results of this study it is concluded that Trixene AS is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study.
Acceptability of Study, Discussion
In all experiments, no precipitation of the test item Trixene AS was observed at any of the tested concentrations up to 5 μL/plate.
In the first experiment, the test item caused no cytotoxicity towards all bacteria strains
In the second experiment, the test item caused cytotoxicity towards all bacteria strains in the different concentrations.
The confirmation tests of the genotype did not show any irregularities. The control of the titre was above the demanded value of 109 bacteria/mL.
Nearly all of the means of all replicates of the spontaneous revertants (in negative and sol-vent controls) were within the range of the historical data of the test facility. Nearly all num-bers of revertant colonies of the positive controls were within the range of the historical data of the laboratory, but all were increased in comparison with the negative controls, which demonstrated the mutagenic po-tential of the diagnostic mutagens.
Since all criteria for acceptability have been met, the study is considered valid.
Any other information on results incl. tables
Survey of the Findings
The mean revertant values of the three replicates are presented in the following table
Mean Revertants First Experiment
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
||||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
Demin. Water |
Mean |
79 |
90 |
43 |
49 |
78 |
77 |
324 |
339 |
13 |
16 |
sd |
13.3 |
16.4 |
5.5 |
9.0 |
3.5 |
3.2 |
45.4 |
19.7 |
1.0 |
4.2 |
|
DMSO |
Mean |
80 |
99 |
44 |
41 |
86 |
86 |
299 |
347 |
16 |
14 |
sd |
24.6 |
5.7 |
4.9 |
9.5 |
12.7 |
7.0 |
37.8 |
19.7 |
3.5 |
3.1 |
|
Positive Controls* |
Mean |
512 |
1125 |
392 |
227 |
1256 |
1312 |
1200 |
1163 |
251 |
224 |
sd |
72.8 |
50.8 |
28.0 |
40.9 |
133.1 |
181.7 |
104.0 |
46.9 |
26.0 |
90.9 |
|
f(l) |
6.40 |
11.36 |
8.91 |
5.54 |
16.10 |
15.26 |
4.01 |
3.35 |
19.31 |
16.00 |
|
5 µL/plate |
Mean |
77 |
79 |
42 |
40 |
75 |
77 |
315 |
264 |
11 |
11 |
sd |
6.7 |
11.2 |
2.1 |
8.1 |
10.5 |
2.6 |
39.5 |
36.7 |
3.5 |
1.2 |
|
f(l) |
0.96 |
0.80 |
0.95 |
0.98 |
0.87 |
0.90 |
1.05 |
0.76 |
0.69 |
0.79 |
|
1.5 µL/plate |
Mean |
81 |
87 |
38 |
40 |
86 |
89 |
251 |
256 |
13 |
15 |
sd |
11.0 |
7.0 |
2.6 |
9.9 |
5.9 |
1.2 |
31.1 |
6.9 |
3.2 |
3.0 |
|
f(l) |
1.01 |
0.88 |
0.86 |
0.98 |
1.00 |
1.03 |
0.84 |
0.74 |
0.81 |
1.07 |
|
0.5 µL/plate |
Mean |
81 |
91 |
38 |
39 |
73 |
93 |
276 |
323 |
13 |
15 |
sd |
15.5 |
18.7 |
2.6 |
5.3 |
11.0 |
16.0 |
62.5 |
45.0 |
1.0 |
3.1 |
|
f(l) |
1.01 |
0.92 |
0.86 |
0.95 |
0.85 |
1.08 |
0.92 |
0.93 |
0.81 |
1.07 |
|
0.15 µL/plate |
Mean |
73 |
91 |
40 |
42 |
85 |
74 |
252 |
272 |
17 |
12 |
sd |
2.3 |
15.3 |
3.1 |
12.5 |
5.0 |
1.0 |
34.9 |
58.9 |
3.0 |
2.1 |
|
f(l) |
0.91 |
0.92 |
0.91 |
1.02 |
0.99 |
0.86 |
0.84 |
0.78 |
1.06 |
0.86 |
|
0.05 µL/plate |
Mean |
80 |
79 |
43 |
38 |
83 |
79 |
284 |
297 |
13 |
18 |
sd |
8.5 |
6.1 |
7.9 |
8.0 |
3.1 |
10.1 |
46.1 |
28.4 |
2.1 |
3.6 |
|
f(l) |
1.00 |
0.80 |
0.98 |
0.93 |
0.97 |
0.92 |
0.95 |
0.86 |
0.81 |
1.29 |
f(l) = increase factor
*Different positive controls were used.
Survey of the Findings
The mean revertant values of the three replicates are presented in the following table
Mean Revertants First Experiment
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
||||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
Demin. Water |
Mean |
79 |
90 |
38 |
33 |
104 |
91 |
395 |
352 |
14 |
15 |
sd |
18.2 |
3.0 |
5.1 |
1.2 |
12.7 |
5.0 |
33.3 |
60.4 |
2.9 |
3.5 |
|
DMSO |
Mean |
71 |
81 |
40 |
36 |
87 |
86 |
419 |
384 |
13 |
15 |
sd |
2.5 |
12.3 |
4.9 |
1.7 |
19.3 |
5.9 |
59.0 |
113.4 |
2.1 |
4.6 |
|
Positive Controls* |
Mean |
465 |
701 |
311 |
135 |
543 |
1251 |
1253 |
1456 |
344 |
181 |
sd |
177.1 |
99.8 |
73.4 |
48.0 |
142.6 |
56.2 |
44.1 |
112.0 |
42.3 |
28.1 |
|
f(l) |
6.55 |
8.65 |
7.78 |
3.75 |
5.22 |
14.55 |
2.99 |
3.79 |
24.57 |
12.07 |
|
5 µL/plate |
Mean |
14 |
15 |
4 |
4 |
34 |
32 |
79 |
50 |
6 |
5 |
sd |
3.5 |
1.5 |
1.7 |
1.0 |
4.9 |
8.5 |
25.7 |
10.6 |
2.3 |
2.1 |
|
f(l) |
0.20 |
0.19 |
0.10 |
0.11 |
0.39 |
0.37 |
0.19 |
0.13 |
0.46 |
0.33 |
|
2.5 µL/plate |
Mean |
12 |
36 |
2 |
12 |
37 |
44 |
331 |
377 |
5 |
4 |
sd |
3.5 |
3.1 |
1.0 |
3.0 |
9.5 |
14.1 |
37.8 |
34.9 |
2.1 |
2.5 |
|
f(l) |
0.17 |
0.44 |
0.05 |
0.33 |
0.43 |
0.51 |
0.79 |
0.98 |
0.37 |
0.27 |
|
1.25 µL/plate |
Mean |
18 |
36 |
42 |
38 |
34 |
42 |
339 |
397 |
14 |
12 |
sd |
18.4 |
10.5 |
4.2 |
1.2 |
17.8 |
8.9 |
114.4 |
75.6 |
3.5 |
0.6 |
|
f(l) |
0.25 |
0.44 |
1.05 |
1.06 |
0.39 |
0.49 |
0.81 |
1.03 |
1.08 |
0.80 |
|
0.63 µL/plate |
Mean |
90 |
87 |
40 |
40 |
91 |
87 |
352 |
411 |
13 |
14 |
sd |
18.0 |
23.4 |
3.1 |
2.3 |
13.0 |
6.4 |
36.7 |
100.0 |
4.0 |
1.5 |
|
f(l) |
1.27 |
1.07 |
1.00 |
1.11 |
1.05 |
1.01 |
0.84 |
1.07 |
1.00 |
0.93 |
|
0.31 µL/plate |
Mean |
78 |
100 |
43 |
40 |
88 |
91 |
395 |
349 |
16 |
17 |
sd |
12.5 |
11.1 |
2.1 |
3.5 |
6.4 |
14.9 |
119.8 |
12.2 |
1.2 |
2.3 |
|
f(l) |
1.10 |
1.23 |
1.09 |
1.11 |
1.01 |
1.06 |
0.94 |
0.91 |
1.23 |
1.13 |
|
0.16 µL/plate |
Mean |
79 |
101 |
37 |
38 |
78 |
84 |
336 |
427 |
13 |
17 |
sd |
7.2 |
9.9 |
4.9 |
7.5 |
5.5 |
12.6 |
94.3 |
30.3 |
2.1 |
1.0 |
|
f(l) |
1.11 |
1.25 |
0.93 |
1.06 |
0.90 |
0.98 |
0.80 |
1.11 |
1.00 |
1.13 |
|
0.08 µL/plate |
Mean |
93 |
113 |
38 |
39 |
86 |
99 |
344 |
360 |
15 |
10 |
sd |
25.3 |
18.0 |
1.0 |
5.3 |
12.8 |
28.6 |
89.1 |
32.0 |
4.0 |
0.6 |
|
f(l) |
1.31 |
1.40 |
0.95 |
1.08 |
0.99 |
1.15 |
0.82 |
0.94 |
1.15 |
0.67 |
|
0.04 µL/plate |
Mean |
79 |
115 |
34 |
44 |
83 |
83 |
397 |
363 |
14 |
13 |
sd |
19.1 |
10.3 |
5.6 |
6.2 |
11.1 |
8.1 |
20.1 |
46.2 |
4.2 |
4.6 |
|
f(l) |
1.11 |
1.42 |
0.85 |
1.22 |
0.95 |
0.97 |
0.95 |
0.95 |
1.08 |
0.87 |
f(l) = increase factor
*Different positive controls were used.
DATA OF THE FIRST EXPERIMENT
Determination of Titre
Criterion: The determination of titre should give a number of at least 109cells/mL, correlating to 100 colonies/plate after dilution.
The criterion was fulfilled, exact values are given in the table below.
Titre Values (colonies per plate)
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
|||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
Repl. 1 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
Repl. 2 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
Mean |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
Stand. Dev. |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
Assessment |
ok |
ok |
ok |
ok |
ok |
ok |
ok |
ok |
ok |
ok |
1001 colonies per plate means the bacteria growth was too strong for counting.
Sterility Control
Criterion: No colony per plate may grow.
Sterility (colonies per plate)
|
Demin. water |
DMSO |
Repl. 1 |
0 |
0 |
Repl. 2 |
0 |
0 |
Repl. 3 |
0 |
0 |
Repl. 4 |
0 |
0 |
Assessment |
ok |
ok |
Spontaneous Revertants
The determined values were within the normal range of the laboratory.
Demin. Water
Spontaneous Revertants demin. water (colonies per plate)
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
|||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
Repl. 1 |
94 |
104 |
47 |
40 |
81 |
81 |
344 |
316 |
12 |
21 |
Repl. 2 |
70 |
94 |
46 |
48 |
74 |
75 |
356 |
348 |
14 |
13 |
Repl. 3 |
72 |
72 |
37 |
58 |
78 |
76 |
272 |
352 |
13 |
15 |
Mean |
79 |
90 |
43 |
49 |
78 |
77 |
324 |
339 |
13 |
16 |
sd |
13.3 |
16.4 |
5.5 |
9.0 |
3.5 |
3.2 |
45.4 |
19.7 |
1.0 |
4.2 |
DMSO
Spontaneous Revertants DMSO (colonies per plate)
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
|||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
Repl. 1 |
61 |
93 |
42 |
40 |
84 |
83 |
256 |
356 |
16 |
17 |
Repl. 2 |
108 |
104 |
41 |
32 |
75 |
81 |
328 |
324 |
12 |
15 |
Repl. 3 |
72 |
101 |
50 |
51 |
100 |
94 |
312 |
360 |
19 |
11 |
Mean |
80 |
99 |
44 |
41 |
86 |
86 |
299 |
347 |
16 |
14 |
sd |
24.6 |
5.7 |
4.9 |
9.5 |
12.7 |
7.0 |
37.8 |
19.7 |
3.5 |
3.1 |
Positive Controls
Without metabolic activation:
4-Nitro-1,2-phenylene diamine (NPD) in DMSO, 20 µg/plate
Sodium azide (Na-azide) in demineralized water, 1 µg/plate
With metabolic activation
2-Amino anthracene (2-AA) in DMSO, 1 µg/plate
Benzo-a-pyrene (BaP) in DMSO, 20 µg/plate
Diagnostic Mutagens (colonies per plate)
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
|||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
Substance |
NPA |
2-AA |
NPD |
BaP |
Na-azide |
2-AA |
NPA |
2-AA |
Na-azide |
2-AA |
Repl. 1 |
596 |
1096 |
424 |
244 |
1160 |
1104 |
1304 |
1144 |
276 |
160 |
Repl. 2 |
468 |
1096 |
380 |
180 |
1408 |
1392 |
1096 |
1128 |
224 |
184 |
Repl. 3 |
472 |
1184 |
372 |
256 |
15200 |
1440 |
1200 |
1216 |
252 |
328 |
Mean |
512 |
1125 |
392 |
227 |
1256 |
1312 |
1200 |
1163 |
251 |
224 |
sd |
72.8 |
50.8 |
28.0 |
40.9 |
133.1 |
181.7 |
104.0 |
46.9 |
26.0 |
90.9 |
f(l) |
6.40 |
11.36 |
8.91 |
5.54 |
16.10 |
15.26 |
4.01 |
3.35 |
19.31 |
16.00 |
Rev. abs. |
432 |
1026 |
348 |
186 |
1178 |
1226 |
901 |
816 |
238 |
210 |
f(l) = increase factor.
Rev. abs. = absolute revertants.
Test Item Trixene AS
Mutagenicity Test
Concentration 5 µL/plate (colonies per plate)
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
|||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
Repl. 1 |
84 |
76 |
44 |
44 |
85 |
75 |
288 |
296 |
13 |
10 |
Repl. 2 |
71 |
69 |
41 |
46 |
75 |
80 |
296 |
224 |
7 |
10 |
Repl. 3 |
75 |
91 |
40 |
31 |
64 |
76 |
360 |
272 |
13 |
12 |
Mean |
77 |
79 |
42 |
40 |
75 |
77 |
315 |
264 |
11 |
11 |
sd |
6.7 |
11.2 |
2.1 |
8.1 |
10.5 |
2.6 |
39.5 |
36.7 |
3.5 |
1.2 |
f(l) |
0.96 |
0.80 |
0.95 |
0.98 |
0.87 |
0.90 |
1.05 |
0.76 |
0.69 |
0.79 |
Rev. abs. |
-3 |
-20 |
-2 |
-1 |
-11 |
-9 |
16 |
-83 |
-5 |
-3 |
Concentration 1.5 µL/plate (colonies per plate)
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
|||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
Repl. 1 |
70 |
80 |
37 |
45 |
93 |
90 |
216 |
260 |
9 |
15 |
Repl. 2 |
92 |
86 |
36 |
29 |
82 |
88 |
260 |
248 |
15 |
12 |
Repl. 3 |
81 |
94 |
41 |
47 |
84 |
90 |
276 |
260 |
14 |
18 |
Mean |
81 |
87 |
38 |
40 |
86 |
89 |
251 |
256 |
13 |
15 |
sd |
11.0 |
7.0 |
2.6 |
9.9 |
5.9 |
1.2 |
31.1 |
6.9 |
3.2 |
3.0 |
f(l) |
1.01 |
0.88 |
0.86 |
0.98 |
1.00 |
1.03 |
0.84 |
0.74 |
0.81 |
1.07 |
Rev. abs. |
1 |
-12 |
-6 |
-1 |
0 |
3 |
-48 |
-91 |
-3 |
1 |
Concentration 0.5 µL/plate (colonies per plate)
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
|||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
Repl. 1 |
94 |
94 |
36 |
41 |
62 |
108 |
348 |
312 |
12 |
12 |
Repl. 2 |
86 |
71 |
41 |
43 |
74 |
76 |
244 |
372 |
14 |
18 |
Repl. 3 |
64 |
108 |
37 |
33 |
84 |
94 |
236 |
284 |
13 |
16 |
Mean |
81 |
91 |
38 |
39 |
73 |
93 |
276 |
323 |
13 |
15 |
sd |
15.5 |
18.7 |
2.6 |
5.3 |
11.0 |
16.0 |
62.5 |
45.0 |
1.0 |
3.1 |
f(l) |
1.01 |
0.92 |
0.86 |
0.95 |
0.85 |
1.08 |
0.92 |
0.93 |
0.81 |
1.07 |
Rev. abs. |
1 |
-8 |
-6 |
-2 |
-13 |
7 |
-23 |
-24 |
-3 |
1 |
Concentration 0.15 µL/plate (colonies per plate)
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
|||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
Repl. 1 |
70 |
104 |
37 |
56 |
84 |
75 |
236 |
284 |
20 |
10 |
Repl. 2 |
74 |
94 |
43 |
38 |
90 |
74 |
292 |
208 |
17 |
11 |
Repl. 3 |
74 |
74 |
41 |
32 |
80 |
73 |
228 |
324 |
14 |
14 |
Mean |
73 |
91 |
40 |
42 |
85 |
74 |
252 |
272 |
17 |
12 |
sd |
2.3 |
15.3 |
3.1 |
12.5 |
5.0 |
1.0 |
34.9 |
58.9 |
3.0 |
2.1 |
f(l) |
0.91 |
0.92 |
0.91 |
1.02 |
0.99 |
0.86 |
0.84 |
0.78 |
1.06 |
0.86 |
Rev. abs. |
-7 |
-8 |
-4 |
1 |
-1 |
-12 |
-47 |
-75 |
1 |
-2 |
Concentration 0.05 µL/plate (colonies per plate)
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
|||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
Repl. 1 |
83 |
78 |
49 |
38 |
86 |
78 |
336 |
292 |
11 |
21 |
Repl. 2 |
86 |
86 |
34 |
30 |
80 |
90 |
248 |
272 |
12 |
14 |
Repl. 3 |
70 |
74 |
46 |
46 |
84 |
70 |
268 |
328 |
15 |
19 |
Mean |
80 |
79 |
43 |
38 |
83 |
79 |
284 |
297 |
13 |
18 |
sd |
8.5 |
6.1 |
7.9 |
8.0 |
3.1 |
10.1 |
46.1 |
28.4 |
2.1 |
3.6 |
f(l) |
1.00 |
0.80 |
0.98 |
0.93 |
0.97 |
0.92 |
0.95 |
0.86 |
0.81 |
1.29 |
Rev. abs. |
0 |
-20 |
-1 |
-3 |
-3 |
-7 |
-15 |
-50 |
-3 |
4 |
DATA OF THE SECOND EXPERIMENT
Determination of Titre
Criterion: The determination of titre should give a number of at least 109cells/mL, correlating to 100 colonies/plate after dilution.
The criterion was fulfilled, exact values are given in the table below.
Titre Values (colonies per plate)
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
|||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
Repl. 1 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
Repl. 2 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
Mean |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
Stand. Dev. |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
Assessment |
ok |
ok |
ok |
ok |
ok |
ok |
ok |
ok |
ok |
ok |
1001 colonies per plate means the bacteria growth was too strong for counting.
Sterility Control
Criterion: No colony per plate may grow.
Sterility (colonies per plate)
|
Demin. water |
DMSO |
Repl. 1 |
0 |
0 |
Repl. 2 |
0 |
0 |
Repl. 3 |
0 |
0 |
Repl. 4 |
0 |
0 |
Assessment |
ok |
ok |
Spontaneous Revertants
The determined values were within the normal range of the laboratory.
Demin. Water
Spontaneous Revertants demin. water (colonies per plate)
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
|||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
Repl. 1 |
82 |
93 |
34 |
32 |
118 |
86 |
432 |
408 |
12 |
11 |
Repl. 2 |
59 |
87 |
37 |
32 |
93 |
90 |
368 |
288 |
12 |
18 |
Repl. 3 |
95 |
90 |
44 |
34 |
102 |
96 |
384 |
360 |
17 |
15 |
Mean |
79 |
90 |
38 |
33 |
104 |
91 |
395 |
352 |
14 |
15 |
sd |
18.2 |
3.0 |
5.1 |
1.2 |
12.7 |
5.0 |
33.3 |
60.4 |
2.9 |
3.5 |
DMSO
Spontaneous Revertants DMSO (colonies per plate)
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
|||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
Repl. 1 |
71 |
95 |
46 |
37 |
83 |
93 |
440 |
256 |
15 |
19 |
Repl. 2 |
69 |
71 |
38 |
37 |
70 |
84 |
464 |
424 |
14 |
16 |
Repl. 3 |
74 |
78 |
37 |
34 |
108 |
82 |
352 |
472 |
11 |
10 |
Mean |
71 |
81 |
40 |
36 |
87 |
86 |
419 |
384 |
13 |
15 |
sd |
2.5 |
12.3 |
4.9 |
1.7 |
19.3 |
5.9 |
59.0 |
113.4 |
2.1 |
4.6 |
Positive Controls
Without metabolic activation:
4-Nitro-1,2-phenylene diamine (NPD) in DMSO, 20 µg/plate
Sodium azide (Na-azide) in demineralized water, 1 µg/plate
With metabolic activation
2-Amino anthracene (2-AA) in DMSO, 1 µg/plate
Benzo-a-pyrene (BaP) in DMSO, 20 µg/plate
Diagnostic Mutagens (colonies per plate)
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
|||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
Substance |
NPA |
2-AA |
NPD |
BaP |
Na-azide |
2-AA |
NPA |
2-AA |
Na-azide |
2-AA |
Repl. 1 |
664 |
740 |
336 |
184 |
528 |
1304 |
1256 |
1408 |
312 |
184 |
Repl. 2 |
324 |
776 |
228 |
134 |
692 |
1192 |
1208 |
1584 |
392 |
208 |
Repl. 3 |
408 |
588 |
368 |
88 |
408 |
1256 |
1296 |
1376 |
328 |
152 |
Mean |
465 |
701 |
311 |
135 |
543 |
1251 |
1253 |
1456 |
344 |
181 |
sd |
177.1 |
99.8 |
73.4 |
48.0 |
142.6 |
56.2 |
44.1 |
112.0 |
42.3 |
28.1 |
f(l) |
6.55 |
8.65 |
7.78 |
3.75 |
5.22 |
14.55 |
2.99 |
3.79 |
24.57 |
12.07 |
Rev. abs. |
394 |
620 |
271 |
99 |
439 |
1165 |
834 |
1072 |
330 |
166 |
f(l) = increase factor.
Rev. abs. = absolute revertants.
Test Item Trixene AS
Mutagenicity Test
Concentration 5 µL/plate (colonies per plate)
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
|||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
Repl. 1 |
14 |
13 |
3 |
4 |
32 |
33 |
108 |
60 |
5 |
7 |
Repl. 2 |
17 |
16 |
6 |
3 |
40 |
23 |
70 |
52 |
9 |
3 |
Repl. 3 |
10 |
15 |
3 |
5 |
31 |
40 |
59 |
39 |
5 |
4 |
Mean |
14 |
15 |
4 |
4 |
34 |
32 |
79 |
50 |
6 |
5 |
sd |
3.5 |
1.5 |
1.7 |
1.0 |
4.9 |
8.5 |
25.7 |
10.6 |
2.3 |
2.1 |
f(l) |
0.20 |
0.19 |
0.10 |
0.11 |
0.39 |
0.37 |
0.19 |
0.13 |
0.46 |
0.33 |
Rev. abs. |
-57 |
-66 |
-36 |
-32 |
-53 |
-54 |
-340 |
-334 |
-7 |
-10 |
Concentration 2.5 µL/plate (colonies per plate)
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
|||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
Repl. 1 |
8 |
33 |
3 |
12 |
26 |
59 |
360 |
348 |
7 |
4 |
Repl. 2 |
14 |
35 |
2 |
9 |
42 |
31 |
288 |
368 |
6 |
2 |
Repl. 3 |
14 |
39 |
1 |
15 |
43 |
42 |
344 |
416 |
3 |
7 |
Mean |
12 |
36 |
2 |
12 |
37 |
44 |
331 |
337 |
5 |
4 |
sd |
3.5 |
3.1 |
1.0 |
3.0 |
9.5 |
14.1 |
37.8 |
34.9 |
2.1 |
2.5 |
f(l) |
0.17 |
0.44 |
0.05 |
0.33 |
0.43 |
0.51 |
0.79 |
0.98 |
0.38 |
0.27 |
Rev. abs. |
-59 |
-45 |
-38 |
-24 |
-50 |
-42 |
-88 |
-7 |
-8 |
-11 |
Concentration 1.25 µL/plate (colonies per plate)
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
|||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
Repl. 1 |
39 |
26 |
41 |
39 |
14 |
52 |
312 |
312 |
17 |
12 |
Repl. 2 |
5 |
47 |
39 |
37 |
48 |
39 |
464 |
424 |
14 |
12 |
Repl. 3 |
10 |
36 |
47 |
39 |
40 |
35 |
240 |
456 |
10 |
11 |
Mean |
18 |
36 |
42 |
38 |
34 |
42 |
339 |
397 |
14 |
12 |
sd |
18.4 |
10.5 |
4.2 |
1.2 |
17.8 |
8.9 |
114.4 |
75.6 |
3.5 |
0.6 |
f(l) |
0.25 |
0.44 |
1.05 |
1.06 |
0.39 |
0.49 |
0.81 |
1.03 |
1.08 |
0.80 |
Rev. abs. |
-53 |
-45 |
2 |
2 |
-53 |
-44 |
-80 |
13 |
1 |
-3 |
Concentration 0.63 µL/plate (colonies per plate)
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
|||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
Repl. 1 |
110 |
108 |
43 |
37 |
84 |
84 |
320 |
512 |
9 |
12 |
Repl. 2 |
75 |
62 |
41 |
41 |
106 |
94 |
392 |
312 |
14 |
15 |
Repl. 3 |
85 |
92 |
37 |
41 |
83 |
82 |
344 |
408 |
17 |
14 |
Mean |
90 |
87 |
40 |
40 |
91 |
87 |
352 |
411 |
13 |
14 |
sd |
18.0 |
23.4 |
3.1 |
2.3 |
13.0 |
6.4 |
36.7 |
100.0 |
4.0 |
1.5 |
f(l) |
1.27 |
1.07 |
1.00 |
1.11 |
1.05 |
1.01 |
0.84 |
1.07 |
1.00 |
0.93 |
Rev. abs. |
19 |
6 |
0 |
4 |
4 |
1 |
-67 |
27 |
0 |
-1 |
Concentration 0.31 µL/plate (colonies per plate)
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
|||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
Repl. 1 |
92 |
90 |
41 |
40 |
81 |
80 |
528 |
360 |
17 |
18 |
Repl. 2 |
69 |
98 |
44 |
36 |
93 |
108 |
360 |
336 |
17 |
18 |
Repl. 3 |
72 |
112 |
45 |
43 |
91 |
85 |
296 |
352 |
15 |
14 |
Mean |
78 |
100 |
43 |
40 |
88 |
91 |
395 |
349 |
16 |
17 |
sd |
12.5 |
11.1 |
2.1 |
3.5 |
6.4 |
14.9 |
119.8 |
12.2 |
1.2 |
2.3 |
f(l) |
1.10 |
1.23 |
1.08 |
1.11 |
1.01 |
1.06 |
0.94 |
0.91 |
1.23 |
1.13 |
Rev. abs. |
7 |
19 |
3 |
4 |
1 |
5 |
-24 |
-35 |
3 |
2 |
Concentration 0.16 µL/plate (colonies per plate)
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
|||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
Repl. 1 |
84 |
106 |
40 |
45 |
81 |
96 |
416 |
392 |
12 |
18 |
Repl. 2 |
71 |
108 |
31 |
30 |
82 |
71 |
360 |
440 |
15 |
17 |
Repl. 3 |
83 |
90 |
39 |
38 |
72 |
86 |
232 |
448 |
11 |
16 |
Mean |
79 |
101 |
37 |
38 |
78 |
84 |
336 |
427 |
13 |
17 |
sd |
7.2 |
9.9 |
4.9 |
7.5 |
5.5 |
12.6 |
94.3 |
30.3 |
2.1 |
1.0 |
f(l) |
1.11 |
1.25 |
0.93 |
1.06 |
0.90 |
0.98 |
0.80 |
1.11 |
1.00 |
1.13 |
Rev. abs. |
8 |
20 |
-3 |
2 |
-9 |
-2 |
-83 |
43 |
0 |
2 |
Concentration 0.08 µL/plate (colonies per plate)
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
|||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
Repl. 1 |
122 |
108 |
38 |
33 |
100 |
83 |
424 |
360 |
15 |
10 |
Repl. 2 |
84 |
98 |
39 |
43 |
83 |
132 |
248 |
328 |
11 |
10 |
Repl. 3 |
74 |
133 |
37 |
41 |
75 |
82 |
360 |
392 |
19 |
11 |
Mean |
93 |
113 |
38 |
39 |
86 |
99 |
344 |
360 |
15 |
10 |
sd |
25.3 |
18.0 |
1.0 |
5.3 |
12.8 |
28.6 |
89.1 |
32.0 |
4.0 |
0.6 |
f(l) |
1.31 |
1.40 |
0.95 |
1.08 |
0.99 |
1.15 |
0.82 |
0.94 |
1.15 |
0.67 |
Rev. abs. |
22 |
32 |
-2 |
3 |
-1 |
13 |
-75 |
-24 |
2 |
-5 |
Concentration 0.04 µL/plate (colonies per plate)
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
|||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
Repl. 1 |
100 |
112 |
29 |
46 |
93 |
74 |
416 |
336 |
19 |
18 |
Repl. 2 |
73 |
106 |
33 |
49 |
84 |
90 |
376 |
336 |
13 |
10 |
Repl. 3 |
63 |
126 |
40 |
37 |
71 |
84 |
400 |
416 |
11 |
10 |
Mean |
79 |
115 |
34 |
44 |
83 |
83 |
397 |
363 |
14 |
13 |
sd |
19.1 |
10.3 |
5.6 |
6.2 |
11.1 |
8.1 |
20.1 |
46.2 |
4.2 |
4.6 |
f(l) |
1.11 |
1.42 |
0.85 |
1.22 |
0.95 |
0.97 |
0.95 |
0.95 |
1.08 |
0.87 |
Rev. abs. |
8 |
34 |
-6 |
8 |
-4 |
-3 |
-22 |
-21 |
1 |
-2 |
DATA OF THE CYTOTOXICITY TEST
The toxicity of the following concentration was tested: 5 µL/plate.
Per strain, 2 plates with and without metabolic activation were incubated with the corresponding dose of the test item on maximal soft agar.
Experiment Parameters
Concentration tested: 5 µL/plate
Incubation time: 48 h
Incubation temperature: 37 ± 1 °C
Tested strains: TA97a, TA98, TA100, TA102, TA1535
Method: Plate incorporation method
Determination of Titre
Criterion: The determination of titre should give a number of at least 109cells/mL, correlating to 100 colonies/plate after dilution.
The criterion was fulfilled, exact values are given in the table below.
Titre Values (colonies per plate)
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
|||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
Repl. 1 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
Repl. 2 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
Mean |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
Stand. Dev. |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
Assessment |
ok |
ok |
ok |
ok |
ok |
ok |
ok |
ok |
ok |
ok |
1001 colonies per plate means the bacteria growth was too strong for counting.
Toxicity Control
The test item is considered non-toxic, if the quotient titre/toxicity is below 2.
5 µL/plate on maximal-soft-agar with culture diluted by 106
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
|||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
Repl. 1 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
Repl. 2 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
Mean |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
1001 |
Stand. Dev. |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
0.0 |
Titre/Tox |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
1.00 |
Assessment |
non-toxic |
non-toxic |
non-toxic |
non-toxic |
non-toxic |
non-toxic |
non-toxic |
non-toxic |
non-toxic |
non-toxic |
1001 colonies per plate means the bacteria growth was too strong for counting.
COMPAIRSON WITH HISTORICAL DATA
In the following table, the history of the spontaneous revertants and positive controls of the performed experiments with these strains up to 28. May 2018 is stated in comparison with the experiments performed within this study. Only experiments which were performed before the performance of the study were considered.
For the historical data, the plate incorporation method and the pre-incubation method were used.
Historical Data of Spontaneous Revertants
Strain |
TA97a |
TA98 |
TA100 |
TA102 |
TA1535 |
||||||
Induction |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
-S9 |
+S9 |
|
Demin. water |
Mean |
88 |
95 |
22 |
25 |
93 |
98 |
280 |
300 |
18 |
18 |
Min |
60 |
63 |
6 |
8 |
51 |
64 |
85 |
67 |
6 |
7 |
|
Max |
144 |
138 |
52 |
51 |
147 |
141 |
425 |
587 |
36 |
40 |
|
SD |
17 |
17 |
12 |
11 |
16 |
15 |
57 |
71 |
6 |
6 |
|
Exp 1 |
79 |
90 |
43 |
49 |
78 |
77 |
324 |
339 |
13 |
16 |
|
Exp 2 |
79 |
90 |
38 |
33 |
104 |
91 |
395 |
352 |
14 |
15 |
|
DMSO |
Mean |
88 |
97 |
22 |
24 |
90 |
93 |
280 |
293 |
18 |
17 |
Min |
58 |
67 |
7 |
8 |
44 |
62 |
79 |
80 |
8 |
6 |
|
Max |
135 |
144 |
47 |
50 |
138 |
199 |
413 |
459 |
35 |
37 |
|
SD |
17 |
16 |
12 |
12 |
16 |
17 |
55 |
60 |
6 |
6 |
|
Exp 1 |
80 |
99 |
44 |
41 |
86 |
86 |
299 |
347 |
16 |
14 |
|
Exp 2 |
71 |
81 |
40 |
36 |
87 |
86 |
419 |
384 |
13 |
15 |
|
Positive Controls* |
Mean |
534 |
526 |
413 |
129 |
487 |
781 |
1093 |
1197 |
263 |
134 |
Min |
264 |
228 |
77 |
39 |
220 |
273 |
491 |
408 |
55 |
45 |
|
Max |
1165 |
1181 |
1001 |
487 |
984 |
1912 |
2331 |
6083 |
515 |
712 |
|
SD |
169 |
168 |
169 |
101 |
155 |
284 |
409 |
558 |
84 |
79 |
|
Exp 1 |
512 |
1125 |
392 |
227 |
1256 |
1312 |
1200 |
1163 |
251 |
224 |
|
Exp 2 |
465 |
701 |
311 |
135 |
543 |
1251 |
1253 |
1456 |
344 |
181 |
*Different positive controls were used
The values which lie outside the range of the historical data are given in bold italics.
Bacteria strains are living biological systems, therefore variations in behavior are not unusual.
Each bacteria strain in this study has its own characteristic spontaneous revertant colony number. Day to day variations in the number of spontaneous revertant colonies are usual.
The variations of the values of all bacteria strains lie in an acceptable range.
Evaluation of the mutagenicity if the test item and validity of the study was not affected by the variations.
No critical impact on the outcome of the study is expected.
Applicant's summary and conclusion
- Conclusions:
- Based on the results of this study it is concluded that Trixene AS is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study.
- Executive summary:
Determination of the mutagenic potential of Trixene AS with the Bacterial Reverse Mutation Test following OECD 471 and EU B.13/14
Findings and Results:
Two valid experiments were performed.
The study procedures described in this report were based on the most recent OECD and EC guidelines.
The test item Trixene AS was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535).
The test was performed in two experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation.
Experiment 1:
In the first experiment, the test item (dissolved in DMSO) was tested up to concentrations of 5 μL/plate in the presence and absence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method.
The test item showed no precipitates on the plates at any of the concentrations.
The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the presence and absence of metabolic activation.
The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.
Experiment 2:
Based on the non- toxic results of the first experiment, the test item was tested up to concentrations of 5 μL/plate in the presence and absence of S9-mix in all bacteria strains using the pre-incubation method.
The test item showed no precipitates on the plates at any of the concentrations.
Signs of toxicity were observed in the following concentrations:
• TA97a: 5, 2.5 and 1.25 μL/plate (decrease in the number of revertants)
• TA98: 5 and 2.5 μL/plate (decrease in the number of revertants)
• TA100: 5, 2.5 and 1.25 μL/plate (decrease in the number of revertants)
• TA102: 5 μL/plate (decrease in the number of revertants)
• TA1535: 5 and 2.5 μL/plate (decrease in the number of revertants)
The bacterial background lawn was observed in all concentrations.
The results of this experiments showed that the test item caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the presence and absence of metabolic activation. The test item did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation.
Based on the results of this study it is concluded that Trixene AS is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the presence and absence of metabolic activation under the experimental conditions in this study.
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