Registration Dossier
Registration Dossier
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EC number: 946-383-6 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Genetic toxicity in vitro-Ames Assay
Trixene AS is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the presence and absence of metabolic activation.
Genetic toxicity in vitro-Chromosome aberration assay
Trixene AS did not induce structural chromosome aberrations in human lymphocytes in vitro in the chosen test concentrations.
Genetic toxicity in vitro-Mammalian cell gene mutation test
Trixene AS is considered to be “non-mutagenic under the conditions of the HPRT assay”.
Link to relevant study records
- 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
- 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
- Specific details on test material used for the study:
- No further details specified in the study report.
- Target gene:
- Histidene
- 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 - 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.
- 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. - 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.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 16 Apr. 2018 to 25 Jun. 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- OECD Guidelines for the Testing of Chemicals, Part 473, adopted 29. Jul. 2016 ”In Vitro Mammalian Chromosomal Aberration Test“
- Deviations:
- yes
- Remarks:
- A cytotoxicity of 55 ± 5% was not achieved. See "Any other information" for further details
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- Commission Regulation (EU) 2017/735 amending Regulation (EC) No. 440/2008, EU Method B.10: “‘In Vitro Mammalian Chromosomal Aberration Test”, dated 14. Feb. 2017
- Deviations:
- yes
- Remarks:
- A cytotoxicity of 55 ± 5% was not achieved. See " Any other information" for further details.
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Specific details on test material used for the study:
- No further details specified in the study report.
- Target gene:
- metaphase cells are analysed for the presence of chromosomal aberrations.
- Species / strain / cell type:
- lymphocytes: Human peripheral blood lymphocytes treated with anti-coagulant (heparin).
- Details on mammalian cell type (if applicable):
- Blood Collection
Blood samples were obtained from young (approximately 18 – 35 years of age) non-smoking donors with no known illness or recent exposures to genotoxic agents (e.g. chemicals, ion-izing radiations) at levels that would increase the background incidence of chromosomal aberrations.
Primary cultures of human peripheral lymphocytes are used for this type of study because of their low and stable background rate of chromosomal aberrations. In addition, human cells are generally the most relevant ones for risk assessment. - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 (liver enzyme mixture used for the test with metabolic activation) was obtained from a specialized company (Trinova Biochem GmbH, Gießen) and stored at – 80 ± 5°C.
Batch no: 3852
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:
- Pre-Experiment without Metabolic Activation: 0.08, 0.16, 0.31, 0.63, 1.3, 2.5 and 5 µL/mL
Experiment I with Metabolic Activation: 0.08, 0.16, 0.31, 0.63, 1.3, 2.5 and 5 µL/mL
Experiment I without Metabolic Activation: 0.001, 0.002, 0.005, 0.01, 0.02, 0.04 and 0.08 µL/mL
Experiment II: 0.001, 0.002, 0.005, 0.01, 0.02, 0.04 and 0.08 µL/mL
Selection of Top Concentration
According to OECD 473, test item concentrations producing excessive cytotoxicity, precipitation in the culture medium or marked changes in pH or osmolality should be avoided.
Where cytotoxicity occurs, the highest test concentration should cause a reduction of the mitotic index to 45 ± 5%, corresponding to 55 ± 5% cytotoxicity. Concentrations with moderate and little or no cytotoxicity should be included in the evaluation.
When solubility is a limiting factor, the maximum concentration, if not limited by cytotoxicity, should produce visible turbidity or precipitates in the cultures at the end of the treatment. Only one concentration with precipitates/turbidity should be analysed. The precipitates should not interfere with the conduct of the test. For insoluble or particulate materials, specific adaptation may be needed.
If neither cytotoxicity nor solubility are limiting factors, the maximum concentration should be 2 μL/mL, 2 mg/mL or 10 mM, whichever is the lowest. Usually, concentration intervals of approximately 2 to 3 fold will be appropriate.
When the test item is a substance of unknown or variable composition, a complex reaction product or of biological origin (UVCB), testing may be started at a higher concentration (e.g. 5 mg/mL) to increase the amount of each of the test item components.
According to these criteria, for Trixene AS testing was started with 5 μL/mL as highest concentration in the test. - Vehicle / solvent:
- DMSO was used as solvent control for the test item in a final concentration of 0.5% in the culture medium.
MCM was used as solvent control for the positive control EMS (final concentrations: exper-iment I 100%, experiment II 10%)
0.9% NaCl was used as solvent control for the positive control CPA in a final concentration of 0.5% in the final culture medium. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- Culture Initiation, Treatment and Preparation
Culture Initiation
For the initial cell proliferation, the blood cultures were set up in defined time intervals within 24 h after collection in sterile cell culture vessels, each vessel containing 1 part of heparinised whole blood and 9 parts of complete culture medium RPMI 1640.
The cultures were then incubated at 37 ± 1°C in a humidified atmosphere with 5.0 ± 0.5% CO2
Cell Treatment Pre-Experiment and Experiment I
72 h after seeding duplicate cultures à 10 mL volume were established for each treatment. These cultures were centrifuged (10 min, 500 * g). The cell pellet was re-suspended in minimal culture medium and solvent control, positive control or the single test item concentrations were added. In the case of metabolic activation, 50 μL S9 mix per mL medium were used. The cell cultures were incubated at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2 for 4 h (exposure period).
After the exposure time of 4 h, the cells were spun down by gentle centrifugation (500 * g) for 5 min. The supernatant was discarded and the cells were re-suspended in 5 mL Saline G for washing. The washing procedure was repeated once as described.
After washing, the cells were re-suspended in complete culture medium and incubated at 37 ± 1 °C in a humidified atmosphere with 5% ± 0.5% CO2 for 20.5 h until preparation.
Cell Treatment Experiment II
72 h after seeding duplicate cultures à 10 mL volume were established for each treatment. These cultures were centrifuged (10 min, 500*g). The cell pellet was re-suspended in fresh complete cell culture medium. Medium control, solvent control, positive control and test item were added. The exposure time was 25 h. Harvesting started directly after exposure.
Chromosome Preparation
Chromosome preparation was done 24.5 h (pre-exp. and exp. I) and 25 h (exp. II) after start of exposure. 3 h before harvesting, Colcemid® was added to the cultures (final concentration: 0.1 μg/mL). Each cell culture was harvested and processed separately. The cells were spun down by gentle centrifugation (10 min, 500 * g). The supernatant was discarded and the cells were re-suspended in 12 mL hypotonic KCl solution. The cell suspension was al-lowed to stand at room temperature for 20 min (pre-exp.) or 15 min (exp. I and exp. II). After removal of the hypotonic solution by centrifugation (10 min, 500 * g), the cell pellet was ex-posed to fixative (3:1 mixture of methanol and glacial acetic acid). After fixation at 2-8°C, minimum 30 min, the cell suspension was spun down by gentle centrifugation (10 min, 500 * g), the supernatant was discarded and the cell pellet was re-suspended in fixative again. The washing procedures were repeated until the cell pellet was white.
Preparation of Slides
The slides were prepared by dropping the cell suspension onto clean microscope slides. The slides were then stained with a 10% solution of Giemsa. All slides were independently coded before microscopic analysis.
Determination of Mitotic Index and Cytotoxicity
In all replicates, the number of metaphases per 1000 cell nuclei was enumerated and thereof the mitotic index in % of the solvent control was determined using the following equations:
Mitotic index in %: MI(%) = number of mitotic cells*100% / total number of cells
MI in % of the solvent control = MI(%) test item or positive control*100% / MI(%) solvent control
Cytotoxicity in % = 100% - MI in % of the solvent control
From these determinations, the test item concentrations which were evaluated for structural chromosomal aberrations, were defined.
Determination of Chromosomal Aberrations
Evaluation was performed using Zeiss microscopes and the automatic slide scanning system “Metafer”, provided by MetaSystems, Altlussheim.
Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded separately and reported, but they were not included in the total aberration frequency. Chromosome aberrations were scored according to the classification of ISCN 2013. At least 300 well spread metaphases per concentration, 150 metaphases per replicate, were scored for cytogenetic damage. Only metaphases with 46 ± 2 centromeric regions were included in the analysis. - Rationale for test conditions:
- In accordance with test guidelines
- Evaluation criteria:
- A test item is classified as non-mutagenic if:
-Neither a statistically significant nor a concentration-related increase of the number of cells with structural chromosomal aberrations in the evaluated test concentrations is ob-served.
-The obtained results lie within the range of the historical laboratory control data for sol-vent controls, considering also e.g. 95.5% control limits where appropriate.
A test item is classified as mutagenic if in any of the experimental conditions:
-At least one concentration shows a number of induced structural chromosome aberra-tions (excluding gaps) lying above the range of the historical laboratory control data for solvent controls, considering also e.g. 95.5% control limits where appropriate.
-A dose-related increase in the number of cells with structural chromosome aberrations is observed.
-A statistically significant increase of structural chromosomal aberrations is found, at least at one concentration of the test item. - Statistics:
- The number of aberrant metaphase cells in each treatment group was compared with the solvent control value using Fisher’s exact test or chi-square-test at the 5% level (p < 0.05).
- Key result
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- This study was performed to assess the mutagenic potential of Trixene AS to induce struc-tural chromosomal aberrations in human lymphocytes cultured in vitro in the absence and the presence of an exogenous metabolic activation system (liver S9 mix from male rats, treated with Aroclor 1254).
Three valid experiments were performed.
In each experiment, all cell cultures were set up in duplicates. In order to assess the toxicity of the test solutions to the cultured human lymphocytes; the mitotic index was calculated for all cultures treated with medium control, solvent control, positive control and test item (as far as evaluable). On the basis of the data from the mitotic index, the concentrations were selected for metaphase analysis.
All positive control compounds caused large, statistically significant increases in the propor-tion of aberrant cells, demonstrating the sensitivity of the test system. In experiment I with metabolic activation and in experiment II, the solvent control DMSO showed values lying slightly above the range and the 95.5% control limits of the historical laboratory control data but well within literature data for solvent controls (< 5%).
In the pre-experiment, 7 concentrations of the test item were used and tested without and with metabolic activation. The 4 highest test item concentrations showed precipitation and haemolysis both in the approaches without and with metabolic activation.
In the approach without metabolic activation, the 3 lowest concentrations showed complete cytotoxicity and could not be evaluated. Only the mitotic index of the solvent and positive controls was determined to show the validity of the experiment. As not any of the test item concentrations could be evaluated, this experimental part was designated as pre-experi-ment without metabolic activation and was repeated with lower test item concentrations (ex-periment I without metabolic activation).
In the experimental approach with metabolic activation, the remaining concentrations (with-out precipitation) did not show cytotoxicity, therefore, according to OECD 473 the lowest concentration with precipitates (0.63 μL/mL) and the 2 following concentrations (without pre-cipitates: 0.31 and 0.16 μL/mL) were evaluated. This experimental part was designated as experiment I with metabolic activation.
In experiment I without metabolic activation, the highest tested concentration (0.08 μL/mL) showed again complete cytotoxicity. The remaining test item concentrations showed neither relevant cytotoxicity nor precipitation. Therefore, the 3 highest evaluable concentrations (0.04, 0.02 and 0.01 μL/mL) were scored for structural chromosomal aberrations.
In experiment II (only without metabolic activation, extended exposure), the highest tested concentration (0.08 μL/mL) caused 35% cytotoxicity compared with the concurrent solvent control. The remaining concentrations did not show relevant cytotoxicity. No precipitation was observed. Therefore the 3 highest tested concentrations were scored for structural chro-mosomal aberrations.
In all evaluated experiments, only in one case a statistically significant increase (p < 0.01) of structural chromosomal aberrations was found (exp. I without metabolic activation, 0.04 μL/mL). This value also lay above the historical laboratory control data (also above the 95.5% control limit) for the solvent control DMSO. However, in experiment II (also without metabolic activation, but with extended exposure time) these findings could not be repro-duced: neither a statistically significant nor a biologically relevant increase of structural chro-mosomal aberrations was observed at the evaluated concentrations. All values lay in the range of the concurrent solvent control DMSO. Due to the longer exposure time in experi-ment II, these results are classified as more significant. In experiment I with metabolic acti-vation, the test item concentrations 0.63 and 0.31 μL/mL showed values of structural aber-rations lying above the historical laboratory control data and the 95.5% control limit for the concurrent solvent control but not being statistically significant increased. The values above the historical laboratory control data were clearly inside the literature data for solvent con-trols (<5%).
In none of the tested conditions, a statistically significant dose-response relationship could be detected.
In none of the tested experimental conditions were the three criteria for a positive result fulfilled.
In conclusion, it can be stated that under the experimental conditions reported, the test item Trixene AS did not induce structural chromosome aberrations in human lymphocytes in vitro in the chosen test concentrations. - Conclusions:
- In conclusion, it can be stated that under the experimental conditions reported, the test item Trixene AS did not induce structural chromosome aberrations in human lymphocytes in vitro in the chosen test concentrations.
- Executive summary:
Determination of the mutagenic potential of Trixene AS with the “In Vitro Mammalian Chromosomal Aberration Test” following OECD 473 and EU B.10
Findings and Results:
This study was performed to assess the mutagenic potential of Trixene AS to induce structural chromosomal aberrations in human lymphocytes cultured in vitro in absence and presence of an exogenous metabolic activation system (liver S9 mix from male rats, treated with Aroclor 1254).
Human peripheral blood lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin and exposed to solvent control, test item and positive control. 3 hours before the end of cultivation, cell division was arrested using Colcemid®. After harvesting, slides were prepared and stained. The metaphase cells were examined for chromosomal damage.
Three valid experiments were performed.
In each experiment, all cell cultures were set up in duplicates. In order to assess the toxicity of the test solutions to the cultured human lymphocytes, the mitotic index was calculated for all cultures treated with medium control, solvent control, positive control and test item (as far as evaluable). On the basis of the data from the mitotic index, the concentrations were selected for metaphase analysis.
All positive control compounds ca
used large, statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system. In experiment I with metabolic activation and in experiment II, the solvent control DMSO showed values lying slightly above the range and the 95.5% control limits of the historical laboratory control data but well within literature data for solvent controls (< 5%).
In the pre-experiment, 7 concentrations of the test item were used and tested without and with metabolic activation. The 4 highest test item concentrations showed precipitation and haemolysis both in the approaches without and with metabolic activation.
In the approach without metabolic activation, the 3 lowest concentrations showed complete cytotoxicity and could not be evaluated. Only the mitotic index of the solvent and positive controls was determined to show the validity of the experiment. As not any of the test item concentrations could be evaluated, this experimental part was designated as pre-experiment without metabolic activation and was repeated with lower test item concentrations (experiment I without metabolic activation).
In the experimental approach with metabolic activation, the remaining concentrations (with-out precipitation) did not show cytotoxicity, therefore, according to OECD 473 the lowest concentration with precipitation (0.63 μL/mL) and the 2 following concentrations (without precipitation: 0.31 and 0.16 μL/mL) were evaluated. This experimental part was designated as experiment I with metabolic activation.
In experiment I without metabolic activation, the highest tested concentration (0.08 μL/mL) again showed complete cytotoxicity. The remaining test item concentrations showed neither relevant cytotoxicity nor precipitation, therefore, the 3 highest evaluable concentrations (0.04, 0.02 and 0.01 μL/mL) were scored for structural chromosomal aberrations.
In experiment II (only without metabolic activation, extended exposure), the highest tested concentration (0.08 μL/mL) caused 35% cytotoxicity compared with the concurrent solvent control. The remaining concentrations did not show relevant cytotoxicity. No precipitation was observed. Therefore, the 3 highest tested concentrations were scored for structural chromosomal aberrations.
In all evaluated experiments, only in one case a statistically significant increase (p < 0.01) of structural chromosomal aberrations was found (exp. I without metabolic activation, 0.04 μL/mL). This value also lay above the historical laboratory control data and above the 95.5% control limit for the solvent control DMSO. However, in experiment II (also without metabolic activation, but with an extended exposure time) these findings could not be reproduced: neither a statistically significant nor a biologically relevant increase of structural chromosomal aberrations was observed at the evaluated concentrations. All values lay in the range of the concurrent solvent control DMSO. Due to the longer exposure time in experiment II, these results are classified as more significant. In experiment I with metabolic activation, the test item concentrations 0.63 and 0.31 μL/mL showed values of structural aberrations lying above the historical laboratory control data for the concurrent solvent control but not being statistically significant increased. The values above the historical laboratory control data were clearly inside the literature data for solvent controls (<5%).
In none of the tested conditions, a statistically significant dose-response relationship could be detected.
In none of the tested experimental conditions were the three criteria for a positive result fulfilled.
In conclusion, it can be stated that under the experimental conditions reported, the test item Trixene AS did not induce structural chromosome aberrations in human lymphocytes in vitro in the chosen test concentrations.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 24 Apr. 2018 to 17 Jul. 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- OECD Guideline for the Testing of Chemicals, Part 476, adopted 29. Jul. 2016 ”In Vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes"
- Deviations:
- yes
- Remarks:
- See "Any other information" for details
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- EU-Method B.17 of the Commission Regulation (EC) No. 440/2008, adopted 30. May 2008: “Mutagenicity – In vitro Mammalian Cell Gene Mutation Test”
- Deviations:
- yes
- Remarks:
- See "Any other information" for details
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Specific details on test material used for the study:
- No further details specified in the study report.
- Target gene:
- induce mutations on the endogenous hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on chromosome X
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- Reasons for the Choice of the Cell Line V79
The V79 cell line has been used successfully in in vitro experiments for many years because of its sensitivity to chemical mutagens. Especially the high proliferation rate (doubling time 12 – 16 h in stock cultures) and a high cloning efficiency of untreated cells both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22 (Bradley et al., 1981). The cells were purchased by CLS (Eppelheim, Germany) and were sold under the name V79-4. The modal chromosome number was analysed and confirmed by the supplier of the cells.
Cell Cultures
Prior to use in the experiments, the cells cultures were cleansed of pre-existing mutant cells by culturing in HAT medium (medium containing Hypoxanthine, Aminopterin and Thymidine). Cleansed and for mycoplasma contamination screened stocks of cells were stored in liquid nitrogen in the cell bank of LAUS GmbH to allow a continuous working stock of cells, which guarantees similar parameters of the experiment and reproducible characteristics of the cells.
The cells were thawed 6 - 8 d prior treatment and cultivated in DMEM complete culture medium with 5 % HS in cell culture flasks at 37.0 ± 1.5 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2. The normal cell cycle time as well as the detection of the spontaneous mutant frequency of the used master cell stock is checked in each experiment. - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 (liver enzyme mixture used for the test with metabolic activation) was not produced by LAUS GmbH, but was obtained from a specialized company (Trinova Biochem GmbH, Gießen) and stored at – 80 °C.
Batch nos: 3833, 3852
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:
- Experiment I (with metabolic activation: 0.078, 0.039, 0.020, 0.010, 0.005 and 0.002 µL/mL
Experiment I (without metabolic activation: 0.156, 0.078, 0.039, 0.020, 0.010 and 0.005 µL/mL
Experiment II (with metabolic activation: 0.156, 0.078, 0.039, 0.020, 0.010 and 0.005 µL/mL
Experiment II (without metabolic activation: 0.0049, 0.0024, 0.0012, 0.0006, 0.0003 and 0.00015 µL/mL
According to OECD 476, the highest concentration should be 0.01 M or 2 mg/mL or 2 μL/mL (whichever is the lowest), unless limited by the solubility or toxicity of the test item.
Relative survival values below 20 % are considered toxic. If the pre-test reveals cytotoxicity, a minimum of one concentration of the test item should reduce the relative survival (RS) to 10- 20 % in the main experiments. When the test chemical is not of defined composition, e.g., substance of unknown or variable composition, complex reaction products or biological materials (i.e., Chemical Substances of Unknown or Variable Composition (UVCBs)), testing may be started at a higher concentration to increase the amount of each of the test item components. In case of precipitates or turbidity in culture medium, the highest evaluated concentration in the experiments should produce turbidity or precipitates. - Vehicle / solvent:
- DMEM without supplements was used as solvent control for the positive control Ethylmethanesulfonate (EMS).
DMSO was used as solvent control for the test item and the positive control 7,12-dimethylbenz(a)anthracene (DMBA). - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- Experimental Performance
Experiment I was conducted with and without metabolic activation. In the experiments II only the approach without metabolic activation was used. Despite that, the experimental performance in experiment I and II are identical except the treatment duration with the test item. In experiment I the test item was incubated for 4 hours (with and without S9) and the cells were afterwards washed twice with PBS Dulbecco (2.5 % HS). In both experiments II the incubation time with the test item was 24 hours without a following washing step. A second experiment is only performed if the result of experiment I is not clearly positive or negative or on demand of sponsor. Experiment II was conducted for a verification of the results of experiment I.
First 1 * 106 cells per 10 cm culture dish and 500 cells (for the determination of the cytotoxicity) per 6 cm culture dish were seeded per tested concentration as well as for the solvent and positive controls and incubated for 24 h. The incubation conditions during the whole assay were 37.0° ± 1.5 °C in 5.0 ± 0.5 % CO2.
The cells were treated with the test item after incubation. Each concentration was prepared in duplicate. Directly after the treatment period the cells were washed with PBS Dulbecco (2.5 % HS) twice (not in experiment II –S9). Fresh complete culture medium (5 % HS) was added to the cells before the following incubation. The further implementation of the experiment was divided into the determination of the survival as well as the viability and the mutagenicity.
For the determination of a cytotoxic effect of the test item, the survival of the cells was measured. For this purpose, the cells in the 6 cm dishes were stained with 0.1 % Löffler’s methylene blue solution in 0.01 % KOH solution after a 7 day-incubation time. The colonies were counted and the cloning efficiency (absolute and relative) was calculated.
For the determination of the second part of the experiment (viability and mutagenicity), the cells in the 10 cm culture dishes were counted and adjusted to 1 * 106 cells per 10 cm culture dish after an incubation time of 69 hours (experiment I), 73 hours and 30 min (experiment II) and 72 hours and 15 min (experiment II-2) and afterwards further incubated. After a total expression time of at least 168 h, the cells were counted again and seeded into 10 cm culture dishes (5 * 105 ± 104 cells) for the evaluation of the mutagenicity in selection medium containing 6-TG (final concentration: 2 μg/mL) and into 6 cm culture dishes (500 ± 10 cells) for the evaluation of the viability in complete culture medium. Both plates were incubated for further 7 days. After this incubation time the cell colonies were stained with 0.1 % Löffler’s methylene blue solution in 0.01 % KOH solution and left to stand at room temperature until they were counted for the calculation of the cloning efficiency II and the mutation frequency. - Rationale for test conditions:
- In accordance with test guidelines.
- Evaluation criteria:
- Providing that the study is acceptable, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
-at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
-the increase is concentration-related when evaluated with an appropriate trend test,
-any of the results are outside the distribution of the historical negative control data.
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
Providing that the study is acceptable, a test chemical is considered clearly negative if, in all experimental conditions examined:
-none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
-there is no concentration-related increase when evaluated with an appropriate trend test,
-all results are inside the distribution of the historical negative control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratories historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study is also taken into consideration.
In cases when the response is neither clearly negative nor clearly positive as described above, or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations. - Statistics:
- Not specified
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- The study was performed to investigate the potential of the test item Trixene AS to induce mutations at the HPRT locus on chromosome X Chinese Hamster V79 cells.
The assay was performed in two independent experiments, using two parallel cultures each (duplicates). In experiment I, 6 concentrations of the test item were tested with and without metabolic activation. The exposure time was 4 hours. The following nominal concentrations of the test item were investigated in experiment I:
+S9: 0.078μL/mL, 0.039 μL/ml, 0.02 μL/mL, 0.01 μL/mL, 0.005 μL/mL, 0.002 μL/mL
-S9: 0.156 μL/mL, 0.078 μL/mL, 0.039 μL/mL, 0.02 μL/mL, 0.01 μL/mL, 0.005 μL/mL
In the approach with metabolic activation turbidity was visible only at the highest test item concentration (0.078 μL/mL). In the approach without metabolic activation precipitates were visible only at the highest test item concentration (0.156 μL/mL).
In experiment II, again 6 concentrations of the test item were used and tested without metabolic activation. The exposure time was 24 hours. The following nominal concentrations of the test item were investigated in experiment II:
-S9: 0.156 μL/mL, 0.078 μL/mL, 0.039 μL/mL, 0.02 μL/mL, 0.01 μL/mL, 0.005 μL/mL
In this experiment precipitates were visible only at the highest test item concentration (0.156 μL/mL).
Since all tested concentrations in experiment II induced a cytotoxic effect, a second experiment II (experiment II-2) was performed with the following nominal concentrations:
-S9: 0.0049 μL/mL, 0.0024 μL/mL, 0.0012 μL/mL, 0.0006 μL/mL, 0.0003 μL/mL, 0.00015 μL/mL
Precipitation or turbidity of the test item was not observed up to the highest tested concentration.
EMS (Exp I: 300 μg/mL, Exp II + Exp II-2: 150 μg/mL) and DMBA (1.5 μg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies, without substantial reduction of the viability whereby the mutant frequency of the positive control DMBA (98 * 10-6) was below the range of 95.5 % control limit of the historical control data (99 – 387 * 10-6). Since the difference is only marginal and the positive control nevertheless induced a very clearly positive result, this is considered as uncritical and the study is still valid.
In experiment I (+S9 and –S9) no cytotoxic effect was observed in any of the test item concentrations.
In experiment II (–S9) strong cytotoxic effects were observed in all test item concentrations. For that reason this experiment could not be evaluated for mutagenicity and was repeated with lower test item concentrations.
In experiment II-2 (-S9) cytotoxicity was only observed at the highest test item concentration. At the next lower test item concentration the viability values were ≥ 95 %.
In experiment I no dose dependent increase in mutant colony numbers was observed. A statistically significant increase in mutant colony numbers was observed at the concentrations 0.02 μL/mL and 0.01 μL/mL in the approach with metabolic activation but only in replicate B as well as at the concentration 0.005 μL/mL in the approach without metabolic activation but only in replicate A. Nevertheless, all values remained within the historical control range of the solvent control.
Since the result of experiment I was not clearly negative, experiment II (with 24 h incubation) was performed. Since all test item concentrations in experiment II induced a cytotoxic effect, the experiment was repeated with lower test item concentrations (experiment II-2).
In experiment II-2 no dose dependent increase in mutant colony numbers was observed. A statistically significant increase in mutant colony numbers was observed at the concentrations 0.0024 μL/mL, 0.0012 μL/mL and 0.0006 μL/mL only in replicate A as well as at the concentrations 0.0012 μL/mL and 0.0006 μL/mL at the mean values. Nevertheless, all values remained within the historical control range of the solvent control.
Finally, no biological relevant increase in the number of mutant colonies was detected in both experiments. - Conclusions:
- In conclusion it can be stated that under the experimental conditions of this study Trixene AS did not induce gene mutations at the HPRT locus in V79 cells in the absence and presence of metabolic activation.
Therefore, the test item Trixene AS is considered to be “non-mutagenic under the conditions of the HPRT assay”. - Executive summary:
Determination of the mutagenic potential of Trixene AS with the In Vitro Mammalian Cell Gene Mutation Test using the Hprt genes following OECD 476
Findings and Results:
This study was performed to investigate the potential of Trixene AS to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese Hamster cells (V79).
The assay comprised two pre-tests and three independent experiments (experiment I and two experiments II). Experiment II (exposure date 07. Jun. 2018) was invalid and therefore repeated (Experiment II-2: exposure date: 28. Jun. 2018). The pre-tests were done to detect a potential cytotoxic effect of the test item. Based on the results of these tests the concentrations for the main experiments were determined.
Experiment I was performed with and without metabolic activation (liver S9 mix from male rats, treated with Aroclor 1254) and a treatment period of 4 h. Both experiments II were performed with a treatment period of 24 hours without metabolic activation.
The highest nominal concentration (experiment I +S9: 0.078 μL/mL; -S9: 0.156 μL/mL; experiment II -S9: 0.156 μL/mL, experiment II-2 –S9: 0.0049 μL/mL) applied was chosen with regard to the solubility of the test item in organic solvents and aqueous media as well as the results of the pre-test or the first experiment II.
In experiment I precipitate (-S9) or turbidity (+S9) of the test item was visible at the highest test item concentrations. In experiment II precipitate of the test item was visible at the highest test item concentration. In experiment II-2 no precipitate was visible in all tested test item concentrations.
Ethylmethane sulfonate (EMS) and 7,12-Dimethylbenzanthracene (DMBA) as appropriate reference mutagens were used as positive controls. Both induced a distinct increase in mutant colonies and thus, showed enough sensitivity of the testing procedure and the activity of the metabolic activation system.
In experiment I no dose dependent increase in mutant colony numbers was observed. A statistically significant increase in mutant colony numbers was observed at the concentrations 0.02 μL/mL and 0.01 μL/mL in the approach with metabolic activation but only in replicate B as well as at the concentration 0.005 μL/mL in the approach without metabolic activation but only in replicate A. Nevertheless, all values remained within the historical control range of the solvent control.
Since the result of experiment I was not clearly negative, experiment II (with 24 h incubation) was performed. Since all test item concentrations in experiment II induced a cytotoxic effect, the experiment was repeated with lower test item concentrations (experiment II-2).
In experiment II-2 no dose dependent increase in mutant colony numbers was observed. A statistically significant increase in mutant colony numbers was observed at the concentrations 0.0024 μL/mL, 0.0012 μL/mL and 0.0006 μL/mL only in replicate A as well as at the concentrations 0.0012 μL/mL and 0.0006 μL/mL at the mean values. Nevertheless, all values remained within the historical control range of the solvent control.
Finally, no biological relevant increase in the number of mutant colonies was detected in both experiments.
Conclusion:
It can be stated that under the experimental conditions of this study Trixene AS did not induce gene mutations at the HPRT locus in V79 cells in the absence and presence of metabolic activation.
Therefore, the test item Trixene AS is considered to be “non-mutagenic under the conditions of the HPRT assay”.
Referenceopen allclose all
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.
Results Pre-Experiment
Results Cytotoxicity Pre-Experiment without Metabolic Activation
Treatment |
Precipitation |
Haemolysis |
Mitotic index in % |
Mitotic Index in % of the solvent control |
Cytotoxicity in % |
Solvent control MCM |
No |
No |
10.4 |
100 |
-- |
Solvent control DMSO 0.5% v/v |
No |
No |
10.0 |
100 |
-- |
Positive control EMS 600 µg/mL |
No |
No |
6.9 |
69 |
31 |
Test item 5 µL/mL |
++ |
++ |
n.e. |
n.e. |
n.e. |
Test item 2.5 µL/mL |
++ |
++ |
n.e. |
n.e. |
n.e. |
Test item 1.3 µL/mL |
++ |
+ |
n.e. |
n.e. |
n.e. |
Test item 0.63 µL/mL |
+ |
+ |
n.e. |
n.e. |
n.e. |
Test item 0.31 µL/mL |
No |
No |
n.e. |
n.e. |
n.e. |
Test item 0.16 µL/mL |
No |
No |
n.e. |
n.e. |
n.e. |
Test item 0.08 µL/mL |
No |
No |
n.e. |
n.e. |
n.e. |
++moderate precipitation/haemolysis
+slight precipitation/haemolysis
n.e. not evaluated
Results Experiment I with Metabolic Activation
Results Cytotoxicity Experiment I with Metabolic Activation
Treatment |
Precipitation |
Haemolysis |
Mitotic index in % |
Mitotic Index in % of the solvent control |
Cytotoxicity in % |
Solvent control DMSO |
No |
+ |
13.5 |
100 |
-- |
Solvent control 0.5% NaCl 0.5% v/v |
No |
No |
14.5 |
100 |
-- |
Positive control CPA 30 µg/mL |
No |
No |
13.0 |
89 |
11 |
Test item 5 µL/mL |
++ |
++ |
n.e. |
n.e. |
n.e. |
Test item 2.5 µL/mL |
++ |
++ |
n.e. |
n.e. |
n.e. |
Test item 1.3 µL/mL |
++ |
+ |
n.e. |
n.e. |
n.e. |
Test item 0.63 µL/mL |
+ |
+ |
11.0 |
81 |
19 |
Test item 0.31 µL/mL |
No |
No |
10.1 |
74 |
26 |
Test item 0.16 µL/mL |
No |
No |
14.3 |
106 |
-6 |
Test item 0.08 µL/mL |
No |
No |
16.3 |
120 |
-20 |
++moderate precipitation/haemolysis
+slight precipitation/haemolysis
n.e. not evaluated
Results Chromosomal Aberration Assay Experiment I with Metabolic Activation
Treatment |
Aberrant cells in % |
||
Incl. gaps+ |
Excl. gaps+ |
With exchange |
|
Solvent control DMSO |
9.0 |
2.3# |
0.0 |
Solvent control 0.9% NaCl 0.5% v/v |
7.7 |
3.0 |
0.7 |
Positive control CPA 30 µg/ml |
39.3 |
27.0** |
6.0 |
Test item 0.63 µL/mL |
7.0 |
2.3# |
0.0 |
Test item 0.31 µL/mL |
12.0 |
4.0# |
0.0 |
Test item 0.16 µL/mL |
4.3 |
1.0 |
0.0 |
+ Inclusive cells carrying exchanges
# value above the historical laboratory control data and the 95.5% control limits of the solvent control DMSO but inside the literature data for solvent controls (which means < 5%)
Asterisks indicate statistically significant differences to solvent control, with * p <0.05, ** p<0.01
Results Experiment I without Metabolic Activation
Results Cytotoxicity Experiment I without Metabolic Activation
Treatment |
Precipitation |
Haemolysis |
Mitotic index in % |
Mitotic Index in % of the solvent control |
Cytotoxicity in % |
Solvent control MCM |
No |
No |
14.4 |
100 |
-- |
Solvent control DMSO 0.5% v/v |
No |
No |
11.4 |
100 |
-- |
Positive control EMS 600 µg/mL |
No |
No |
6.5 |
57 |
43 |
Test item 0.08 µL/mL |
No |
No |
n.e. |
n.e. |
n.e. |
Test item 0.04 µL/mL |
No |
No |
11.0 |
76 |
24 |
Test item 0.02 µL/mL |
No |
No |
12.9 |
90 |
10 |
Test item 0.01 µL/mL |
No |
No |
12.4 |
86 |
14 |
Test item 0.005 µL/mL |
No |
No |
10.8 |
75 |
25 |
Test item 0.002 µL/mL |
No |
No |
11.0 |
76 |
24 |
Test item 0.001 µL/mL |
No |
No |
9.8 |
68 |
32 |
n.e. not evaluated
Results Chromosomal Aberration Assay Experiment I without Metabolic Activation
Treatment |
Aberrant cells in % |
||
Incl. gaps+ |
Excl. gaps+ |
With exchange |
|
Solvent control MCM |
6.3 |
2.7 |
0.3 |
Solvent control DMSO 0.5% v/v |
7.0 |
1.3 |
0.0 |
Positive control EMS 600 µg/ml |
27.3 |
19.3** |
3.0 |
Test item 0.04 µL/mL |
8.3 |
4.7**# |
0.7 |
Test item 0.02 µL/mL |
9.3 |
3.0 |
0.3 |
Test item 0.01 µL/mL |
9.0 |
2.7 |
0.0 |
+ Inclusive cells carrying exchanges
# value above the historical laboratory control data of the solvent control DMSO but inside the literature data for solvent controls (which means < 5%)
Asterisks indicate statistically significant differences to solvent control, with * p <0.05, ** p<0.01
Results Experiment II
Results Cytotoxicity Experiment II
Treatment |
Precipitation |
Haemolysis |
Mitotic index in % |
Mitotic Index in % of the solvent control |
Cytotoxicity in % |
Solvent control MCM 10% v/v |
No |
No |
13.4 |
100 |
-- |
Solvent control DMSO 0.5% v/v |
No |
No |
9.8 |
100 |
-- |
Positive control EMS 300 µg/mL |
No |
No |
5.0 |
51 |
49 |
Test item 0.08 µL/mL |
No |
No |
8.7 |
65 |
35 |
Test item 0.04 µL/mL |
No |
+ |
11.8 |
88 |
12 |
Test item 0.02 µL/mL |
No |
No |
11.3 |
84 |
16 |
Test item 0.01 µL/mL |
No |
No |
9.8 |
73 |
27 |
Test item 0.005 µL/mL |
No |
No |
10.5 |
79 |
21 |
Test item 0.002 µL/mL |
No |
No |
10.3 |
77 |
23 |
Test item 0.001 µL/mL |
No |
No |
10.7 |
80 |
20 |
+slight precipitation/haemolysis
Results Chromosomal Aberration Assay Experiment II
Treatment |
Aberrant cells in % |
||
Incl. gaps+ |
Excl. gaps+ |
With exchange |
|
Solvent control MCM 10% v/v |
6.3 |
1.7 |
0.3 |
Solvent control DMSO 0.5% v/v |
6.3 |
3.3# |
0.0 |
Positive control EMS 300 µg/ml |
27.0 |
14.3** |
1.7 |
Test item 0.08 µL/mL |
4.3 |
2.7 |
0.0 |
Test item 0.04 µL/mL |
3.7 |
1.7 |
0.0 |
Test item 0.02 µL/mL |
9.0 |
2.3 |
0.3 |
+ Inclusive cells carrying exchanges
# value above the historical laboratory control data but inside the literature data for solvent controls (which means < 5%)
Asterisks indicate statistically significant differences to solvent control, with * p <0.05, ** p<0.01
Results
Summary of Results of Experiment I
|
Concentration |
S9 mix |
Treatment time |
Culture |
Relative Survival |
Mutant frequency per 106cells |
Mutant frequency per 106cells |
|
[µL/mL] |
|
[h] |
|
[%] |
|
Mean |
Solvent Control Test Item |
- |
+ |
4 |
A |
- |
12 |
9 |
B |
- |
6 |
|||||
Solvent Control DMBA |
- |
+ |
4 |
A |
- |
9 |
6 |
B |
- |
3 |
|||||
Positive Control (DMBA) |
1.5 µg/mL |
+ |
4 |
A |
109.9% |
78** |
98** |
B |
83.7% |
118** |
|||||
Test item |
0.078(1) |
+ |
4 |
A |
40.8% |
2 |
2 |
B |
43.3% |
3 |
|||||
Test item |
0.039 |
+ |
4 |
A |
112.5% |
7 |
9 |
B |
89.4% |
12 |
|||||
Test item |
0.020 |
+ |
4 |
A |
119.3% |
14 |
15 |
B |
101.6% |
16* |
|||||
Test item |
0.010 |
+ |
4 |
A |
124.3% |
4 |
12 |
B |
120.7% |
19** |
|||||
Test item |
0.005 |
+ |
4 |
A |
152.7% |
4 |
9 |
B |
118.7% |
14 |
|||||
Test item |
0.002 |
+ |
4 |
A |
137.1% |
7 |
7 |
B |
179.2% |
7 |
|||||
Solvent Control Test Item |
- |
- |
4 |
A |
- |
7 |
8 |
B |
- |
9 |
|||||
Solvent Control EMS |
- |
- |
4 |
A |
- |
9 |
7 |
B |
- |
6 |
|||||
Positive Control (EMS) |
300 µg/mL |
- |
4 |
A |
98.6% |
61** |
77** |
B |
137.6% |
93** |
|||||
Test Item |
0.156(2) |
- |
4 |
A |
49.6% |
10 |
8 |
B |
99.0% |
5 |
|||||
Test item |
0.078 |
- |
4 |
A |
61.3% |
8 |
7 |
B |
91.8% |
5 |
|||||
Test item |
0.039 |
- |
4 |
A |
60.0% |
14 |
9 |
B |
96.0% |
3 |
|||||
Test item |
0.020 |
- |
4 |
A |
42.9% |
13 |
9 |
B |
120.6% |
6 |
|||||
Test item |
0.010 |
- |
4 |
A |
61.2% |
14 |
9 |
B |
108.0% |
5 |
|||||
Test item |
0.005 |
- |
4 |
A |
50.5% |
21** |
14 |
B |
102.4% |
6 |
Asterisks indicate statistically significant differences to solvent control, with *p<0.05, **p<0.01
(1)= Turbidity visible at the end of treatment
(2)= Precipitates visible at the end of treatment
Summary of Results of Experiment II
|
Concentration |
S9 mix |
Treatment time |
Culture |
Relative Survival |
Mutant frequency per 106cells |
Mutant frequency per 106cells |
|
[µL/mL] |
|
[h] |
|
[%] |
|
Mean |
Solvent Control Test Item |
- |
- |
24 |
A |
- |
20 |
28 |
B |
- |
35 |
|||||
Solvent Control EMS |
- |
- |
24 |
A |
- |
13 |
14 |
B |
- |
15 |
|||||
Positive Control (EMS) |
150 µg/mL |
- |
24 |
A |
138.2% |
299** |
286** |
B |
90.0% |
273** |
|||||
Test Item |
0.156(1) |
- |
24 |
A |
0.0% |
0 |
17 |
B |
0.0% |
33 |
|||||
Test item |
0.078 |
- |
24 |
A |
0.0% |
15 |
8 |
B |
0.0% |
0 |
|||||
Test item |
0.039 |
- |
24 |
A |
0.0% |
11 |
10 |
B |
0.0% |
9 |
|||||
Test item |
0.020 |
- |
24 |
A |
0.0% |
9 |
8 |
B |
0.0% |
7 |
|||||
Test item |
0.010 |
- |
24 |
A |
0.4% |
15 |
15 |
B |
1.4% |
15 |
|||||
Test item |
0.005 |
- |
24 |
A |
11.1% |
8 |
11 |
B |
7.9% |
13 |
Asterisks indicate statistically significant differences to solvent control, with **p<0.01
(1)= Precipitates visible at the end of treatment
Italics written values were not considered for evaluation because of cytotoxicity
Summary of Results of Experiment II-2
|
Concentration |
S9 mix |
Treatment time |
Culture |
Relative Survival |
Mutant frequency per 106cells |
Mutant frequency per 106cells |
|
[µL/mL] |
|
[h] |
|
[%] |
|
Mean |
Solvent Control Test Item |
- |
- |
24 |
A |
- |
4 |
4 |
B |
- |
4 |
|||||
Solvent Control EMS |
- |
- |
24 |
A |
- |
11 |
12 |
B |
- |
13 |
|||||
Positive Control (EMS) |
150 µg/mL |
- |
24 |
A |
106.6% |
210** |
174** |
B |
114.4% |
137** |
|||||
Test Item |
0.0049 |
- |
24 |
A |
0.0% |
14 |
8 |
B |
0.8% |
3 |
|||||
Test item |
0.0024 |
- |
24 |
A |
95.2% |
12* |
9 |
B |
104.3% |
7 |
|||||
Test item |
0.0012 |
- |
24 |
A |
78.9% |
17** |
12* |
B |
75.8% |
6 |
|||||
Test item |
0.0006 |
- |
24 |
A |
90.9% |
17** |
12* |
B |
77.4% |
8 |
|||||
Test item |
0.0003 |
- |
24 |
A |
100.7% |
4 |
7 |
B |
70.6% |
6 |
|||||
Test item |
0.00015 |
- |
24 |
A |
87.3% |
n/e |
n/e |
B |
105.3% |
n/e |
Asterisks indicate statistically significant differences to solvent control, with **p<0.01
Italics written values were not considered for evaluation because of cytotoxicity.
n/e = not evaluated because the OECD 476 guideline requires only 4 concentrations.
List of Abbreviations
In the following table, the abbreviations that are used are presented.
List of Abbreviations
CE |
Cloning efficient |
Conc. |
Concentration |
DMBA |
7,12-dimethylbenz(a)anthracene |
DMEM |
Dulbecco’s Modified Eagle’ Medium |
DMSO |
Dimethyl sulfoxide |
EDTA |
Ethylenediaminetetraacetic acid |
EMS |
Ethylmethane sulfonate |
HPRT |
Hypoxanthine-guanine phosphoribosyl transferase |
HS |
Horse serum |
MF |
Mutation frequency |
NAPD |
Nicotinamide ademine dinucleotise phosphate |
OECE |
Organisation for Economic Co-operation and Development |
PBS Dulbecco |
Dulbecco’s Phosphate buffered saline |
RS |
Relative survival |
6-TG |
6-thioguanine |
PRE-TEST I – DETAILED DATA
Note: All given values except the counts are rounded values. For the calculation with the validated calculation sheet the unrounded values were used.
The cell number of the respective cell suspension was measured using the cell counter.
For the determination of the corresponding cells/mL of the suspension the factor 100 was taken into account.
Cell Numbers
Cell Number for Treatment Pre-Test I with and without Metabolic Activation
Count 1:10 Dilution of Original Cell Suspension |
Cells/mL in Original Cell Suspension |
Cells seeded/6 cm dish for survival |
1380 |
1380000 |
500* |
*Note: This number of cells was added into all single culture dishes (7 test item concentrations + 2 solvent controls + 1 positive control: Replicate A and B)
PRE-TEST II – DETAILED DATA
Note: All given values except the counts are rounded values. For the calculation with the validated calculation sheet the unrounded values were used.
The cell number of the respective cell suspension was measured using the cell counter.
For the determination of the corresponding cells/mL of the suspension the factor 100 was taken into account.
Cell Numbers
Cell Number for Treatment Pre-Test II with and without Metabolic Activation
Count 1:10 Dilution of Original Cell Suspension |
Cells/mL in Original Cell Suspension |
Cells seeded/6 cm dish for survival |
923 |
92300 |
500* |
*Note: This number of cells was added into all single culture dishes (7 test item concentrations + 2 solvent controls + 1 positive control: Replicate A and B)
Survival
The survival is given by the CE I value. This corresponds to the number of cells at the end of treatment divided by the total number of cells at the beginning of treatment.
Survival Pre-Test II with Metabolic Activation
|
Conc. |
Culture |
Number of colonies per dish |
CE I absolute |
CE I relative |
CE I relative Mean |
|
|
[µL/mL] |
|
I |
II |
|
|
|
Solvent Control Test Item |
- |
A |
338 |
348 |
0.69 |
- |
- |
Solvent Control Test Item |
- |
B |
289 |
298 |
0.59 |
- |
|
Solvent Control DMBA |
- |
A |
294 |
271 |
0.57 |
- |
- |
Solvent Control DMBA |
- |
B |
269 |
298 |
0.57 |
- |
|
Positive Control (DMBA) |
1.5 µg/mL |
A |
241 |
307 |
0.55 |
97.0% |
90.5% |
Positive Control (DMBA) |
1.5 µg/mL |
B |
254 |
222 |
0.48 |
84.0% |
|
Test item |
0.078 |
A |
38 |
35 |
0.07 |
10.6% |
13.3% |
Test Item |
0.078 |
B |
43 |
51 |
.009 |
16.0% |
|
Test item |
0.039 |
A |
181 |
182 |
0.36 |
52.9% |
57.2% |
Test item |
0.039 |
B |
181 |
180 |
0.366 |
16.5% |
|
Test item |
0.02 |
A |
263 |
286 |
0.55 |
80.0% |
74.3% |
Test item |
0.02 |
B |
192 |
211 |
0.40 |
68.7% |
|
Test item |
0.01 |
A |
303 |
296 |
0.60 |
87.3% |
94.9% |
Test item |
0.01 |
B |
315 |
286 |
0.60 |
102.4% |
|
Test item |
0.005 |
A |
298 |
289 |
0.59 |
85.6% |
91.9% |
Test item |
0.005 |
B |
294 |
283 |
0.58 |
98.3% |
|
Test item |
0.002 |
A |
265 |
297 |
0.56 |
81.9% |
89.5% |
Test item |
0.002 |
B |
311 |
259 |
0.57 |
97.1% |
|
Test item |
0.001 |
A |
309 |
238 |
0.55 |
79.7% |
82.0% |
Test item |
0.001 |
B |
249 |
246 |
0.50 |
84.3% |
|
Survival Pre-Test II without Metabolic Activation
|
Conc. |
Culture |
Number of colonies per dish |
CE I absolute |
CE I relative |
CE I relative Mean |
|
|
[µL/mL] |
|
I |
II |
|
|
|
Solvent Control Test Item |
- |
A |
252 |
224 |
0.48 |
- |
- |
Solvent Control Test Item |
- |
B |
353 |
357 |
0.71 |
- |
|
Solvent Control DMBA |
- |
A |
236 |
273 |
0.51 |
- |
- |
Solvent Control DMBA |
- |
B |
382 |
338 |
0.72 |
- |
|
Positive Control (DMBA) |
300 µg/mL |
A |
281 |
227 |
0.51 |
99.8% |
97.3% |
Positive Control (DMBA) |
300 µg/mL |
B |
374 |
308 |
0.68 |
94.7% |
|
Test item |
0.078 |
A |
138 |
142 |
0.28 |
58.8% |
48.3% |
Test Item |
0.078 |
B |
133 |
135 |
0.27 |
37.7% |
|
Test item |
0.039 |
A |
194 |
174 |
0.37 |
77.3% |
68.6% |
Test item |
0.039 |
B |
205 |
220 |
0.43 |
59.9% |
|
Test item |
0.02 |
A |
171 |
255 |
0.43 |
89.5% |
73.8% |
Test item |
0.02 |
B |
209 |
204 |
0.41 |
58.2% |
|
Test item |
0.01 |
A |
204 |
210 |
0.41 |
87.0% |
84.8% |
Test item |
0.01 |
B |
304 |
282 |
0.59 |
82.5% |
|
Test item |
0.005 |
A |
274 |
245 |
0.52 |
109.0% |
98.5% |
Test item |
0.005 |
B |
285 |
339 |
0.62 |
87.9% |
|
Test item |
0.002 |
A |
256 |
233 |
0.49 |
102.7% |
95.8% |
Test item |
0.002 |
B |
312 |
319 |
0.63 |
88.9% |
|
Test item |
0.001 |
A |
252 |
272 |
0.52 |
110.1% |
97.9% |
Test item |
0.001 |
B |
312 |
297 |
0.61 |
85.8% |
|
EXPERIMENT I – DETAILED DATA
Note: All given values except the counts are rounded values. For the calculation with the validated calculation sheet the unrounded values were used.
The cell number of the respective cell suspension was measured using the cell counter.
For the determination of the corresponding cells/mL of the suspension the factor 100 was taken into account.
Cell Numbers Experiment I
Cell Number for Treatment Experiment I with and without Metabolic Activation
Count 1:10 Dilution of Original Cell Suspension |
Cells/mL in Original Cell Suspension |
Control Count after adjustment to 107Cells |
Cells seeded/10 cm dish for Viability and Mutagenicity |
Cells seeded/6 cm dish for survival |
20892 |
2089200 |
1056 |
1056000* |
500* |
*Note: This number of cells was added into all single culture dishes (6 test item concentrations + 2 solvent controls + 1 positive control: Replicate A and B)
Cell Passage 72h after Starting of Expression Time. Approach with Metabolic Activation
|
Conc. |
Culture |
Cells counted |
Cells/mL counted |
Adjusted cells/ culture dish |
|
[µL/mL] |
|
|
|
|
Solvent Control Test Item |
- |
A |
16485 |
1648500 |
10000640 |
Solvent Control Test Item |
- |
B |
18845 |
1844500 |
1000670 |
Solvent Control DMBA |
- |
A |
19388 |
1938800 |
1000421 |
Solvent Control DMBA |
- |
B |
18578 |
1587800 |
999496 |
Positive Control (DMBA) |
1.5 µg/mL |
A |
16624 |
1662400 |
1000765 |
Positive Control (DMBA) |
1.5 µg/mL |
B |
18642 |
1864200 |
999211 |
Test item |
0.078 |
A |
19817 |
1981700 |
1000759 |
Test item |
0.078 |
B |
18974 |
1897400 |
999930 |
Test item |
0.039 |
A |
18562 |
1856200 |
1000492 |
Test item |
0.039 |
B |
18285 |
1828500 |
1000190 |
Test item |
0.02 |
A |
18822 |
1882200 |
999448 |
Test item |
0.02 |
B |
18247 |
1824700 |
999936 |
Test item |
0.01 |
A |
23081 |
2308100 |
999407 |
Test item |
0.01 |
B |
17365 |
1736500 |
1000224 |
Test item |
0.05 |
A |
15156 |
1515600 |
1000296 |
Test item |
0.05 |
B |
18157 |
1815700 |
1000451 |
Test item |
0.02 |
A |
18240 |
1824000 |
999552 |
Test item |
0.02 |
B |
18738 |
1373800 |
1000609 |
Cell Passage 72h after Starting of Expression Time. Approach without Metabolic Activation
|
Conc. |
Culture |
Cells counted |
Cells/mL counted |
Adjusted cells/ culture dish |
|
[µL/mL] |
|
|
|
|
Solvent Control Test Item |
- |
A |
22113 |
2211300 |
999508 |
Solvent Control Test Item |
- |
B |
24794 |
2479400 |
999198 |
Solvent Control EMS |
- |
A |
22702 |
2270200 |
998888 |
Solvent Control EMS |
- |
B |
20206 |
2020600 |
1000197 |
Positive Control (EMS) |
300 µg/mL |
A |
21019 |
2101900 |
1000504 |
Positive Control (EMS) |
300 µg/mL |
B |
20731 |
2073100 |
999234 |
Test item |
0.156 |
A |
21390 |
2139000 |
1001052 |
Test item |
0.156 |
B |
20920 |
2092000 |
999976 |
Test item |
0.078 |
A |
21709 |
2170900 |
1000785 |
Test item |
0.078 |
B |
21140 |
2114000 |
999922 |
Test item |
0.039 |
A |
23657 |
2365700 |
10000691 |
Test item |
0.039 |
B |
21724 |
2172400 |
999304 |
Test item |
0.02 |
A |
24495 |
2449500 |
999396 |
Test item |
0.02 |
B |
22973 |
2297300 |
999326 |
Test item |
0.01 |
A |
20975 |
2097500 |
1000508 |
Test item |
0.01 |
B |
22054 |
2205400 |
999046 |
Test item |
0.005 |
A |
22479 |
2247900 |
1000316 |
Test item |
0.005 |
B |
20188 |
2018800 |
999306 |
Seeding for Mutagenicity and Viability. AT least 168 h after Starting of Expression Time. Approach with Metabolic Activation
|
Conc. |
Culture |
Cells counted |
Cells/mL counted |
Adjusted cells/ culture dish (Mutagenicity) |
Adjusted cells/ culture dish I and II (Viability) |
|
[µL/mL] |
|
|
|
|
|
Solvent Control Test Item |
- |
A |
13415 |
1341500 |
499559 |
499 |
Solvent Control Test Item |
- |
B |
12921 |
1292100 |
500122 |
502 |
Solvent Control DMBA |
- |
A |
13747 |
1374700 |
499891 |
500 |
Solvent Control DMBA |
- |
B |
12786 |
1278600 |
500271 |
502 |
Positive Control (DMBA) |
1.5 µg/mL |
A |
15619 |
1561900 |
500447 |
500 |
Positive Control (DMBA) |
1.5 µg/mL |
B |
14212 |
1421200 |
499686 |
501 |
Test item |
0.078 |
A |
12910 |
1291000 |
499697 |
502 |
Test item |
0.078 |
B |
13197 |
1319700 |
499268 |
499 |
Test item |
0.039 |
A |
11922 |
1192200 |
500633 |
499 |
Test item |
0.039 |
B |
11928 |
1192800 |
500885 |
499 |
Test item |
0.02 |
A |
13573 |
1357300 |
500694 |
499 |
Test item |
0.02 |
B |
12760 |
1276000 |
801485 |
502 |
Test item |
0.01 |
A |
13473 |
1347300 |
499363 |
499 |
Test item |
0.01 |
B |
14248 |
1424800 |
499703 |
501 |
Test item |
0.05 |
A |
13591 |
1359100 |
498979 |
499 |
Test item |
0.05 |
B |
12691 |
1269100 |
500991 |
502 |
Test item |
0.02 |
A |
13810 |
1381000 |
500004 |
500 |
Test item |
0.02 |
B |
13763 |
1376300 |
499990 |
500 |
Seeding for Mutagenicity and Viability. At least 168h after Starting of Expression Time. Approach without Metabolic Activation
|
Conc. |
Culture |
Cells counted |
Cells/mL counted |
Adjusted cells/ culture dish (Mutagenicity) |
Adjusted cells/ culture dish I and II (Viability) |
|
[µL/mL] |
|
|
|
|
|
Solvent Control Test Item |
- |
A |
14402 |
1440200 |
499799 |
502 |
Solvent Control Test Item |
- |
B |
14604 |
1460400 |
500142 |
502 |
Solvent Control EMS |
- |
A |
14543 |
1454300 |
500097 |
502 |
Solvent Control EMS |
- |
B |
14804 |
1480400 |
500488 |
498 |
Positive Control (EMS) |
300 µg/mL |
A |
14583 |
1458300 |
500192 |
502 |
Positive Control (EMS) |
300 µg/mL |
B |
15390 |
1539000 |
500153 |
499 |
Test item |
0.156 |
A |
12940 |
1294000 |
500858 |
502 |
Test item |
0.156 |
B |
13756 |
1375600 |
499977 |
500 |
Test item |
0.078 |
A |
13738 |
1373800 |
499564 |
500 |
Test item |
0.078 |
B |
13813 |
1381300 |
500112 |
500 |
Test item |
0.039 |
A |
14002 |
1400200 |
500326 |
501 |
Test item |
0.039 |
B |
14777 |
1477700 |
500355 |
498 |
Test item |
0.02 |
A |
14180 |
1418000 |
499555 |
501 |
Test item |
0.02 |
B |
13302 |
1330200 |
500912 |
499 |
Test item |
0.01 |
A |
14885 |
1488500 |
499439 |
498 |
Test item |
0.01 |
B |
14363 |
1436300 |
499957 |
502 |
Test item |
0.005 |
A |
12729 |
1572900 |
499460 |
500 |
Test item |
0.005 |
B |
15354 |
1535400 |
500064 |
499 |
Survival Experiment I
The survival is given by the CE I value. This corresponds to the number of cells at the end of treatment divided by the total number of cells at the beginning of treatment.
Survival Experiment I with Metabolic Activation
|
Conc. |
Culture |
Number of colonies per dish |
CE I absolute |
CE I relative |
CE I relative Mean |
|
|
[µL/mL] |
|
I |
II |
|
|
|
Solvent Control Test Item |
- |
A |
234 |
261 |
0.50 |
- |
- |
Solvent Control Test Item |
- |
B |
257 |
257 |
0.51 |
- |
|
Solvent Control DMBA |
- |
A |
256 |
249 |
0.51 |
- |
- |
Solvent Control DMBA |
- |
B |
248 |
261 |
0.51 |
- |
|
Positive Control (DMBA) |
1.5 µg/mL |
A |
241 |
243 |
0.48 |
95.8% |
97.4% |
Positive Control (DMBA) |
1.5 µg/mL |
B |
235 |
269 |
0.50 |
99.0% |
|
Test item |
0.078 |
A |
90 |
79 |
0.17 |
34.1% |
31.7% |
Test Item |
0.078 |
B |
63 |
87 |
0.15 |
29.2% |
|
Test item |
0.039 |
A |
219 |
215 |
0.43 |
87.7% |
85.7% |
Test item |
0.039 |
B |
220 |
210 |
0.43 |
83.7% |
|
Test item |
0.02 |
A |
236 |
248 |
0.48 |
97.8% |
94.9% |
Test item |
0.02 |
B |
241 |
232 |
0.47 |
92.0% |
|
Test item |
0.01 |
A |
253 |
266 |
0.52 |
104.8% |
104.5% |
Test item |
0.01 |
B |
262 |
273 |
0.54 |
104.1% |
|
Test item |
0.005 |
A |
239 |
269 |
0.51 |
102.6% |
100.4% |
Test item |
0.005 |
B |
247 |
258 |
0.51 |
98.2% |
|
Test item |
0.002 |
A |
240 |
251 |
0.49 |
99.2% |
101.7% |
Test item |
0.002 |
B |
270 |
266 |
0.54 |
104.3% |
|
Survival Experiment I without Metabolic Activation
|
Conc. |
Culture |
Number of colonies per dish |
CE I absolute |
CE I relative |
CE I relative Mean |
|
|
[µL/mL] |
|
I |
II |
|
|
|
Solvent Control Test Item |
- |
A |
247 |
229 |
0.48 |
- |
- |
Solvent Control Test Item |
- |
B |
240 |
231 |
0.47 |
- |
|
Solvent Control EMS |
- |
A |
235 |
236 |
0.47 |
- |
- |
Solvent Control EMS |
- |
B |
228 |
242 |
0.47 |
- |
|
Positive Control (EMS) |
300 µg/mL |
A |
234 |
256 |
0.48 |
104.0% |
104.5% |
Positive Control (EMS) |
300 µg/mL |
B |
260 |
233 |
0.49 |
104.9% |
|
Test item |
0.156 |
A |
193 |
171 |
0.36 |
76.5& |
77.4% |
Test Item |
0.156 |
B |
188 |
181 |
0.37 |
78.3% |
|
Test item |
0.078 |
A |
203 |
207 |
0.41 |
86.1% |
88.7% |
Test item |
0.078 |
B |
209 |
221 |
0.43 |
91.3% |
|
Test item |
0.039 |
A |
247 |
218 |
0.47 |
97.7% |
100.1% |
Test item |
0.039 |
B |
232 |
251 |
0.48 |
102.5% |
|
Test item |
0.02 |
A |
235 |
260 |
0.50 |
104.0% |
105.5% |
Test item |
0.02 |
B |
249 |
255 |
0.50 |
107.0% |
|
Test item |
0.01 |
A |
261 |
239 |
0.50 |
105.0% |
104.9% |
Test item |
0.01 |
B |
222 |
271 |
0.49 |
104.7% |
|
Test item |
0.005 |
A |
230 |
231 |
0.46 |
96.8% |
98.3% |
Test item |
0.005 |
B |
217 |
253 |
0.47 |
99.8% |
|
Viability Experiment I
The viability was determined by the Relative Survival (RS). This corresponds to the adjusted CE in treated culture divided by the adjusted CE in corresponding solvent control multiplied by 100.
Relative Survival Experiment I with Metabolic Activation
|
Conc. |
Culture |
Cells seeded |
Number of colonies per dish |
|
CE II absolute |
CE II relative |
Adjusted CE |
RS |
|
[µg/mL] |
|
I + II |
I |
II |
|
|
|
|
Solvent Control Test Item |
- |
A |
998 |
346 |
313 |
0.660 |
- |
0.327 |
- |
Solvent Control Test Item |
- |
B |
1004 |
257 |
255 |
0.510 |
- |
0.262 |
- |
Solvent Control DMBA |
- |
A |
1000 |
339 |
331 |
0.670 |
- |
0.338 |
- |
Solvent Control DMBA |
- |
B |
1003 |
381 |
361 |
0.740 |
- |
0.376 |
- |
Positive Control (DMBA) |
1.5 µg/mL |
A |
999 |
384 |
384 |
0.769 |
114.7% |
0.372 |
109.9% |
Positive Control (DMBA) |
1.5 µg/mL |
B |
1002 |
328 |
299 |
0.626 |
84.6% |
0.315 |
83.7% |
Test item |
0.078 |
A |
1004 |
423 |
360 |
0.789 |
119.5% |
0.133 |
40.8% |
Test item |
0.078 |
B |
997 |
372 |
382 |
0.756 |
148.2% |
0.113 |
43.3% |
Test item |
0.039 |
A |
998 |
422 |
424 |
0.847 |
128.4% |
0.368 |
112.5% |
Test item |
0.039 |
B |
998 |
264 |
280 |
0.545 |
106.9% |
0.234 |
89.4% |
Test item |
0.02 |
A |
999 |
364 |
440 |
0.805 |
122.0% |
0.390 |
119.3% |
Test item |
0.02 |
B |
1003 |
257 |
308 |
0.563 |
110.4% |
0.266 |
101.6% |
Test item |
0.01 |
A |
998 |
339 |
442 |
0.783 |
118.5% |
0.406 |
124.3% |
Test item |
0.01 |
B |
1003 |
302 |
291 |
0.592 |
116.0% |
0.316 |
120.7% |
Test item |
0.005 |
A |
999 |
491 |
490 |
0.982 |
148.8% |
0.499 |
152.7% |
Test item |
0.005 |
B |
1003 |
322 |
296 |
0.616 |
120.8% |
0.311 |
118.7% |
Test item |
0.002 |
A |
1001 |
458 |
455 |
0.912 |
138.2% |
0.448 |
137.1% |
Test item |
0.002 |
B |
1000 |
455 |
421 |
0.876 |
171.8% |
0.470 |
179.2% |
Relative Survival Experiment I without Metabolic Activation
|
Conc. |
Culture |
Cells seeded |
Number of colonies per dish |
|
CE II absolute |
CE II relative |
Adjusted CE |
RS |
|
[µg/mL] |
|
I + II |
I |
II |
|
|
|
|
Solvent Control Test Item |
- |
A |
1004 |
486 |
444 |
0.927 |
- |
0.441 |
- |
Solvent Control Test Item |
- |
B |
1004 |
347 |
394 |
0.738 |
- |
0.348 |
- |
Solvent Control EMS |
- |
A |
1004 |
336 |
319 |
0.653 |
- |
0.307 |
- |
Solvent Control EMS |
- |
B |
996 |
323 |
313 |
0.639 |
- |
0.300 |
- |
Positive Control (EMS) |
300 µg/mL |
A |
1004 |
321 |
300 |
0.619 |
94.8% |
0.303 |
98.6% |
Positive Control (EMS) |
300 µg/mL |
B |
999 |
427 |
410 |
0.838 |
131.2% |
0.413 |
137.6% |
Test item |
0.156 |
A |
1004 |
287 |
317 |
0.601 |
64.9% |
0.219 |
49.6% |
Test item |
0.156 |
B |
100 |
431 |
502 |
0.933 |
126.4% |
0.344 |
99.0% |
Test item |
0.078 |
A |
1000 |
362 |
298 |
0.660 |
71.2% |
0.271 |
61.3% |
Test item |
0.078 |
B |
1000 |
381 |
361 |
0.742 |
100.5% |
0.319 |
91.8% |
Test item |
0.039 |
A |
1002 |
267 |
303 |
0.569 |
61.4% |
0.265 |
60.0% |
Test item |
0.039 |
B |
995 |
348 |
340 |
0.691 |
93.6% |
0.334 |
96.0% |
Test item |
0.02 |
A |
1003 |
189 |
194 |
0.382 |
41.2% |
0.189 |
42.9% |
Test item |
0.02 |
B |
998 |
415 |
415 |
0.832 |
112.7% |
0.419 |
120.6% |
Test item |
0.01 |
A |
996 |
277 |
261 |
0.540 |
58.3% |
0.270 |
61.2% |
Test item |
0.01 |
B |
1003 |
411 |
353 |
0.761 |
103.2% |
0.375 |
108.0% |
Test item |
0.005 |
A |
1000 |
249 |
234 |
0.483 |
52.1% |
0.223 |
50.5% |
Test item |
0.005 |
B |
998 |
387 |
369 |
0.758 |
102.6% |
0.356 |
102.4% |
Mutagenicity Experiment I
The mutagenicity was determined by the mutant colonies per 106cells. This corresponds to the CEMUTdivided by the CE in non-selective medium (≥ CR IIabs).
Mutagenicity in Experiment I with Metabolic Activation
|
Conc. |
Culture |
Cell seeded |
Number of colonies per dish |
CEMUT |
MF |
MF |
MF Mean |
||||
|
[µL/mL] |
|
Total |
I |
II |
III |
IV |
V |
|
|
Per 106cells |
|
Solvent Control Test Item |
- |
A |
2497797 |
3 |
4 |
3 |
4 |
5 |
0.00001 |
0.00001 |
12 |
9 |
Solvent Control Test item |
- |
B |
2500611 |
1 |
3 |
1 |
0 |
3 |
0.00000 |
0.00001 |
6 |
|
Solvent Control DMBA |
- |
A |
2499455 |
1 |
3 |
3 |
4 |
4 |
0.00001 |
0.00001 |
9 |
6 |
Solvent Control DMBA |
- |
B |
2501356 |
3 |
0 |
0 |
2 |
1 |
0.00000 |
0.00000 |
3 |
|
Positive Control (DMBA) |
1.5 µg/mL |
A |
2502234 |
32 |
35 |
30 |
31 |
22 |
0.00006 |
0.00008 |
78 |
98 |
Positive Control (DMBA) |
1.5 µg/mL |
B |
2498430 |
41 |
33 |
45 |
34 |
32 |
0.00007 |
0.00012 |
118 |
|
Test item |
0.078 |
A |
2498483 |
2 |
0 |
2 |
0 |
0 |
0.00000 |
0.00000 |
2 |
2 |
Test item |
0.078 |
B |
2496340 |
1 |
1 |
1 |
0 |
2 |
0.00000 |
0.00000 |
3 |
|
Test item |
0.039 |
A |
2503165 |
1 |
1 |
5 |
4 |
3 |
0.00001 |
0.00001 |
7 |
9 |
Test item |
0.039 |
B |
2504425 |
4 |
2 |
2 |
5 |
3 |
0.00001 |
0.00001 |
12 |
|
Test item |
0.02 |
A |
2503470 |
5 |
8 |
5 |
4 |
6 |
0.00001 |
0.00001 |
14 |
15 |
Test item |
0.02 |
B |
2507424 |
6 |
3 |
5 |
3 |
6 |
0.00001 |
0.00001 |
16 |
|
Test item |
0.01 |
A |
2496813 |
0 |
1 |
2 |
2 |
3 |
0.00000 |
0.00000 |
4 |
12 |
Test item |
0.01 |
B |
2498514 |
3 |
5 |
3 |
8 |
9 |
0.00001 |
0.00002 |
19 |
|
Test item |
0.005 |
A |
2494895 |
2 |
2 |
3 |
2 |
2 |
0.00000 |
0.00000 |
4 |
9 |
Test item |
0.005 |
B |
2504955 |
5 |
3 |
6 |
3 |
5 |
0.00001 |
0.00001 |
14 |
|
Test item |
0.002 |
A |
2500019 |
4 |
2 |
4 |
2 |
4 |
0.00001 |
0.00001 |
7 |
7 |
Test item |
0.002 |
B |
2499952 |
3 |
3 |
3 |
5 |
2 |
0.00001 |
0.00001 |
7 |
|
Mutagenicity in Experiment I without Metabolic Activation
|
Conc. |
Culture |
Cell seeded |
Number of colonies per dish |
CEMUT |
MF |
MF |
MF Mean |
||||
|
[µL/mL] |
|
Total |
I |
II |
III |
IV |
V |
|
|
Per 106cells |
|
Solvent Control Test Item |
- |
A |
2498993 |
3 |
4 |
3 |
1 |
5 |
0.00001 |
0.00001 |
7 |
8 |
Solvent Control Test item |
- |
B |
2500712 |
3 |
5 |
5 |
3 |
1 |
0.00001 |
0.00001 |
9 |
|
Solvent Control EMS |
- |
A |
2500483 |
4 |
4 |
2 |
2 |
3 |
0.00001 |
0.00001 |
9 |
7 |
Solvent Control EMS |
- |
B |
2502441 |
0 |
1 |
3 |
3 |
2 |
0.00000 |
0.00001 |
6 |
|
Positive Control (EMS) |
300 µg/mL |
A |
2500958 |
12 |
20 |
22 |
18 |
22 |
0.00004 |
0.00006 |
61 |
77 |
Positive Control (EMS) |
300 µg/mL |
B |
2500767 |
44 |
37 |
37 |
35 |
41 |
0.00008 |
0.00009 |
93 |
|
Test item |
0.156 |
A |
2504289 |
2 |
4 |
4 |
3 |
2 |
0.00001 |
0.00001 |
10 |
8 |
Test item |
0.156 |
B |
2499886 |
1 |
2 |
2 |
4 |
3 |
0.00000 |
0.00001 |
5 |
|
Test item |
0.078 |
A |
2497818 |
5 |
2 |
1 |
24 |
0 |
0.00001 |
0.00001 |
8 |
7 |
Test item |
0.078 |
B |
2500562 |
0 |
2 |
2 |
3 |
2 |
0.00000 |
0.00000 |
5 |
|
Test item |
0.039 |
A |
2501631 |
8 |
6 |
4 |
0 |
2 |
0.00001 |
0.00001 |
14 |
9 |
Test item |
0.039 |
B |
2501774 |
1 |
0 |
1 |
1 |
3 |
0.00000 |
0.00000 |
3 |
|
Test item |
0.02 |
A |
2497776 |
2 |
3 |
2 |
3 |
2 |
0.00000 |
0.00001 |
13 |
9 |
Test item |
0.02 |
B |
2504561 |
1 |
3 |
2 |
3 |
3 |
0.00000 |
0.00001 |
6 |
|
Test item |
0.01 |
A |
2497193 |
4 |
4 |
5 |
2 |
4 |
0.00000 |
0.00000 |
4 |
9 |
Test item |
0.01 |
B |
2499786 |
3 |
2 |
1 |
3 |
0 |
0.00000 |
0.00000 |
5 |
|
Test item |
0.005 |
A |
2497302 |
3 |
6 |
6 |
5 |
5 |
0.00001 |
0.00002 |
21 |
14 |
Test item |
0.005 |
B |
2500320 |
2 |
3 |
4 |
1 |
2 |
0.00000 |
0.00001 |
6 |
|
EXPERIMENT II – DETAILED DATA
Note: All given values except the counts are rounded values. For the calculation with the validated calculation sheet the unrounded values were used.
The cell number of the respective cell suspension was measured using the cell counter.
For the determination of the corresponding cells/mL of the suspension the factor 100 was taken into account.
Cell Numbers Experiment II
Cell Number for Treatment Experiment II without Metabolic Activation
Count 1:10 Dilution of Original Cell Suspension |
Cells/mL in Original Cell Suspension |
Control Count after adjustment to 107Cells |
Cells seeded/10 mL medium for Viability and Mutagenicity |
Cells seeded/10 mL medium for survival |
20353 |
2035300 |
1063 |
1063000* |
500* |
*Note: This number of cells was added into all single culture dishes (6 test item concentrations + 2 solvent controls + 1 positive control: Replicate A and B)
Cell Passage 72h after Starting of Expression Time. Approach without Metabolic Activation
|
Conc. |
Culture |
Cells counted |
Cells/mL counted |
Adjusted cells/ culture dish |
|
[µL/mL] |
|
|
|
|
Solvent Control Test Item |
- |
A |
15688 |
1568800 |
999326 |
Solvent Control Test Item |
- |
B |
13927 |
1382700 |
999959 |
Solvent Control EMS |
- |
A |
14669 |
146600 |
1000426 |
Solvent Control EMS |
- |
B |
13859 |
1385900 |
1000620 |
Positive Control (EMS) |
150 µg/mL |
A |
15022 |
1502200 |
1000465 |
Positive Control (EMS) |
150 µg/mL |
B |
14518 |
1451800 |
1000290 |
Test item |
0.156 |
A |
2959 |
295900 |
1000142 |
Test item |
0.156 |
B |
2383 |
238300 |
1000860 |
Test item |
0.078 |
A |
2423 |
242300 |
1000699 |
Test item |
0.078 |
B |
3161 |
316100 |
998876 |
Test item |
0.039 |
A |
5339 |
533900 |
999995 |
Test item |
0.039 |
B |
6594 |
659400 |
1000310 |
Test item |
0.02 |
A |
15581 |
1558100 |
1000300 |
Test item |
0.02 |
B |
16426 |
1642600 |
1000343 |
Test item |
0.01 |
A |
19344 |
1934400 |
1000085 |
Test item |
0.01 |
B |
17442 |
1744200 |
999427 |
Test item |
0.005 |
A |
18170 |
1717000 |
999350 |
Test item |
0.005 |
B |
12825 |
1282500 |
1000350 |
Seeding for Mutagenicity and Viability. At least 168h after Starting of Expression Time. Approach without Metabolic Activation
|
Conc. |
Culture |
Cells counted |
Cells/mL counted |
Adjusted cells/ culture dish (Mutagenicity) |
Adjusted cells/ culture dish I and II (Viability) |
|
[µL/mL] |
|
|
|
|
|
Solvent Control Test Item |
- |
A |
13670 |
1367000 |
499486 |
500 |
Solvent Control Test Item |
- |
B |
11944 |
1194400 |
499476 |
499 |
Solvent Control EMS |
- |
A |
16673 |
1667300 |
499442 |
502 |
Solvent Control EMS |
- |
B |
10527 |
1052700 |
499463 |
498 |
Positive Control (EMS) |
150 µg/mL |
A |
12724 |
1272400 |
500070 |
502 |
Positive Control (EMS) |
150 µg/mL |
B |
12039 |
1203900 |
499253 |
500 |
Test item |
0.156 |
A |
11486 |
1148600 |
499210 |
502 |
Test item |
0.156 |
B |
11143 |
1114300 |
500695 |
501 |
Test item |
0.078 |
A |
11681 |
1168100 |
499551 |
498 |
Test item |
0.078 |
B |
11259 |
1125900 |
499127 |
501 |
Test item |
0.039 |
A |
11185 |
1118500 |
500639 |
501 |
Test item |
0.039 |
B |
9857 |
985700 |
500110 |
499 |
Test item |
0.02 |
A |
13682 |
1368200 |
499924 |
500 |
Test item |
0.02 |
B |
13904 |
1390400 |
500238 |
500 |
Test item |
0.01 |
A |
12801 |
1280100 |
499948 |
502 |
Test item |
0.01 |
B |
11444 |
1144400 |
499375 |
502 |
Test item |
0.005 |
A |
12141 |
1214100 |
501366 |
500 |
Test item |
0.005 |
B |
11749 |
1174900 |
499503 |
498 |
Survival Experiment II
The survival is given by the CE I value. This corresponds to the number of cells at the end of treatment divided by the total number of cells at the beginning of treatment.
Survival Experiment II without Metabolic Activation
|
Conc. |
Culture |
Number of colonies per dish |
CE I absolute |
CE I relative |
CE I relative Mean |
|
|
[µL/mL] |
|
I |
II |
|
|
|
Solvent Control Test Item |
- |
A |
349 |
341 |
0.69 |
- |
- |
Solvent Control Test Item |
- |
B |
346 |
374 |
0.72 |
- |
|
Solvent Control EMS |
- |
A |
340 |
307 |
0.65 |
- |
- |
Solvent Control EMS |
- |
B |
315 |
389 |
0.70 |
- |
|
Positive Control (EMS) |
150 µg/mL |
A |
360 |
358 |
0.72 |
111.0% |
99.7% |
Positive Control (EMS) |
150 µg/mL |
B |
290 |
333 |
0.62 |
88.5% |
|
Test item |
0.156 |
A |
0 |
0 |
0.00 |
0.0% |
0.0% |
Test Item |
0.156 |
B |
0 |
0 |
0.00 |
0.0% |
|
Test item |
0.078 |
A |
0 |
0 |
0.00 |
0.0% |
0.0% |
Test item |
0.078 |
B |
0 |
0 |
0.00 |
0.0% |
|
Test item |
0.039 |
A |
0 |
0 |
0.00 |
0.0% |
0.0% |
Test item |
0.039 |
B |
0 |
0 |
0.00 |
0.0% |
|
Test item |
0.02 |
A |
0 |
0 |
0.00 |
0.0% |
0.1% |
Test item |
0.02 |
B |
0 |
1 |
0.00 |
0.1% |
|
Test item |
0.01 |
A |
2 |
1 |
0.00 |
0.4% |
0.8% |
Test item |
0.01 |
B |
4 |
5 |
0.01 |
1.3% |
|
Test item |
0.005 |
A |
28 |
44 |
0.07 |
10.4% |
8.9% |
Test item |
0.005 |
B |
23 |
30 |
0.05 |
7.4% |
|
Viability Experiment II
The viability was determined by the Relative Survival (RS). This corresponds to the adjusted CE in treated culture divided by the adjusted CE in corresponding solvent control multiplied by 100.
Relative Survival Experiment II without Metabolic Activation
|
Conc. |
Culture |
Cells seeded |
Number of colonies per dish |
|
CE II absolute |
CE II relative |
Adjusted CE |
RS |
|
[µg/mL] |
|
I + II |
I |
II |
|
|
|
|
Solvent Control Test Item |
- |
A |
1000 |
231 |
233 |
0.464 |
- |
0.320 |
- |
Solvent Control Test Item |
- |
B |
998 |
223 |
206 |
0.430 |
- |
0.309 |
- |
Solvent Control EMS |
- |
A |
1004 |
190 |
189 |
0.377 |
- |
0.244 |
- |
Solvent Control EMS |
- |
B |
996 |
219 |
233 |
0.454 |
- |
0.319 |
- |
Positive Control (EMS) |
150 µg/mL |
A |
1004 |
242 |
230 |
0.470 |
124.6% |
0.338 |
138.2% |
Positive Control (EMS) |
150 µg/mL |
B |
999 |
226 |
235 |
0.461 |
101.7% |
0.287 |
90.0% |
Test item |
0.156 |
A |
1004 |
232 |
233 |
0.463 |
99.8% |
0.000 |
0.0% |
Test item |
0.156 |
B |
1001 |
206 |
217 |
0.423 |
98.3% |
0.000 |
0.0% |
Test item |
0.078 |
A |
997 |
224 |
252 |
0.478 |
102.9% |
0.000 |
0.0% |
Test item |
0.078 |
B |
1002 |
239 |
214 |
0.452 |
105.2% |
0.000 |
0.0% |
Test item |
0.039 |
A |
1001 |
227 |
251 |
0.477 |
102.9% |
0.000 |
0.0% |
Test item |
0.039 |
B |
999 |
211 |
215 |
0.427 |
99.3% |
0.000 |
0.0% |
Test item |
0.02 |
A |
1000 |
259 |
240 |
0.499 |
107.6% |
0.000 |
0.0% |
Test item |
0.02 |
B |
1001 |
234 |
221 |
0.455 |
105.8% |
0.000 |
0.1% |
Test item |
0.01 |
A |
1003 |
245 |
236 |
0.479 |
103.3% |
0.001 |
0.4% |
Test item |
0.01 |
B |
1003 |
247 |
239 |
0.484 |
112.7% |
0.004 |
1.4% |
Test item |
0.005 |
A |
999 |
232 |
262 |
0.494 |
106.5% |
0.036 |
11.1% |
Test item |
0.005 |
B |
996 |
229 |
232 |
0.463 |
107.6% |
0.025 |
7.9% |
Mutagenicity Experiment II
The mutagenicity was determined by the mutant colonies per 106cells. This corresponds to the CEMUTdivided by the CE in non-selective medium (≥ CR IIabs).
Mutagenicity in Experiment II without Metabolic Activation
|
Conc. |
Culture |
Cell seeded |
Number of colonies per dish |
CEMUT |
MF |
MF |
MF Mean |
||||
|
[µL/mL] |
|
Total |
I |
II |
III |
IV |
V |
|
|
Per 106cells |
|
Solvent Control Test Item |
- |
A |
2497428 |
6 |
2 |
6 |
5 |
4 |
0.00001 |
0.00002 |
20 |
28 |
Solvent Control Test item |
- |
B |
2497382 |
10 |
7 |
5 |
7 |
9 |
0.00002 |
0.00004 |
35 |
|
Solvent Control EMS |
- |
A |
2497209 |
3 |
2 |
2 |
2 |
3 |
0.00000 |
0.00001 |
13 |
14 |
Solvent Control EMS |
- |
B |
2497316 |
4 |
2 |
6 |
4 |
1 |
0.00001 |
0.00002 |
15 |
|
Positive Control (EMS) |
150 µg/mL |
A |
2500349 |
71 |
70 |
72 |
66 |
73 |
0.00014 |
0.00030 |
299 |
286 |
Positive Control (EMS) |
150 µg/mL |
B |
2496264 |
71 |
55 |
65 |
60 |
63 |
0.00013 |
0.00027 |
273 |
|
Test item |
0.156 |
A |
2496048 |
0 |
0 |
0 |
0 |
0 |
0.00000 |
0.00000 |
0 |
17 |
Test item |
0.156 |
B |
2503476 |
9 |
5 |
4 |
8 |
9 |
0.00001 |
0.00003 |
33 |
|
Test item |
0.078 |
A |
2497757 |
3 |
4 |
3 |
3 |
5 |
0.00001 |
0.00002 |
15 |
8 |
Test item |
0.078 |
B |
2495636 |
0 |
0 |
0 |
0 |
0 |
0.00000 |
0.00000 |
0 |
|
Test item |
0.039 |
A |
2503193 |
5 |
0 |
1 |
2 |
5 |
0.00001 |
0.00001 |
11 |
10 |
Test item |
0.039 |
B |
2500548 |
1 |
2 |
4 |
2 |
1 |
0.00000 |
0.00001 |
9 |
|
Test item |
0.02 |
A |
2499620 |
3 |
0 |
3 |
3 |
2 |
0.00000 |
0.00001 |
9 |
8 |
Test item |
0.02 |
B |
2501192 |
1 |
1 |
2 |
2 |
2 |
0.00000 |
0.00001 |
7 |
|
Test item |
0.01 |
A |
2499741 |
3 |
8 |
5 |
0 |
2 |
0.00001 |
0.00002 |
15 |
15 |
Test item |
0.01 |
B |
2496873 |
5 |
4 |
3 |
4 |
2 |
0.00001 |
0.00001 |
15 |
|
Test item |
0.005 |
A |
2506828 |
1 |
2 |
0 |
4 |
3 |
0.00000 |
0.00001 |
8 |
11 |
Test item |
0.005 |
B |
2497516 |
0 |
5 |
4 |
3 |
3 |
0.00001 |
0.00001 |
13 |
|
EXPERIMENT II-2 – DETAILED DATA
Note: All given values except the counts are rounded values. For the calculation with the validated calculation sheet the unrounded values were used.
The cell number of the respective cell suspension was measured using the cell counter.
For the determination of the corresponding cells/mL of the suspension the factor 100 was taken into account.
Cell Numbers Experiment II-2
Cell Number for Treatment Experiment II without Metabolic Activation
Count 1:10 Dilution of Original Cell Suspension |
Cells/mL in Original Cell Suspension |
Control Count after adjustment to 107Cells |
Cells seeded/10 mL medium for Viability and Mutagenicity |
Cells seeded/10 mL medium for survival |
20015 |
2001500 |
999 |
999000* |
500* |
*Note: This number of cells was added into all single culture dishes (6 test item concentrations + 2 solvent controls + 1 positive control: Replicate A and B)
Cell Passage 72h after Starting of Expression Time. Approach without Metabolic Activation
|
Conc. |
Culture |
Cells counted |
Cells/mL counted |
Adjusted cells/ culture dish |
|
[µL/mL] |
|
|
|
|
Solvent Control Test Item |
- |
A |
12221 |
1222100 |
999678 |
Solvent Control Test Item |
- |
B |
11743 |
1174300 |
1000504 |
Solvent Control EMS |
- |
A |
12150 |
1215000 |
999945 |
Solvent Control EMS |
- |
B |
10919 |
1091900 |
1000180 |
Positive Control (EMS) |
150 µg/mL |
A |
13633 |
1363300 |
1000662 |
Positive Control (EMS) |
150 µg/mL |
B |
12453 |
1245300 |
999976 |
Test item |
0.0049 |
A |
11779 |
1177900 |
1000037 |
Test item |
0.0049 |
B |
11212 |
1121200 |
1000110 |
Test item |
0.0024 |
A |
11913 |
11916300 |
999501 |
Test item |
0.0024 |
B |
13820 |
1382000 |
1000568 |
Test item |
0.0012 |
A |
13875 |
1387500 |
1000388 |
Test item |
0.0012 |
B |
13334 |
1333400 |
1000050 |
Test item |
0.0006 |
A |
10701 |
1070100 |
999473 |
Test item |
0.0006 |
B |
14686 |
1468600 |
1000117 |
Test item |
0.0003 |
A |
10894 |
1089400 |
1000069 |
Test item |
0.0003 |
B |
12618 |
1261800 |
1000607 |
Test item |
0.00015 |
A |
11028 |
1102800 |
1000240 |
Test item |
0.00015 |
B |
12759 |
1275900 |
1000306 |
Seeding for Mutagenicity and Viability. At least 168h after Starting of Expression Time. Approach without Metabolic Activation
|
Conc. |
Culture |
Cells counted |
Cells/mL counted |
Adjusted cells/ culture dish (Mutagenicity) |
Adjusted cells/ culture dish I and II (Viability) |
|
[µL/mL] |
|
|
|
|
|
Solvent Control Test Item |
- |
A |
13359 |
1335900 |
500719 |
499 |
Solvent Control Test Item |
- |
B |
14756 |
1475600 |
500422 |
498 |
Solvent Control EMS |
- |
A |
12778 |
1277800 |
499958 |
502 |
Solvent Control EMS |
- |
B |
11923 |
1192300 |
500675 |
499 |
Positive Control (EMS) |
150 µg/mL |
A |
13358 |
1335800 |
500682 |
499 |
Positive Control (EMS) |
150 µg/mL |
B |
14655 |
1465500 |
500087 |
503 |
Test item |
0.0049 |
A |
12638 |
123800 |
501107 |
501 |
Test item |
0.0049 |
B |
12980 |
1298000 |
500138 |
498 |
Test item |
0.0024 |
A |
12517 |
1251700 |
500680 |
501 |
Test item |
0.0024 |
B |
14912 |
1491200 |
499559 |
499 |
Test item |
0.0012 |
A |
12612 |
1261200 |
500076 |
501 |
Test item |
0.0012 |
B |
14970 |
1497000 |
499662 |
499 |
Test item |
0.0006 |
A |
11590 |
1159000 |
499696 |
498 |
Test item |
0.0006 |
B |
13700 |
1370000 |
500582 |
500 |
Test item |
0.0003 |
A |
12387 |
1238700 |
499803 |
501 |
Test item |
0.0003 |
B |
9976 |
997600 |
500978 |
500 |
Test item |
0.00015 |
A |
12044 |
1204400 |
499460 |
499 |
Test item |
0.00015 |
B |
13770 |
1377000 |
500003 |
500 |
Survival Experiment II-2
The survival is given by the CE I value. This corresponds to the number of cells at the end of treatment divided by the total number of cells at the beginning of treatment.
Survival Experiment II without Metabolic Activation
|
Conc. |
Culture |
Number of colonies per dish |
CE I absolute |
CE I relative |
CE I relative Mean |
|
|
[µL/mL] |
|
I |
II |
|
|
|
Solvent Control Test Item |
- |
A |
405 |
384 |
0.79 |
- |
- |
Solvent Control Test Item |
- |
B |
412 |
409 |
0.82 |
- |
|
Solvent Control EMS |
- |
A |
383 |
385 |
0.77 |
- |
- |
Solvent Control EMS |
- |
B |
393 |
432 |
0.83 |
- |
|
Positive Control (EMS) |
150 µg/mL |
A |
403 |
525 |
0.93 |
120.8% |
115.1% |
Positive Control (EMS) |
150 µg/mL |
B |
420 |
483 |
0.90 |
109.5% |
|
Test item |
0.0049 |
A |
0 |
0 |
0.00 |
0.0% |
0.4% |
Test Item |
0.0049 |
B |
4 |
3 |
0.01 |
0.9% |
|
Test item |
0.0024 |
A |
363 |
357 |
0.72 |
91.3% |
98.5% |
Test item |
0.0024 |
B |
462 |
406 |
0.87 |
105.7% |
|
Test item |
0.0012 |
A |
354 |
403 |
0.76 |
95.9% |
100.5% |
Test item |
0.0012 |
B |
425 |
438 |
0.86 |
105.1% |
|
Test item |
0.0006 |
A |
408 |
390 |
0.80 |
101.1% |
104.9% |
Test item |
0.0006 |
B |
459 |
433 |
0.89 |
108.6% |
|
Test item |
0.0003 |
A |
399 |
375 |
0.77 |
98.1% |
100.4% |
Test item |
0.0003 |
B |
420 |
423 |
0.84 |
102.7% |
|
Test item |
0.00015 |
A |
413 |
489 |
0.90 |
114.3% |
117.4% |
Test item |
0.00015 |
B |
496 |
493 |
0.99 |
120.5% |
|
Viability Experiment II-2
The viability was determined by the Relative Survival (RS). This corresponds to the adjusted CE in treated culture divided by the adjusted CE in corresponding solvent control multiplied by 100.
Relative Survival Experiment II without Metabolic Activation
|
Conc. |
Culture |
Cells seeded |
Number of colonies per dish |
|
CE II absolute |
CE II relative |
Adjusted CE |
RS |
|
[µg/mL] |
|
I + II |
I |
II |
|
|
|
|
Solvent Control Test Item |
- |
A |
997 |
552 |
512 |
1.067 |
- |
0.842 |
- |
Solvent Control Test Item |
- |
B |
995 |
646 |
601 |
1.253 |
- |
1.029 |
- |
Solvent Control EMS |
- |
A |
1004 |
514 |
512 |
1.022 |
- |
0.785 |
- |
Solvent Control EMS |
- |
B |
998 |
560 |
570 |
1.132 |
- |
0.934 |
- |
Positive Control (EMS) |
150 µg/mL |
A |
997 |
455 |
444 |
0.901 |
88.2% |
0.837 |
106.6% |
Positive Control (EMS) |
150 µg/mL |
B |
1005 |
617 |
572 |
1.183 |
104.5% |
1.068 |
114.4% |
Test item |
0.0049 |
A |
1002 |
541 |
472 |
1.011 |
94.7% |
0.000 |
0.0% |
Test item |
0.0049 |
B |
995 |
611 |
573 |
1.190 |
95.0% |
0.008 |
0.8% |
Test item |
0.0024 |
A |
1002 |
566 |
549 |
1.113 |
104.3% |
0.801 |
95.2% |
Test item |
0.0024 |
B |
997 |
625 |
607 |
1.236 |
98.6% |
1.073 |
104.3% |
Test item |
0.0012 |
A |
1002 |
449 |
430 |
0.877 |
82.2% |
0.664 |
78.9% |
Test item |
0.0012 |
B |
997 |
460 |
441 |
0.904 |
72.1% |
0.780 |
75.8% |
Test item |
0.0006 |
A |
995 |
485 |
469 |
0.958 |
89.8% |
0.765 |
90.9% |
Test item |
0.0006 |
B |
1000 |
405 |
487 |
0.892 |
71.2% |
0.796 |
77.4% |
Test item |
0.0003 |
A |
1001 |
510 |
586 |
1.095 |
102.6% |
0.847 |
100.7% |
Test item |
0.0003 |
B |
1000 |
447 |
415 |
0.862 |
68.8% |
0.727 |
70.6% |
Test item |
0.00015 |
A |
998 |
412 |
401 |
0.814 |
76.3% |
0.734 |
87.3% |
Test item |
0.00015 |
B |
1000 |
573 |
522 |
1.095 |
87.4% |
1.083 |
105.3% |
Mutagenicity Experiment II-2
The mutagenicity was determined by the mutant colonies per 106cells. This corresponds to the CEMUTdivided by the CE in non-selective medium (≥ CR IIabs).
Mutagenicity in Experiment II without Metabolic Activation
|
Conc. |
Culture |
Cell seeded |
Number of colonies per dish |
CEMUT |
MF |
MF |
MF Mean |
||||
|
[µL/mL] |
|
Total |
I |
II |
III |
IV |
V |
|
|
Per 106cells |
|
Solvent Control Test Item |
- |
A |
2503597 |
3 |
0 |
1 |
3 |
4 |
0.00000 |
0.00000 |
4 |
4 |
Solvent Control Test item |
- |
B |
2502109 |
2 |
4 |
3 |
1 |
3 |
0.00001 |
0.00000 |
4 |
|
Solvent Control EMS |
- |
A |
2499791 |
3 |
8 |
5 |
4 |
9 |
0.00001 |
0.00001 |
11 |
12 |
Solvent Control EMS |
- |
B |
2503375 |
5 |
13 |
8 |
2 |
8 |
0.00001 |
0.00001 |
13 |
|
Positive Control (EMS) |
150 µg/mL |
A |
2503410 |
83 |
92 |
103 |
103 |
94 |
0.00019 |
0.00021 |
210 |
174 |
Positive Control (EMS) |
150 µg/mL |
B |
2500435 |
85 |
82 |
82 |
79 |
78 |
0.00016 |
0.00014 |
137 |
|
Test item |
0.0049 |
A |
2505533 |
7 |
6 |
9 |
11 |
3 |
0.00001 |
0.00001 |
14 |
8 |
Test item |
0.0049 |
B |
2500691 |
1 |
0 |
2 |
4 |
1 |
0.00000 |
0.00000 |
2 |
|
Test item |
0.0024 |
A |
2503400 |
77 |
6 |
5 |
8 |
5 |
0.00001 |
0.00001 |
12 |
9 |
Test item |
0.0024 |
B |
2497795 |
6 |
6 |
5 |
4 |
0 |
0.00001 |
0.00001 |
7 |
|
Test item |
0.0012 |
A |
2500378 |
7 |
9 |
8 |
8 |
6 |
0.00002 |
0.00002 |
17 |
12 |
Test item |
0.0012 |
B |
2498309 |
2 |
3 |
5 |
1 |
2 |
0.00001 |
0.00001 |
6 |
|
Test item |
0.0006 |
A |
2498481 |
14 |
5 |
10 |
3 |
8 |
0.00002 |
0.00001 |
17 |
12 |
Test item |
0.0006 |
B |
2502909 |
5 |
2 |
3 |
4 |
4 |
0.00001 |
0.00001 |
8 |
|
Test item |
0.0003 |
A |
2499013 |
5 |
4 |
2 |
5 |
3 |
0.00001 |
0.00001 |
7 |
7 |
Test item |
0.0003 |
B |
2504891 |
5 |
2 |
4 |
2 |
1 |
0.00001 |
0.00001 |
6 |
|
Test item |
0.00015 |
A |
2497301 |
n/e |
n/e |
n/e |
n/e |
n/e |
n/e |
n/e |
n/e |
n/e |
Test item |
0.00015 |
B |
2500017 |
n/e |
n/e |
n/e |
n/e |
n/e |
n/e |
n/e |
n/e |
|
n/e = not evaluated because the OECD 476 guideline prescribes only 4 concentrations
HISTORICAL DATA
In the following table, the historical data of the solvent controls and positive controls are presented.
Historical Data of the Solvent Controls
|
Number of mutant colonies per 106cells |
||
4 h treatment / with metabolic activation |
|||
|
Solvent control |
Positive control (DMBA) |
|
|
DMSO |
|
|
Mean value |
15 |
243 |
|
Standard deviation |
9.2 |
72.2 |
|
Range |
2 – 39 |
100 – 461 |
|
95.5 % control limit |
-4 – 33 |
99 – 387 |
|
99.7 % control limit |
-13 – 42 |
26 – 459 |
|
∑ |
52 |
35 |
|
Study no. 17100909G865 |
9, 6 |
98* |
|
4 h treatment / without metabolic activation |
|||
|
Solvent control |
Positive control (EMS) |
|
|
DMSO |
DMEM |
|
Mean value |
12 |
15 |
127 |
Standard deviation |
8.0 |
9.0 |
33.4 |
Range |
2 – 28 |
2 – 32 |
71 – 207 |
95.5 % control limit |
-4 – 28 |
-3 – 33 |
60 – 194 |
99.7 % control limit |
-12 – 36 |
-12 – 42 |
27 – 227 |
∑ |
22 |
44 |
33 |
Study no. 17100909G865 |
8 |
7 |
77 |
24 h treatment / without metabolic activation |
|||
|
Solvent control |
Positive control (EMS) |
|
|
DMSO |
DMEM |
|
Mean value |
15 |
12 |
237 |
Standard deviation |
10.4 |
6.6 |
97.0 |
Range |
2 – 40 |
4 – 27 |
35 – 517 |
95.5 % control limit |
-6 – 36 |
-2 – 25 |
43 – 431 |
99.7 % control limit |
-16 – 46 |
-8 – 32 |
-54 – 528 |
∑ |
24 |
32 |
28 |
Study no. 17100909G865 |
4 |
12 |
171 |
*value is outside the range of 95.5% control limit if the historical control data (99 – 387 * 10-6).
Since the difference is only marginal and the positive control nevertheless induced a very clearly positive result, this is considered as uncritical and the study is still valid.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Genetic toxicity in vitro-Ames assay
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.
Genetic toxicity in vitro-Chromosome aberration assay
The study was performed to assess the mutagenic potential of Trixene AS to induce structural chromosomal aberrations in human lymphocytes cultured in vitro in absence and presence of an exogenous metabolic activation system (liver S9 mix from male rats, treated with Aroclor 1254).
Human peripheral blood lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin and exposed to solvent control, test item and positive control. 3 hours before the end of cultivation, cell division was arrested using Colcemid®. After harvesting, slides were prepared and stained. The metaphase cells were examined for chromosomal damage.
In each experiment, all cell cultures were set up in duplicates. In order to assess the toxicity of the test solutions to the cultured human lymphocytes, the mitotic index was calculated for all cultures treated with medium control, solvent control, positive control and test item (as far as evaluable). On the basis of the data from the mitotic index, the concentrations were selected for metaphase analysis.
In all evaluated experiments, only in one case a statistically significant increase (p < 0.01) of structural chromosomal aberrations was found (exp. I without metabolic activation, 0.04 μL/mL). This value also lay above the historical laboratory control data and above the 95.5% control limit for the solvent control DMSO. However, in experiment II (also without metabolic activation, but with an extended exposure time) these findings could not be reproduced: neither a statistically significant nor a biologically relevant increase of structural chromosomal aberrations was observed at the evaluated concentrations. All values lay in the range of the concurrent solvent control DMSO. Due to the longer exposure time in experiment II, these results are classified as more significant. In experiment I with metabolic activation, the test item concentrations 0.63 and 0.31 μL/mL showed values of structural aberrations lying above the historical laboratory control data for the concurrent solvent control but not being statistically significant increased. The values above the historical laboratory control data were clearly inside the literature data for solvent controls (<5%).
In conclusion, it can be stated that under the experimental conditions reported, the test item Trixene AS did not induce structural chromosome aberrations in human lymphocytes in vitro in the chosen test concentrations.
Genetic toxicity in vitro - Mammalian cell gene mutation assay
This study was performed to investigate the potential of Trixene AS to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese Hamster cells (V79).
The assay comprised two pre-tests and three independent experiments (experiment I and two experiments II). Experiment II (exposure date 07. Jun. 2018) was invalid and therefore repeated (Experiment II-2: exposure date: 28. Jun. 2018). The pre-tests were done to detect a potential cytotoxic effect of the test item. Based on the results of these tests the concentrations for the main experiments were determined.
In experiment I no dose dependent increase in mutant colony numbers was observed. A statistically significant increase in mutant colony numbers was observed at the concentrations 0.02 μL/mL and 0.01 μL/mL in the approach with metabolic activation but only in replicate B as well as at the concentration 0.005 μL/mL in the approach without metabolic activation but only in replicate A. Nevertheless, all values remained within the historical control range of the solvent control.
Since the result of experiment I was not clearly negative, experiment II (with 24 h incubation) was performed. Since all test item concentrations in experiment II induced a cytotoxic effect, the experiment was repeated with lower test item concentrations (experiment II-2).
In experiment II-2 no dose dependent increase in mutant colony numbers was observed. A statistically significant increase in mutant colony numbers was observed at the concentrations 0.0024 μL/mL, 0.0012 μL/mL and 0.0006 μL/mL only in replicate A as well as at the concentrations 0.0012 μL/mL and 0.0006 μL/mL at the mean values. Nevertheless, all values remained within the historical control range of the solvent control.
Finally, no biological relevant increase in the number of mutant colonies was detected in both experiments.
Conclusion:
It can be stated that under the experimental conditions of this study Trixene AS did not induce gene mutations at the HPRT locus in V79 cells in the absence and presence of metabolic activation. Therefore, the test item Trixene AS is considered to be “non-mutagenic under the conditions of the HPRT assay”.
Justification for classification or non-classification
Based on the negative results obtained in the in vitro mutagenicity studies with the test substance, the substance is not considered to be mutagenic under Regulation (EC) 1272/2008.
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