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EC number: 618-780-1 | CAS number: 916809-14-8
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
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- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
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- Stability in organic solvents and identity of relevant degradation products
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- Stability: thermal, sunlight, metals
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- Endpoint summary
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- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Ames test: not mutagenic (BASF, 2008)
in vitro Micronucelus assay: negative (BASF, 2010)
HPRT: negative (BASF, 2011)
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:
- October - November 2007
- 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:
- 21 Jul 1997
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- (from the competent authority) Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Batch No.of test material: 8712 / 056
- Purity: 99.6 g / 100 g
- Date of production: 01 Oct 2007
- Physical state and appearance: solid, white
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerator - 20 °C
- Stability under test conditions: The stability of the test substance at room temperature in the vehicle water over a period of 4 hours was verified analytically.
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The substance was dissolved in water. To achieve a solution of the test substance in the vehicle, the test substance preparation was treated with ultrasonic waves and was shaken thoroughly. All test substance formulations were prepared immediately before administration. - Target gene:
- his, trp
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor induced rat liver S9 fraction
- Test concentrations with justification for top dose:
- 1st Experiment (Standard Plate Test): 0; 20; 100; 500; 2500 and 5000 µg/plate
2nd Experiment (Preincubation Test): 0; 312.5; 625; 1250; 2500 and 5000 µg/plate
In agreement with the recommendations of current guidelines 5 mg/plate or 5 µL/plate are generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose will be tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 µL/plate might also be tested in repeat experiments for further calculation / substantiation. - Vehicle / solvent:
- - Vehicle used: water
- Justification for choice of vehicle: Due to the good solubility of the test substance in water, water was used as vehicle. - Untreated negative controls:
- yes
- Remarks:
- sterility control
- Negative solvent / vehicle controls:
- yes
- Remarks:
- vehicle control
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- other: - 2-aminoanthracene (2-AA): with S9 mix, 2.5 µg/plate dissolved in DMSO (TA 1535, TA 100, TA 1537, TA 98) or 60 µg/plate dissolved in DMSO (E.coli WP2 uvrA)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation); preincubation
DURATION
- Exposure duration: 48 - 72 hours in the dark at 37 °C
DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn (= reduced his- or trp- background growth), reduction in the titer - Rationale for test conditions:
- Bacterial reverse mutation assays using amino-acid requiring strains of Salmonella typhimurium and Escherichia coli are commonly employed as initial screening methods for detecting a point mutagenic activity of chemical substances and are used to screen for possible mammalian mutagens and carcinogens.
- Evaluation criteria:
- - Mutagenicity: Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments. In general, five doses of the test substance are tested with a maximum of 5 mg/plate, and triplicate plating is used for all test groups at least in the 1st Experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments are based on the findings of the 1st Experiment.
- Titer: The titer is generally determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.
- Toxicity: Toxicity detected by a decrease in the number of revertants, clearing or diminution of the background lawn (= reduced his- or trp- background growth), reduction in the titer is recorded for all test groups both with and without S9 mix in all experiments and indicated in the tables.
- Solubility: Precipitation of the test material is recorded and indicated in the tables. As long as precipitation does not interefere with the colony scoring, 5 mg/plate is generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at least in the 1st Experiment even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate might also be tested in repeat experiments for further clarification / substantiation. - Statistics:
- Acceptance criteria
Generally, the experiment is considered valid if the following criteria are met:
- The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
- The sterility controls revealed no indication of bacterial contamination.
- The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
- The titer of viable bacteria was >= 10^8 / mL.
Assessment criteria
The test chemical is considered positive in this assay if the following criteria are met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance is generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other. - Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- in preincubation test only at 5000 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test substance precipitation was found.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see tables below
- Negative (solvent/vehicle) historical control data: see tables below - Conclusions:
- Under the experimental conditions chosen here, it is concluded that the test substance is not a mutagenic substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.
- Executive summary:
The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.
Strains: TA 1535, TA 100, TA 1537, TA 98 and E.coli WP2 uvrA
Dose Range: 20 µg - 5000 µg/plate (SPT), 312.5 µg - 5000 µg/plate (PIT)
Test Conditions: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (Aroclor-induced rat liver S9 mix).
Solubility: No precipitation of the test substance was found.
Toxicity: A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions at 5000 µg/plate.
Mutagenicity: A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.
According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium / Escherichia coli reverse mutation assay under the experimental conditions chosen here.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- May - August 2010
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD 487 (DRAFT)
- Version / remarks:
- Version 5, 02 Nov 2009
- Deviations:
- no
- GLP compliance:
- yes
- Remarks:
- (from the competent authority) Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz
- Type of assay:
- in vitro mammalian cell micronucleus test
- Target gene:
- N/A
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MEM (minimal essential medium with Earle's salts) containing a L-glutamine source
supplemented with
− 10% (v/v) fetal calf serum (FCS)
− 1% (v/v) penicillin/streptomycin (10 000 IU / 10 000 μg/mL)
− 1% (v/v) amphotericine B (250 μg/mL)
During exposure to the test substance for 4 hours only, MEM medium was used without FCS
supplementation.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S-9 mix
- Test concentrations with justification for top dose:
- 1st Experiment
4 hours exposure; 24 hours harvest time; without S9 mix:
0; 362.5; 725.0; 1 450.0; 2 900.0; 4 350.0; 5 800.0 μg/mL
4 hours exposure, 24 hours harvest time, with S9 mix:
0; 362.5; 725.0; 1 450.0; 2 900.0; 4 350.0; 5 800.0 μg/mL
2nd Experiment
24 hours exposure, 24 hours harvest time, without S9 mix
0; 250.0; 500.0; 1 000.0; 1 500.0; 2 000 μg/mL
4 hours exposure, 24 hours harvest time, with S9 mix
0; 250.0; 500.0; 1 000.0; 2 000.0; 3 000.0 μg/mL
Following the requirements of the current draft guideline a test substance should be tested up to a maximum concentration of 5 mg/mL, 5 µL/mL or 10 mM, which is the lowest. In case of toxicity, the top concentration should produce 55 % +/- 5 % cytotoxicity (cell number and / or proliferation index) compared to the respective vehicle control. For relatively insoluble test substances at least one concentration should be scored showing no precipitation in culture medium at the end of exposure period.
In the pretest the parameters pH value and osmolarity were not relevant influenced by the addition of the test substance preparation to the culture medium at the concentrations measured.
In addition, no test substance precipitation in the vehicle acetone was observed up to the highest applied concentration of 5800 µg/mL (stock solution). In culture medium test substance precipitation was observed at the end of treatment at 5800 µg/mL after 4 and 24 hours treatment in the absence and the presence of S9 mix.
After 4 hours treatment in the absence and the presence of S9 mix cytotoxicity indicated by reduced cell numbers of about or below 55 % +/- 5 % of control was observed at the highest applied concentration of 5800 µg/mL. Besides, in the pretest with 24 hours continuous treatment in the absence of S9 mix, the cell numbers were clearly reduced after treatment with 1450 µg/mL and above. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, acetone was selected as vehicle,
which had been demonstrated to be suitable in the in vitro mircronucleus test and for which historical control data are available. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 and 24 hours
- Recovery time: 0 or 20 hours
- Harverst time: 24 hours
NUMBER OF REPLICATIONS: The cell cycle of the untreated V79 cells lasts for about 12 - 14 hours under the selected
culture conditions (last measurement based on the BrdU method of Speit et al. [ 7]: 12 hours; Feb 2009). Thus, the selected harvest time of 24 hours is about 2 times the normal cell cycle length.
NUMBER OF CELLS EVALUATED: A sample of at least 1 000 cells for each culture were analyzed for micronuclei, i.e. at least
2 000 cells for each test group.
DETERMINATION OF CYTOTOXICITY
- Method: cell count - Evaluation criteria:
- A test substance is considered "positive" if the following criteria are met:
- A significant, dose-related and reproducible increase in the number of cells containing micronuclei.
- The number of micronucleated cells exceeds both the value of the concurrent negative/vehicle control and the range of the historical negative control data.
A test substance generally is considered "negative" if the following criteria are met:
- The number of micronucleated cells in the dose groups is not significant increased above the concurrent vehicle control value and is within the range of the historical negative control data. - Statistics:
- The statistical evaluation of the data was carried out using the MUVIKE program system (BASF SE). The proportion of cells containing micronuclei was calculated for each group. A comparison of each dose group with the concurrent negative/vehicle control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test is Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided.
If the results of this test were statistically significant compared with the respective negative/vehicle control, labels (* p ≤ 0.05, ** p ≤ 0.01) have been be printed in the tables. - Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- In this study, clearly reduced cell counts of about or below 55% ± 5% of control were observed at all experimental parts at least at the highest concentration scored for the occurrence of micronuclei.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- Under the experimental conditions chosen here, the conclusion is drawn that the test substance has not the potential to induce micronuclei (clastogenic and / or aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.
- Executive summary:
The test substance was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity) both in the absence and the presence of a metabolizing system.
According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following doses were tested and the test groups in bold type were evaluated:
1st Experiment
4 hours exposure, 24 hours harvest time, without S9 mix
0; 362.5; 725.0; 1450.0; 2900.0; 4350.0; 5800.0 µg/mL
4 hours exposure, 24 hours harvest time, with S9 mix
0; 362.5; 725.0; 1450.0; 2900.0; 4350.0; 5800.0 µg/mL
2nd Experiment
24 hours exposure, 24 hours harvest time, without S9 mix
0; 250.0; 500.0; 1000.0; 1500.0; 200.0 µg/mL
4 hours exposure, 24 hours harvest time, with S9 mix
0; 250.0; 500.0; 1000.0; 2000.0; 3000.0 µg/mL
A sample of at least 1000 cells for each culture were analyzed for micronuclei, i.e. at least 2000 cells for each test group.
The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for V79 cells. Both positive control substances, EMS and cyclophosphamide, led to a statistically significant and biologically relevant increase in the number of cells containing micronuclei.
On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other. The single statistically significant outlier value observed in the 2nd Experiment in the presence of metabolic activation after 4 hours treatment with 1000 µg/mL has to be considered biologically irrelevant due to missing dose-dependency and lacking confirmation in the 1st Experiment at a comparable concentration range.
Thus, under the experimental conditions described, the test substance is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- October 2010 - February 2011
- 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:
- 21 Jul 1997
- Deviations:
- no
- GLP compliance:
- yes
- Remarks:
- (from the competent authority) Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- - Type and identity of media:
All media were supplemented with:
- 1% (v/v) penicillin/streptomycin (stock solution: 10 000 IU / 10 000 μg/mL)
- 1% (v/v) amphotericine B (stock solution: 250 μg/mL)
Treatment medium (4-hour exposure period)
Ham's F12 medium containing stable glutamine and hypoxanthine (Biochrom; Cat. No.
FG 0815).
Culture medium and Treatment medium (24-hour exposure)
Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10%
(v/v) fetal calf serum (FCS).
Pretreatment medium ("HAT" medium)
Ham's F12 medium supplemented with:
- hypoxanthine (13.6 x 10-3 mg/mL)
- aminopterin (0.18 x 10-3 mg/mL)
- thymidine (3.88 x 10-3 mg/mL)
- 10% (v/v) fetal calf serum (FCS)
Selection medium ("TG" medium)
Hypoxanthine-free Ham's F12 medium supplemented with:
- 6-thioguanine (10 μg/mL)
- 1% (v/v) stable glutamine (200 mM)
- 10% (v/v) fetal calf serum (FCS)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- phenobarbital and ß-naphthoflavone induced rat liver S-9 mix
- Test concentrations with justification for top dose:
- 1st Experiment (invalid due to technical error)
without S9 mix (4-hour exposure period)
0; 93.8; 187.5; 375.0; 750.0; 1 500.0; 3 000.0; 6 000.0 μg/mL
with S9 mix (4-hour exposure period)
0; 93.8; 187.5; 375.0; 750.0; 1 500.0; 3 000.0; 6 000.0 μg/mL
2nd Experiment
without S9 mix (24-hour exposure period) (failed recommendations of OECD 476)
0; 125.0; 250.0; 500.0; 1 000.0; 2 000.0; 4 000.0; 6 000.0 μg/mL
with S9 mix (4-hour exposure period)
0; 250.0; 500.0; 1 000.0; 2 000.0; 4 000.0; 6 000.0 μg/mL
3rd Experiment
without S9 mix (24-hour exposure period) (failed recommendations of OECD 476)
0; 3.13; 6.25; 12.5; 25.0; 50.0; 100.0; 200.0 μg/mL
4th Experiment
without S9 mix (24-hour exposure period)
0; 7.8; 15.6; 31.3; 62.5; 125.0; 250.0; 500.0; 1 000.0; 2 000.0; 4 000.0 μg/mL
5th Experiment
without S9 mix (4-hour exposure period)
0; 46.9; 93.8; 187.5; 375.0; 750.0; 1 500.0; 3 000.0; 6 000.0 μg/mL
with S9 mix (4-hour exposure period)
0; 93.8; 187.5; 375.0; 750.0; 1 500.0; 3 000.0; 6 000.0 μg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test subtance in water, acetone was selected as vehicle, which had been demonstrated to be suitable in the CHO/HPRT assay and for which historical data are available. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- other: Methylcholanthrene (MCA): with S9 mix, 20 µg/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 20-24 hours
- Exposure duration: 4 or 24 hours
- Expression time (cells in growth medium): 7-9 days
- Selection time (if incubation with a selection agent): 6-7 days
NUMBER OF CELLS EVALUATED: 10^5 cells
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency - Evaluation criteria:
- A finding is assessed as positive if the following criteria are met:
- Increase of the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range.
- Evidence of reproducibility of any increase in mutant frequencies.
- A statistically significant increase in mutant frequencies and the evidence of a doseresponse
relationship.
Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 106 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient
evidence of mutagenicity.
The test substance is considered non-mutagenic according to the following criteria:
- The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significant increased above the concurrent negative control and is within our historical negative control data range. - Statistics:
- Due to the clearly negative findings, a statistical evaluation was not carried out.
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- see any other information on results
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- Under the experimental conditions chosen here, the conclusion is drawn that the test substance is not a mutagenic substance in the HPRT locus assay using CHO cells in the absence and the presence of metabolic activation.
- Executive summary:
The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese Hamster Ovary (CHO) cells in vitro. Five independent experiments were carried out with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation).
Based on the observations and the toxicity data of a previously performed pretest for an in vitro micronucleus assay and taking into account the cytotoxicity actually found in the main experiments, the following doses were tested and the doses in bold type were evaluated:
1st Experiment (invalid due to technical error)
without S9 mix (4 -hour exposure period)
0; 93.8; 187.5; 375.0; 750.0; 1500.0; 3000.0; 6000.0 µg/mL
with S9 mix (4 -hour exposure period)
0; 93.8; 187.5; 375.0; 750.0; 1500.0; 3000.0; 6000.0 µg/mL
2nd Experiment
without S9 mix (24 -hour exposure period) (failed recommendations of OECD 476)
0; 125.0; 250.0; 500.0; 1000.0; 2000.0; 4000.0; 6000.0 µg/mL
with S9 mix ((4 -hour exposure period)
0; 250.0; 500.0; 1000.0; 2000.0; 4000.0; 6000.0 µg/mL
3rd Experiment
without S9 mix (24 -hour exposure period) (failed recommendations of OECD 476)
0; 3.13; 6.25; 12.5; 25.0; 50.0; 100.0; 200.0 µg/mL
4th Experiment
without S9 mix (24 -hour exposure period)
0; 7.8; 15.6; 31.3; 62.5; 125.0; 250.0; 500.0; 1000.0; 2000.0; 4000.0 µg/mL
5th Experiment
without S9 mix (4 -hour exposure period)
0; 46.9; 93.8; 187.5; 375.0; 750.0; 1500.0; 3000.0; 6000.0 µg/mL
with S9 mix (4 -hour exposure)
0; 93.8; 187.5; 375.0; 750.0; 1500.0; 3000.0; 6000.0 µg/mL
After an attachment period of 20 - 24 hours and a treatment period of 4 hours both with and without metabolic activation and 24 hours without metabolic activation, an expression phase of about 6 - 8 days and a selection period of about 1 week followed. The colonies of each test group were fixed with methanol, stained with Giemsa and counted.
The vehicle controls gave mutant frequencies within the range expected for the CHO cell line.
Both positive control substances, EMS and MCA, led to the expected increase in the frequencies of forward mutations.
Due to a technical error in the 1st Experiment in the absence and presence of S9 mix the data obtained has to be regarded as invalid and, therefore, they are not reported.
In the 2nd and 3rd Experiment after 24 hours treatment in the absence of metabolic activation the recommendations of the current OECD Guideline 476 were not fulfilled due to excessive cytotoxicity or lacking cytotoxicity, respectively. Therefore, these experimental parts were discontinued.
In all experimental parts assessed as valid regarding the current OECD Guideline 476 and evaluated for gene mutations the highest concentrations applied were clearly cytotoxic.
On the basis from the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after adding a metabolizing system in five experiments performed independently of each other.
Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.
Referenceopen allclose all
Table 1: Historical Negative Control Data TA 1535
Method |
S9 mix |
Negative control |
Period |
No. of plates |
Min* |
Max* |
Mean* |
SD |
SPT |
- |
Water |
01.00 – 02.07 |
400 |
10 |
24 |
17 |
2 |
SPT |
- |
DMSO |
11.04 – 02.07 |
400 |
10 |
25 |
17 |
2 |
SPT |
- |
Acetone |
01.96 – 11.06 |
259 |
11 |
25 |
18 |
2 |
SPT |
1 : 9 |
Water |
01.00 – 01.07 |
400 |
12 |
24 |
18 |
2 |
SPT |
1 : 9 |
DMSO |
11.04 – 03.07 |
400 |
10 |
24 |
17 |
2 |
SPT |
1 : 9 |
Acetone |
01.96 – 11.06 |
258 |
11 |
25 |
19 |
2 |
PIT |
- |
Water |
04.98 – 02.07 |
400 |
10 |
25 |
18 |
2 |
PIT |
- |
DMSO |
07.03 – 03.07 |
400 |
10 |
25 |
17 |
2 |
PIT |
- |
Acetone |
01.96 – 11.06 |
216 |
10 |
25 |
18 |
3 |
PIT |
1 : 9 |
Water |
11.97 – 02.07 |
400 |
11 |
25 |
18 |
2 |
PIT |
1 : 9 |
DMSO |
06.03 – 03.07 |
400 |
10 |
24 |
16 |
2 |
PIT |
1 : 9 |
Acetone |
01.96 – 11.06 |
201 |
10 |
24 |
18 |
2 |
*: revertants/plate
Table 2: Historical Negative Control Data TA 100
Method |
S9 mix |
Negative control |
Period |
No. of plates |
Min* |
Max* |
Mean* |
SD |
SPT |
- |
Water |
12.99 – 02.07 |
400 |
82 |
150 |
110 |
9 |
SPT |
- |
DMSO |
11.04 – 02.07 |
400 |
80 |
135 |
109 |
9 |
SPT |
- |
Acetone |
01.96 – 11.06 |
306 |
91 |
160 |
117 |
14 |
SPT |
1 : 9 |
Water |
12.99 – 01.07 |
400 |
88 |
152 |
115 |
2 |
SPT |
1 : 9 |
DMSO |
11.04 – 03.07 |
400 |
81 |
154 |
111 |
11 |
SPT |
1 : 9 |
Acetone |
01.96 – 11.06 |
301 |
85 |
160 |
119 |
15 |
PIT |
- |
Water |
04.98 – 02.07 |
400 |
90 |
160 |
115 |
13 |
PIT |
- |
DMSO |
09.03 – 03.07 |
400 |
81 |
153 |
109 |
10 |
PIT |
- |
Acetone |
01.96 – 11.06 |
223 |
91 |
160 |
116 |
14 |
PIT |
1 : 9 |
Water |
03.98 – 02.07 |
400 |
92 |
159 |
120 |
15 |
PIT |
1 : 9 |
DMSO |
07.03 – 03.07 |
400 |
81 |
157 |
110 |
10 |
PIT |
1 : 9 |
Acetone |
05.96 – 11.06 |
198 |
96 |
160 |
118 |
15 |
*: revertants/plate
Table 3: Historical Negative Control Data TA 1537
Method |
S9 mix |
Negative control |
Period |
No. of plates |
Min* |
Max* |
Mean* |
SD |
SPT |
- |
Water |
11.99 – 02.07 |
400 |
5 |
16 |
10 |
2 |
SPT |
- |
DMSO |
10.04 – 03.07 |
400 |
5 |
16 |
10 |
2 |
SPT |
- |
Acetone |
01.96 – 11.06 |
261 |
5 |
18 |
10 |
2 |
SPT |
1 : 9 |
Water |
10.99 – 01.07 |
400 |
6 |
17 |
11 |
2 |
SPT |
1 : 9 |
DMSO |
09.04 – 03.07 |
400 |
5 |
16 |
10 |
2 |
SPT |
1 : 9 |
Acetone |
01.96 – 11.06 |
261 |
5 |
20 |
11 |
2 |
PIT |
- |
Water |
04.98 – 02.07 |
400 |
5 |
16 |
10 |
2 |
PIT |
- |
DMSO |
07.03 – 03.07 |
400 |
5 |
17 |
10 |
2 |
PIT |
- |
Acetone |
01.96 – 11.06 |
213 |
6 |
17 |
10 |
2 |
PIT |
1 : 9 |
Water |
11.97 – 02.07 |
400 |
6 |
20 |
11 |
2 |
PIT |
1 : 9 |
DMSO |
07.03 – 03.07 |
400 |
5 |
17 |
10 |
2 |
PIT |
1 : 9 |
Acetone |
01.96 – 11.06 |
204 |
5 |
20 |
11 |
2 |
*: revertants/plate
Table 4: Historical Negative Control Data TA 98
Method |
S9 mix |
Negative control |
Period |
No. of plates |
Min* |
Max* |
Mean* |
SD |
SPT |
- |
Water |
01.00 – 02.07 |
400 |
17 |
44 |
29 |
5 |
SPT |
- |
DMSO |
10.04 – 03.07 |
400 |
15 |
40 |
29 |
4 |
SPT |
- |
Acetone |
01.96 – 11.06 |
263 |
19 |
50 |
29 |
5 |
SPT |
1 : 9 |
Water |
01.00 – 01.07 |
400 |
20 |
49 |
36 |
5 |
SPT |
1 : 9 |
DMSO |
10.04 – 03.07 |
400 |
21 |
50 |
35 |
5 |
SPT |
1 : 9 |
Acetone |
01.96 – 11.06 |
262 |
25 |
50 |
38 |
5 |
PIT |
- |
Water |
01.98 – 02.07 |
400 |
18 |
44 |
28 |
4 |
PIT |
- |
DMSO |
07.03 – 03.07 |
400 |
17 |
40 |
29 |
4 |
PIT |
- |
Acetone |
01.96 – 11.06 |
216 |
19 |
45 |
28 |
5 |
PIT |
1 : 9 |
Water |
11.97 – 02.07 |
400 |
17 |
49 |
35 |
2 |
PIT |
1 : 9 |
DMSO |
07.03 – 03.07 |
400 |
20 |
50 |
33 |
5 |
PIT |
1 : 9 |
Acetone |
01.96 – 11.06 |
203 |
20 |
50 |
36 |
6 |
*: revertants/plate
Table 5: Historical Negative Control Data E.coli WP2 uvrA
Method |
S9 mix |
Negative control |
Period |
No. of plates |
Min* |
Max* |
Mean* |
SD |
SPT |
- |
Water |
09.99 – 01.07 |
400 |
25 |
55 |
33 |
5 |
SPT |
- |
DMSO |
09.04 – 03.07 |
400 |
25 |
59 |
34 |
5 |
SPT |
- |
Acetone |
04.96 – 11.06 |
234 |
25 |
55 |
34 |
5 |
SPT |
1 : 9 |
Water |
09.99 – 01.07 |
400 |
25 |
56 |
36 |
6 |
SPT |
1 : 9 |
DMSO |
09.04 – 03.07 |
400 |
25 |
60 |
38 |
6 |
SPT |
1 : 9 |
Acetone |
04.96 – 11.06 |
242 |
25 |
59 |
37 |
6 |
PIT |
- |
Water |
10.97 – 02.07 |
400 |
25 |
52 |
32 |
5 |
PIT |
- |
DMSO |
06.03 – 03.07 |
400 |
25 |
54 |
33 |
5 |
PIT |
- |
Acetone |
01.96 – 11.06 |
191 |
25 |
54 |
32 |
5 |
PIT |
1 : 9 |
Water |
11.97 – 02.07 |
400 |
25 |
57 |
37 |
6 |
PIT |
1 : 9 |
DMSO |
07.03 – 03.07 |
400 |
25 |
56 |
36 |
5 |
PIT |
1 : 9 |
Acetone |
01.96 – 11.06 |
197 |
25 |
52 |
36 |
6 |
*: revertants/plate
Table 6: Historical Positive Control Data TA 1535
Method |
S9 mix |
Positive control µg/plate |
Period |
No. of plates |
Min* |
Max* |
Mean* |
SD |
SPT |
- |
MNNG 5.0 |
03.05 – 02.07 |
400 |
509 |
1602 |
752 |
187 |
SPT |
1 : 9 |
2-AA 2.5 |
02.05 – 03.07 |
400 |
77 |
342 |
132 |
28 |
PIT |
- |
MNNG 5.0 |
06.04 – 03.07 |
400 |
512 |
1317 |
708 |
150 |
PIT |
1 : 9 |
2-AA 2.5 |
03.04 – 03.07 |
400 |
83 |
398 |
130 |
33 |
*: revertants/plate
Table 7: Historical Positive Control Data TA 100
Method |
S9 mix |
Positive control µg/plate |
Period |
No. of plates |
Min* |
Max* |
Mean* |
SD |
SPT |
- |
MNNG 5.0 |
02.05 – 03.07 |
400 |
500 |
1574 |
823 |
201 |
SPT |
1 : 9 |
2-AA 2.5 |
02.05 – 03.07 |
400 |
521 |
1878 |
876 |
205 |
PIT |
- |
MNNG 5.0 |
06.04 – 03.07 |
400 |
520 |
1302 |
797 |
141 |
PIT |
1 : 9 |
2-AA 2.5 |
04.04 – 03.07 |
400 |
517 |
1497 |
829 |
174 |
*: revertants/plate
Table 8: Historical Positive Control Data TA 1537
Method |
S9 mix |
Positive control µg/plate |
Period |
No. of plates |
Min* |
Max* |
Mean* |
SD |
SPT |
- |
AAC 100 |
02.05 – 03.07 |
400 |
212 |
1172 |
441 |
109 |
SPT |
1 : 9 |
2-AA 2.5 |
02.05 – 03.07 |
400 |
72 |
200 |
129 |
22 |
PIT |
- |
AAC 100 |
05.04 – 03.07 |
400 |
221 |
1028 |
429 |
100 |
PIT |
1 : 9 |
2-AA 2.5 |
03.04 – 03.07 |
400 |
80 |
198 |
123 |
19 |
*: revertants/plate
Table 9: Historical Positive Control Data TA 98
Method |
S9 mix |
Positive control µg/plate |
Period |
No. of plates |
Min* |
Max* |
Mean* |
SD |
SPT |
- |
NOPD 10 |
02.05 – 03.07 |
400 |
332 |
1316 |
616 |
145 |
SPT |
1 : 9 |
2-AA 2.5 |
02.05 – 03.07 |
400 |
502 |
1414 |
697 |
160 |
PIT |
- |
NOPD 10 |
06.04 – 03.07 |
400 |
388 |
1430 |
630 |
169 |
PIT |
1 : 9 |
2-AA 2.5 |
03.04 – 03.07 |
400 |
510 |
1223 |
663 |
120 |
*: revertants/plate
Table 10: Historical Positive Control Data E.coli WP2 uvrA
Method |
S9 mix |
Positive control µg/plate |
Period |
No. of plates |
Min* |
Max* |
Mean* |
SD |
SPT |
- |
4-NQO 5.0 |
01.05 – 03.07 |
400 |
500 |
1587 |
653 |
188 |
SPT |
1 : 9 |
2-AA 60.0 |
01.05 – 03.07 |
400 |
150 |
383 |
231 |
30 |
PIT |
- |
4-NQO 5.0 |
05.04 – 03.07 |
400 |
500 |
1339 |
593 |
102 |
PIT |
1 : 9 |
2-AA 60.0 |
05.04 – 03.07 |
400 |
150 |
307 |
226 |
25 |
*: revertants/plate
RESULTS
MICRONUCLEUS ANALYSIS
1st Experiment
In the 1st Experiment after 4 hours exposure with and without metabolic activation no
relevant increase in the number of micronucleated cells was found. In the experimental part
without S9 mix a single, statistically significant increase in the number of micronucleated cells
(0.9%) exceeding the concurrent vehicle control value (0.2% micronucleated cells) was
observed at 725 μg/mL. However, in the 1st Experiment all values after treatment with the
test substance (0.2 – 1.3% micronucleated cells) were close to the concurrent vehicle control
values (0.2% and 0.8% micronucleated cells without and with S9 mix, respectively) and
clearly within our historical negative control data range (0.3 - 1.8% micronucleated cells).
2nd Experiment
In the 2nd Experiment after 24 hours exposure without S9 mix no relevant increase in the
number of micronucleated cells was found. The values after treatment with the test
substance (0.1 – 0.6% micronucleated cells) were close to the concurrent vehicle control
value (0.5% micronucleated cells) and clearly within our historical negative control data range
(0.3 - 1.8% micronucleated cells).
Besides after 4 hours exposure in the presence of metabolic activation a single, statistically
significant increase in the number of micronucleated cells was found at an intermediate
concentration of 1 000 μg/mL (2.2% micronucleated cells). Due to inhomogeneous data this
value was confirmed by scoring an increased sample of 2 000 micronucleated cells per test
group. This value was slightly above our historical negative control data range (0.3 - 1.8%
micronucleated cells). However, this increase of micronucleated cells occurred at an
intermediate concentration, means no dose-dependency was observed in this experiment.
PROLIFERATION INDEX
The proliferation index (PI) is based on the scoring of at least 1 000 cells per culture (2 000
cells per test group) for the different test groups without and with metabolic activation and
includes the measurement of colony size.
In this study, in the absence and the presence of S9 mix no cytotoxicity indicated by reduced
PI values was observed. In the 2nd Experiment after 4 hours exposure with S9 mix a sample
of 2 000 cells was scored at 1 000 μg/mL due to inhomogeneous genotoxicity data.
CELL COUNT
In this study, clearly reduced cell counts of about or below 55% ± 5% of control were
observed at all experimental parts at least at the highest concentration scored for the
occurrence of micronuclei.
In detail, in the absence of S9 mix in the 1st Experiment after 4 hours treatment clearly
reduced cell numbers were obtained at 1 450 μg/mL and above (51.9% of control). In the
2nd Experiment after 24 hours treatment cytotoxicity was observed at 1 000 μg/mL and
above (50.5% of control). In the presence of S9 mix after 4 hours in the 1st Experiment
growth inhibition indicated by reduced cell counts was observed at 2 900 μg/mL and above
(27.0% of control). In the 2nd Experiment in the presence of S9 mix reduced cell numbers
occurred at 2 000 μg/mL and above (60.6% of control).
CELL MORPHOLOGY
In this study, cell attachment and/or cell quality was influenced from about 3 000 μg/mL
onward after 4 hours treatment in the absence and presence of metabolic activation. After
24 hours test substance treatment without S9 mix cell attachment and/or cell quality was
strongly influenced from 1 500 μg/mL onward.
TREATMENT CONDITIONS
Osmolarity and pH values were not influenced by test substance treatment.
Test item precipitation in culture medium at the end of treatment period was observed from
about 2 900 μg/mL onward in the absence and presence of metabolic activation.
DISCUSSION
According to the results of the present in vitro micronucleus assay, the test substance
LIMUS-Sambaydestillation did not lead to a biologically relevant increase in the number of
micronucleated cells either without S9 mix or after the addition of a metabolizing system in
two experiments performed independently of each other. The frequencies of micronuclei after
test substance treatment were close to the range of the concurrent vehicle control values at
both exposure times and nearby the range of our historical negative control data.
In the 1st Experiment after 4 hours treatment in the absence of metabolic activation a single,
statistically significant increase in the number of micronucleated cells (0.9%) was obtained at
the lowest concentration scored for the occurrence of micronucleated cells (725 μg/mL).
However, no dose-dependency was obtained and the value was clearly within the range of
our historical negative control data. Therefore, this observation has to be regarded as
biologically irrelevant.
In the 2nd Experiment in the presence of metabolic activation a single, statistically significant
increase in the number of micronucleated cells (2.2%) was obtained after 4 hours exposure
with an intermediate concentration (1 000 μg/mL). Due to inhomogeneous data this value
was confirmed by scoring an increased sample of 2 000 micronucleated cells per test group.
This value slightly exceeded our historical negative control data range (0.3 - 1.8%
micronucleated cells). Due to missing dose-dependency and lacking confirmation in the
1st Experiment after 4 hours treatemnt in the presence of metabolic activation at a
comparable concentration range (725, 1 450 and 2 900 μg/mL) this single finding has to be
regarded as biologically irrelevant.
The number of micronucleated cells in the vehicle control groups were within our historical
negative control data range and, thus, fulfilled the acceptance criteria of this study .
The increase in the frequencies of micronuclei induced by the positive control substances
EMS and CPP demonstrated the sensitivity of the test system and of the metabolic activity of
the S9 mix employed. Although the values in the 1st Experiment in the absence and
presence of metabolic activation (2.4% micronucleated cells, each) were slightly below the
range of the historical positive control data (without S9 mix: 2.6 – 8.3% micronucleated cells;
with S9 mix: 2.5 - 23.5% micronucleated cells; see APPENDIX 7) the rates of micronucleated
cells were clearly increased compared to the concurrent vehicle control groups (0.8% and
0.2%, respectively). Thus, the positive controls fulfilled the acceptance criteria of this study.
CONCLUSION
Thus, under the experimental conditions chosen here, the conclusion is drawn that LIMUSSambaydestillation
has not the potential to induce micronuclei (clastogenic and/or
aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of
metabolic activation.
RESULTS
Unfortunately, in the 1st Experiment a false test substance was used which had both, a
similar test substance name and a similar test substance number. Due to this technical error
the data obtained in the 1st Experiment in the absence and presence of S9 mix has to be
regarded as invalid and, thus, the data are neither reported nor discussed (for details see raw
data).
In addition, the 2nd and 3rd Experiment in the absence of metabolic activation did not fulfill
the recommendations of the current OECD Guideline 476 due to excessive cytotoxicity or
lacking cytotoxicity, respectively. Both experimental parts were discontinued after the
1st subcultivation.
MUTANT FREQUENCY
In this study, no relevant increase in the number of mutant colonies was observed either
without S9 mix or after the addition of a metabolizing system. In all experiments after 4 and
24 hours treatment with the test substance the values for the corrected mutation frequencies
(MFcorr.: 0.00 – 8.82 per 106 cells) were close to the respective vehicle control values (MFcorr.:
1.18 – 6.81 per 106 cells) and clearly within the range of our historical negative control data
(without S9 mix: MFcorr.: 0.00 – 15.95 per 106 cells; with S9 mix: MFcorr.: 0.00 – 12.62 per
106 cells).
The positive control substances EMS (without S9 mix; 300 μg/mL) and MCA (with S9 mix;
20 μg/mL) induced clearly increased mutant frequencies as expected. The values of the
corrected mutant frequencies (without S9 mix: MFcorr.: 186.96 – 561.60 per 106 cells; with
S9 mix: MFcorr.: 61.36 – 79.80 per 106 cells) were clearly within our historical positive control
data range (without S9 mix: MFcorr.: 48.83 – 999.46 per 106 cells; with S9 mix: MFcorr.: 26.29 –
413.54 per 106 cells).
CYTOTOXICITY
Cytotoxic effects indicated by clearly reduced cloning efficiencies of below 20% of control or
extremely low cell numbers at the 1st subcultivation were observed in the 2nd, 4th and
5th Experiment in the absence and presence of S9 mix in the highest applied concentrations.
In detail, in the 2nd Experiment in the absence of S9 mix, there was a decrease in the
number of colonies from 125.0 μg/mL onward after an exposure period of 24 hours. In the
presence of S9 mix there was a decrease in the number of colonies from 4 000.0 μg/mL
onward after an exposure period of 4 hours.
In contrary, in the 3rd Experiment in the absence of S9 mix when applying concentrations up
to 200 μg/mL no decrease in the number of colonies was obtained.
In the 5th Experiment in the absence of S9 mix there was a decrease in the number of
colonies from 1 500.0 μg/mL onward after an exposure period of 4 hours. In addition, in the
presence of S9 mix after 4 hours exposure there was a decrease in the number of colonies at
an intermediate concentration of 750.0 μg/mL: Test groups from 3 000.0 μg/mL onward could
nor be determined due to low cell numbers at 1st subcultivation.
It is remarkable that in all experimental parts assessed for gene mutations clearly reduced
colony counts in term of survival (CE1) were obtained at intermediate concentrations around
500.0 μg/mL. However, no adverse effects were obtained by means of viability (CE2) when
subcultivation of the test groups was feasible. These findings occurred far below the border
of saturation which started at about 4 000.0 μg/mL. Or described otherwise, just before
reaching total cytotoxicity an increase in colony numbers (only CE1) was observed.
CELL MORPHOLOGY
Details on the observations on cell morphology (cell attachment) are given in Appendix 3.
In all experimental parts with 4 and 24 hours treatment in the absence and presence of
S9 mix the morphology and attachment of the cells was adversely influenced at the highest
applied concentrations, except when using only 200.0 μg/mL as top concentration in the
3rd Experiment in the absence of S9 mix.
TREATMENT CONDITIONS
Osmolarity and pH values were not influenced by test substance treatment.
In all experimental parts of this study in the absence and presence of S9 mix test substance
precipitation in culture medium determined at the end of treatment period was observed from
about 4 000 μg/mL onward.
DISCUSSION
The 1st Experiment of this study was cancelled because of testing a false test substance.
However, the data are recorded and included in the raw data of this study.
According to the results of the present in vitro study, the test substance LIMUSSambaydestillation
did not lead to a relevant increase in the number of mutant colonies
either without S9 mix or after the addition of a metabolizing system in five experiments
performed independently of each other. The mutant frequencies at any concentration were
within the range of the concurrent vehicle control values and within the range of our historical
negative control data (see Appendix 4).
The mutation frequencies of the vehicle control groups were within our historical negative
control data range including all vehicles used in our laboratory and, thus, fulfilled the
acceptance criteria of this study (see Appendix 4)
The increase in the frequencies of mutant colonies induced by the positive control
substances EMS and MCA clearly demonstrated the sensitivity of the test method and of the
metabolic activity of the S9 mix employed. The values were within the range of the historical
positive control data and, thus, fulfilled the acceptance criteria of this study (see Appendix 5).
CONCLUSION
Thus, under the experimental conditions chosen here, the conclusion is drawn that
LIMUS-Sambaydestillation is not a mutagenic substance in the HPRT locus assay using
CHO cells in the absence and the presence of metabolic activation.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
In vitro:
Ames test:
There is no Ames-Test for NPPT available but the following data is suitable for a read across:
The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.
Strains: TA 1535, TA 100, TA 1537, TA 98 and E.coli WP2 uvrA
Dose Range: 20 µg - 5000 µg/plate (SPT), 312.5 µg - 5000 µg/plate (PIT)
Test Conditions: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (Aroclor-induced rat liver S9 mix).
Solubility: No precipitation of the test substance was found.
Toxicity: A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions at 5000 µg/plate.
Mutagenicity: A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.
According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium / Escherichia coli reverse mutation assay under the experimental conditions chosen here.
Micronucleus Assay:
There is no micronucleus assay for NPPT available but the following data is suitable for a read across:
The substance LIMUS-Sambaydestillation was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity) both in the absence and the presence of a metabolizing system based on OECD guideline 487 and GLP (BASF, 2010).
According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following doses were tested and the test groups in bold type were evaluated:
1st Experiment
4 hours exposure; 24 hours harvest time; without S9 mix:
0; 362.5; 725.0; 1 450.0; 2 900.0; 4 350.0; 5 800.0 μg/mL
4 hours exposure, 24 hours harvest time, with S9 mix:
0; 362.5; 725.0; 1 450.0; 2 900.0; 4 350.0; 5 800.0 μg/mL
2nd Experiment
24 hours exposure, 24 hours harvest time, without S9 mix
0; 250.0; 500.0; 1 000.0; 1 500.0; 2 000 μg/mL
4 hours exposure, 24 hours harvest time, with S9 mix
0; 250.0; 500.0; 1 000.0; 2 000.0; 3 000.0 μg/mL
A sample of at least 1 000 cells for each culture were analyzed for micronuclei, i.e. at least 2 000 cells for each test group.
The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for V79 cells. Both positive control substances, EMS and cyclophosphamide, led to a statistically significant and biologically relevant increase in the number of cells containing micronuclei.
On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other. The single statistically significant outlier value observed in the 2nd Experiment in the presence of metabolic activation after 4 hours treatment with 1 000 μg/mL has to be considered biologically irrelevant due to missing dose-dependency and lacking confirmation in the 1st Experiment at a comparable concentration range.
Thus, under the experimental conditions described, LIMUS-Sambaydestillation is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the
absence and the presence of metabolic activation.
HPRT:
There is no HPRT-Test for NPPT available but the following data is suitable for a read across:
The substance LIMUS-Sambaydestillation was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro according ot OECD 476 guideline and GLP (BASF, 2011). Five independent experiments were carried out with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). Based on the observations and the toxicity data of a previously performed pretest for an in
vitro micronucleus assay and taking into account the cytotoxicity actually found in the main experiments, the following doses were tested and the doses in bold type were evaluated:
1st Experiment (invalid due to technical error)
without S9 mix (4-hour exposure period)
0; 93.8; 187.5; 375.0; 750.0; 1 500.0; 3 000.0; 6 000.0 μg/mL
with S9 mix (4-hour exposure period)
0; 93.8; 187.5; 375.0; 750.0; 1 500.0; 3 000.0; 6 000.0 μg/mL
2nd Experiment
without S9 mix (24-hour exposure period) (failed recommendations of OECD 476)
0; 125.0; 250.0; 500.0; 1 000.0; 2 000.0; 4 000.0; 6 000.0 μg/mL
with S9 mix (4-hour exposure period)
0; 250.0; 500.0; 1 000.0; 2 000.0; 4 000.0; 6 000.0 μg/mL
3rd Experiment
without S9 mix (24-hour exposure period) (failed recommendations of OECD 476)
0; 3.13; 6.25; 12.5; 25.0; 50.0; 100.0; 200.0 μg/mL
4th Experiment
without S9 mix (24-hour exposure period)
0; 7.8; 15.6; 31.3; 62.5; 125.0; 250.0; 500.0; 1 000.0; 2 000.0; 4 000.0 μg/mL
5th Experiment
without S9 mix (4-hour exposure period)
0; 46.9; 93.8; 187.5; 375.0; 750.0; 1 500.0; 3 000.0; 6 000.0 μg/mL
with S9 mix (4-hour exposure period)
0; 93.8; 187.5; 375.0; 750.0; 1 500.0; 3 000.0; 6 000.0 μg/mL
After an attachment period of 20 - 24 hours and a treatment period of 4 hours both with and without metabolic activation and 24 hours without metabolic activation, an expression phase of about 6 - 8 days and a selection period of about 1 week followed. The colonies of each test group were fixed with methanol, stained with Giemsa and counted.
The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, EMS and MCA, led to the expected increase in the frequencies of forward mutations.
Due to a technical error in the 1st Experiment in the absence and presence of S9 mix the
data obtained has to be regarded as invalid and, therefore, they are not reported.
In the 2nd and 3rd Experiment after 24 hours treatment in the absence of metabolic activation the recommendations of the current OECD Guideline 476 were not fulfilled due to excessive cytotoxicity or lacking cytotoxicity, respectively. Therefore, these experimental parts were discontinued.
In all experimental parts assessed as valid regarding the current OECD Guideline 476 and evaluated for gene mutations the highest concentrations applied were clearly cytotoxic.
On the basis from the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after adding a metabolizing system in five experiments performed independently of each other.
Thus, under the experimental conditions of this study, the test substance LIMUSSambaydestillation is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.
Read Across justification:
LIMUS-Sambaydestillation is a reaction mass of phosphorothioic
triamide, N-butyl- (NBPT) and phosphorothioic triamide, N-propyl-
(NPPT), the subject of this registration. The relation of NBPT and NPPT
in the reaction mass is ca. 3:1 and the overall total content of NBPT
and NPPT in the reaction mass is up to 85 %. Thus, as the reaction mass
contains a certain amount of NPPT (up to ca. 21 %) and based on the
structural similarities between NBPT and NPPT (NPPT is one CH2 -group
shorter) the reaction mass is suitable for a read across and thus for
filling the data gaps of NPPT.
Justification for classification or non-classification
Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No indication of genotoxicity was observed in the Ames test (OECD 471, GLP), the HPRT test (OECD 476, GLP) and the in vitro Micronucleus test (OECD 487 (draft), GLP). As a result, the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the tenth time in Regulation (EC) No. 2017/776.
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