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EC number: 813-604-1 | CAS number: 68533-01-7
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
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- Ecotoxicological Summary
- Aquatic toxicity
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- Short-term toxicity to fish
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- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
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- Toxicity to microorganisms
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- Toxicological Summary
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Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
A genotoxic potential was not detected in three in vitro tests according to OECD TGs 471, 473 and 476, respectively.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study conducted according to OECD Guideline 471 and GLP
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his-, trp-
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fraction from rats treated with phenobarbital (i.p.) and β-naphthoflavone (orally) each on 3 consecutive days
- Test concentrations with justification for top dose:
- 0; 33; 100; 333; 1 000; 2 500 and 5 000 μg/plate
- Vehicle / solvent:
- ethanol (due to insolubility of the test substance in ultrapure water)
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); 4-nitro-o-phenylenediamine (NOPD); 2-aminoanthracene (2-AA)
- Remarks:
- All positive control substances were used without S9 mix, except for 2-AA (with S9 mix)
- Details on test system and experimental conditions:
- TEST SYSTEM:
Deep-frozen bacterial cultures (Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA) are thawed at room temperature,
and 0.1 mL of this bacterial suspension is inoculated in nutrient broth solution (8 g/L Difco nutrient broth + 5 g/L NaCl) and incubated in the shaking water bath at 37°C for about 12 - 16 hours. As a rule, a germ density of ≥ 108 bacteria/mL is reached. These cultures grown overnight are kept in iced water from the beginning of the experiment until the end in order to prevent further growth.
SALMONELLA TYPHIMURIUM:
The rate of induced back mutations of several bacteria mutants from histidine auxotrophy (his-) to histidine prototrophy (his+) is determined. The tester strains TA 1535, TA 1537, TA 98 and TA 100 are derivatives of Salmonella typhimurium LT2 and have GC base pairs at the primary reversion site. All strains have a defective excision repair system (uvrB), which prevents the repair of lesions which are induced in the DNA, and this deficiency results in greatly enhanced sensitivity of some mutagens. Furthermore, all strains show a considerably reduced hydrophilic polysaccharide
layer (rfa), which leads to an increase in permeability to lipophilic substances.
The strains TA 1535 and TA 100 are derived from histidine-prototrophic Salmonella strains by the substitution mutation his G 46 and are used to detect base pair substitutions. TA 1537 and TA 98 are strains for the detection of frameshift mutagens. These strains carry different frameshift markers, i.e. the +1 mutant his C 3076 in the case of TA 1537 and the +2 type his D 3052 in the case of TA 98. The strains TA 98 and TA 100 carry an R factor plasmid pKM 101 (4) and, in addition to having genes resistant to antibiotics, they have a modified postreplication DNA repair system,
which increases the mutation rate by inducing a defective repair in the DNA; this again leads to a considerable increase in sensitivity.
ESCHERICHIA COLI:
Escherichia coli WP2 uvrA which has an AT base pair at the primary reversion site is a derivative of E. coli WP2 with a deficient excision repair and is used to detect substances which induce base pair substitutions. The rate of induced back mutations from tryptophan auxotrophy (trp-) to tryptophan independence (trp+) is determined.
SCOPE OF TESTS AND TEST CONDITIONS:
All experiments were conducted with and without S9 mix, with 3 test plates per dose or per control, and applying the test substance at the following concentrations: 0; 33; 100; 333; 1 000; 2 500 and 5 000 μg/plate.
1. Experiment: Standard plate test in TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA
2. Experiment: Preincubation test in TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA (Reason: No mutagenicity was observed in the standard plate test)
3. Experiment: Preincubation test in TA 1537 (Reason: Concerning the bacteriotoxicity inconclusive values were observed in the 2nd Experiment)
4. Experiment: Preincubation test in TA 1537 (Reason: Due to technical reason, an evaluation of the TA 1537 in the 3rd Experiment was not possible.)
STANDARD PLATE TEST:
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution [minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin (S. typhimurium); or 0.5 mM tryptophan (E. coli)] are kept in a water bath at about 42 - 45°C, and the remaining components are added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples are poured onto minimal glucose agar plates within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants or trp+ revertants) are counted.
PREINCUBATION TEST:
0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) are incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar is added and, after mixing, the samples are poured onto the agar plates within approx. 30 seconds. After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies are counted.
TITER DETERMINATION:
The titer was 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. In the standard plate test, 0.1 mL of the overnight cultures is diluted to 10-6 in each case. Test tubes containing 2-mL portions of soft agar containing maximal amino acid solution (5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) are kept in a water bath at about 42 - 45°C, and the remaining components are added in the following order:
0.1 mL vehicle (without and with test substance)
0.1 mL fresh bacterial culture (dilution: 10-6)
0.5 mL S9 mix
In the preincubation test, 0.1 mL of the overnight cultures is diluted to 10-6 in each case. 0.1 mL vehicle (with and without test substance), 0.1 mL bacterial suspension and 0.5 mL S9 mix are incubated at 37°C for about 20 minutes using a shaker. Subsequently, 2 mL of soft agar containing maximal amino acid solution for titer determination (5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) is added. After mixing, the samples are poured onto the agar plates within approx. 30 seconds. After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies are counted. - Evaluation criteria:
- 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.
• Fresh bacterial culture containing approximately 109 cells per mL were used. For approval the titer of viable bacteria was ≥ 108 colonies per mL.
The test substance is considered positive 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, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- weak bacteriotoxicity in preincubation test at 5 000 μg/plate only
- Vehicle 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
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- MUTAGENICITY:
No relevant increase in the number of his+ or trp+ revertants was observed in any of the strains in either test system.
TOXICITY:
No bacteriotoxic effect (reduced his- or trp- background growth, slight decrease in the number of his+ or trp+ revertants, slight reduction in the titer) was observed in the standard plate test up to the highest required concentration.
In the preincubation assay a weak bacteriotoxicity (slight decrease in the number of his+ revertants) was observed depending on the strain and test conditions at 5 000 μg/plate. The inconclusive Data observed in the 2nd Experiment was not confirmed in a repeat experiment and has to be considered as not relevant.
SOLUBILITY:
No test substance precipitation was found with and without S9 mix. - Conclusions:
- Interpretation of results (migrated information):
negative
Under the experimental conditions chosen here, it is concluded that NM01 is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation. - Executive summary:
The test substance NM01 was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several strains of Salmonella typhimurium and Escherichia coli (TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA), in a reverse mutation assay. A standard plate test and a preincubation test were conducted, both with and without metabolic activation (liver S9 mix from induced rats), and both covering a dose range of 33 μg - 5 000 μg/plate. No precipitation of the test substance was found with and without S9 mix. A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions at 5 000 μg/plate. A biologically 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.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study conducted according to OECD Guideline 473 and GLP
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/β-naphthoflavone induced rat liver S9
- Test concentrations with justification for top dose:
- 8.2, 16.4, 32.8, 65.6, 131.3, 262.5, 525.0, 1050.0, 2100.0 μg/mL
- Vehicle / solvent:
- deionised water
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Remarks:
- EMS: without metabolic activation; CPA: with metabolic activation
- Details on test system and experimental conditions:
- CHINESE HAMSTER V79 CELLS CULTURE CONDITIONS:
Thawed stock cultures were propagated at 37 °C in 80 cm2 plastic flasks. About 5 x 105 cells per flask were seeded in 15 mL of MEM (minimal essential medium) containing Hank’s salts, glutamine and Hepes (25 mM). Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL) and 10 % (v/v) fetal bovine serum (FBS). The cells were sub-cultured twice a week.
Exponentially growing stock cultures more than 50 % confluent were rinsed with Ca-Mg-free salt solution containing 8000 mg/L NaCl, 200 mg/L KCl, 200 mg/L KH2PO4 and 150 mg/L Na2HPO4. Afterwards the cells were treated with trypsin-EDTA-solution at 37 °C for approx. 5 minutes. Then, by adding complete culture medium including 10 % (v/v) FBS the enzymatic treatment was stopped and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.25 % (w/v) in Ca-Mg-free salt solution. For experimental performance the cells were seeded into Quadriperm dishes containing microscopic slides. Into each chamber 1 x 104 – 6 x 104 cells were seeded. All incubations were done at 37 °C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % air).
MAMMALIAN MICROSOMAL FRACTION S9 MIX:
Due to the limited capacity for metabolic activation of potential mutagens in in vitro methods an exogenous metabolic activation system was used.
Phenobarbital/β-naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared and stored according to the currently valid version of the Harlan CCR SOP for rat liver S9 preparation. Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test. An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4). The protein concentration of the S9 preparation used for this study was 35.0 mg/mL (Lot no. 310114).
EXPERIMENTAL SET-UP:
see table 1 in section "any other information on materials and methods incl. tables"
DOSE SELECTION:
With regard to the molecular weight and the purity (95 % preliminary information at study start) of the test item, 2100.0 μg/mL (approx. 10 mM) were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 8.2 to 2100.0 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, precipitation of the test item was observed at the end of treatment at 1050.0 μg/mL and above. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I. Using reduced mitotic indices/cell numbers as an indicator for toxicity, no cytotoxic effects were observed in Experiment I after 4 hours treatment in the absence and presence of S9 mix. Therefore, 2100.0 μg/mL was chosen as top treatment concentration for Experiment II.
PRE-EXPERIMENT:
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterized by the percentages of mitotic suppression and/or reduction in cell number in comparison to the controls by counting 1000 cells per culture in duplicate. The experimental conditions in this pre-test phase were identical to those required and described below for the main experiment. The pre-test was performed with 9 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 18 hrs after start of the exposure.
CYTOGENETIC EXPERIMENTS:
Pulse exposure (with S9 mix): The culture medium of exponentially growing cell cultures was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 μL S9 mix per mL culture medium was added. After 4 hours the cultures were washed twice with "Saline G" (pH 7.2) containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose x H2O, 192 mg/L Na2HPO4 x 2 H2O and 150 mg/L KH2PO4. The cells were then cultured in complete medium containing 10 % (v/v) FBS for the remaining culture time of 14 and 24 hours.
Continuous exposure (without S9 mix): The culture medium of exponentially growing cell cultures was replaced with complete medium containing 10 % (v/v) FBS including the test item. The medium was not changed until preparation of the cells.
Preparation of metaphases: Cultures were treated with the metaphase-arresting substance colcemid (final concentration: 0.2 μg/mL) approximately two to three hours before the requested harvest time. The cells were treated on the slides in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37 °C. After incubation in the hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid (3+1 parts, respectively). The slides were stained with Giemsa, mounted after drying and covered with a slid. All slides were labeled with a computer-generated random code to prevent scorer bias.
Evaluation of cytotoxicity and cytogenetic damage: Cytotoxicity is characterized by the percentages of mitotic suppression in comparison with the controls by counting 1000 cells per culture in duplicate. Additionally, the cell numbers were determined microscopically by counting 10 defined fields per coded slide. The cell number of the treatment groups are given in percentage compared to the respective solvent control. 100 well-spread metaphases were evaluated per culture for structural aberrations, except for the positive controls in Experiment II, in the absence of S9 mix, where only 50 metaphases were evaluated. Only metaphases containing a number of centromeres equal to a number of 22 ± 1 were included in the analysis. Breaks, fragments, deletions, exchanges and chromosomal disintegrations are recorded as structural chromosomal aberrations. Gaps were recorded as well, but they are not included in the calculation of the aberration rates since gaps are achromatic lesions of unknown biological relevance for which a clear relationship to treatment cannot be established. In addition, the number of polyploid cells in 500 metaphase cells per culture (% polyploid metaphases) was evaluated. - Evaluation criteria:
- Many experiments with V79 cells have established a range of aberration frequencies acceptable for control cultures. The current historical data range together with the statistical significance, confirmed by the Fisher’s exact test (p < 0.05), should be considered for classification of the test item. The chromosomal aberration assay will be considered acceptable if it meets the following criteria:
a) The rate of chromosomal aberrations in the solvent controls falls within the historical laboratory control data range.
b) The rate of chromosomal aberrations in the positive controls is statistically significant increased.
A test item can be classified as non-clastogenic if:
− the number of induced structural chromosomal aberrations in all evaluated dose groups is in the range of the historical laboratory control data and
− no statistically significant increase of the rate of structural chromosomal aberrations is observed in comparison to the respective solvent control.
A test item can be classified as clastogenic if:
− the number of induced structural chromosomal aberrations is not in the range of the historical laboratory control data and
− either a concentration-related or a statistically significant increase in the number of cells carrying structural chromosomal aberrations is observed. - Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- in Experiment II without S9 after 28 hours
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- PRECIPITATION:
Visible precipitation of the test item in the culture medium was observed at 1050.0 μg/mL and above in the absence and presence of S9 mix.
CYTOTOXICITY:
In Experiment II in the absence of S9 mix after 28 hours continuous treatment clear cytotoxicity, indicated as reduced cell numbers, was observed at the highest evaluated concentration. In all other experimental parts no clear cytotoxicity was observed up to the highest applied concentration.
CHROMOSOME ABERRATION:
In the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.0 – 3.5 % aberrant cells, excluding gaps) exceeded the range of the solvent control values (0.5 – 2.5 % aberrant cells, excluding gaps), but were within the range of the laboratory historical solvent control data (see Appendix 2). However, in Experiment II in the presence of S9 mix one single statistically significant increase (3.5 % aberrant cells, excluding gaps), within the range of the laboratory historical solvent control data (0.0 – 3.5% aberrant cells, excluding gaps) was observed after treatment with 525.0 μg/mL (Table 17).
In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (1.6 – 4.5 %) as compared to the rates of the solvent controls (2.1 – 4.4 %). - Conclusions:
- Interpretation of results (migrated information):
negative
NM01 is considered to be non-clastogenic in this chromosome aberration test, when tested up to cytotoxic and/or precipitating concentrations. - Executive summary:
The test item NM01, suspended in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. In each experimental group two parallel cultures were analysed. Per culture 100 metaphases were evaluated for structural chromosomal aberrations, except for the positive controls in Experiment II, in the absence of S9 mix, where only 50 metaphases were evaluated. The highest applied concentration in this study (2100.0 μg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight and the purity (95% preliminary information at study start) of the test item and with respect to the current OECD Guideline 473. Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 473.
In Experiment II in the absence of S9 mix after 28 hours continuous treatment clear cytotoxicity, indicated as reduced cell numbers, was observed at the highest evaluated concentration. In all other experimental parts no clear cytotoxicity was observed up to the highest applied concentration. In both independent experiments, no relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. However, in Experiment II in the presence of S9 mix one single statistically significant increase (3.5% aberrant cells, excluding gaps), within the range of the laboratory historical solvent control data (0.0 – 3.5 % aberrant cells, excluding gaps) was observed after treatment with 525.0 μg/mL. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study conducted according to OECD Guideline 476 and GLP
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- hprt
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/β-naphthoflavone induced rat liver S9
- Test concentrations with justification for top dose:
- see table 1 in section "any other information on materials and methods incl. tables"
- Vehicle / solvent:
- deionised water
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Remarks:
- EMS: without metabolic activation; DMBA: with metabolic activation
- Details on test system and experimental conditions:
- TEST SYSTEM:
The V79 cells are exposed to the test item both with and without exogenous metabolic activation. At a defined time interval after treatment the descendants of the treated original population are monitored for the loss of functional HPRT enzyme.
HPRT (hypoxanthine-guanine phosphoribosyl transferase) catalyzes the conversion of the nontoxic 6-TG (6-thioguanine) to its toxic ribophosphorylated derivative. Therefore, cells deficient in HPRT due to a forward mutation are resistant to 6-TG. These cells are able to proliferate in the presence of 6-TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic ex-pression is achieved by allowing exponential growth of the cells for 7 - 9 days. The expression period is terminated by adding 6-TG to the culture medium.
Mutant frequency is determined by seeding known numbers of cells in medium containing the selective agent to detect mutant cells, and in medium without selective agent to determine the surviving cells. After a suitable period the colonies are counted. Mutant frequencies are calculated from the number of mutant colonies corrected for cell survival.
In order to establish a concentration response effect of the test item at least four concentration levels are tested. These concentration levels should yield a concentration related toxic effect. The highest concentration level should induce a reduced level of survival.
CELL CULTURE CONDITIONS:
Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing; Technical University, Germany) are stored in liquid nitrogen in the cell bank of Harlan CCR allowing the repeated use of the same cell culture batch in experiments. Before freezing, the level of spontaneous mutants was depressed by treatment with HAT-medium. Each batch is screened for mycoplasm contamination and checked for karyotype stability and spontaneous mutant frequency. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.
Thawed stock cultures are propagated at 37 °C in 75 cm2 plastic flasks. About 5×105 cells were seeded into each flask with 15 mL of MEM (minimal essential medium) containing Hank’s salts supplemented with 10 % foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1 %). The cells were sub-cultured twice weekly. The cell cultures were incubated at 37°C in a 1.5 % carbon dioxide atmosphere (98.5 % air).
PRE-TEST ON TOXICITY AND DOSE SELECTION:
A pre-test was performed in order to determine the concentration range for the mutagenicity experiments. The general culture conditions and experimental conditions in this pre-test were the same as described for the mutagenicity experiment below. In this pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE).The pre-experiment was performed in the presence and absence (4 hours treatment) of metabolic activation. Test item concentrations between 16.4 μg/mL and 2100 μg/mL (equal to a molar concentration of approximately 10 mM) were used. The highest concentration in the pre-experiment was chosen with regard to the purity (>95%, preliminary information at study start) and the molecular weight (200.24 g/mol) of the test item. No relevant toxic effect occurred up to the maximum concentration tested with and without metabolic activation following 4 hours of treatment.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 hours) prior to removal to the test item. Precipitation occurred at 262.5 μg/mL and above after 4 hours treatment without metabolic activation and at 525.0 μg/mL after 4 hours treatment with metabolic activation.
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
The dose range of the first experiment was set according to the data on precipitation generated in the pre-experiment. In the first main experiment the individual concentrations were spaced by a factor of 2.0. Narrower spacing (factor 1.75) was used in the second experiment.
To overcome problems with possible deviations in toxicity the main experiments were started with more than four concentrations (see table 1 in section "any other information on materials and methods incl. tables".
CULTURE MEDIUM:
For seeding and treatment of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, 10% FBS (except during 4 hour treatment), neomycin (5 μg/mL) and amphotericin B (1%). For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2.
SEEDING:
Two to three days after sub-cultivation stock cultures were trypsinized at 37 °C for 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10 % FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2 % in PBS.
The PBS is composed as follows (per litre): NaCl 8000 mg; KCl 200 mg; KH2PO4 200 mg; Na2HPO4 150 mg
Prior to the trypsin treatment the cells were rinsed with PBS buffer containing 200 mg/l EDTA (ethylene diamine tetraacetic acid). Approximately 1.5×106 (single culture) and 5×102 cells (in duplicate) were seeded in plastic culture flasks. The cells were grown for 24 hours prior to treatment.
TREATMENT:
After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 μl/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. After 4 hours this medium was replaced with complete medium following two washing steps with "saline G". In the second experiment the cells were exposed to the test item for 24 hours in complete medium, supplemented with 10 % FBS, in the absence of metabolic activation.
The "saline G" solution had the following constituents (per litre): NaCl 8000 mg; KCl 400 mg; Glucose 1100 mg; Na2HPO4×2H2O 192 mg; KH2PO4 150 mg
The pH was adjusted to 7.2 The colonies used to determine the cloning efficiency (survival) were fixed and stained approx. 7 days after treatment as described below. Three or four days after treatment 1.5×106 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3 - 5×105 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution. The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope. - Evaluation criteria:
- A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
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 laboratory´s 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 was also taken into consideration. - Statistics:
- A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- 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
- Positive controls validity:
- valid
- Additional information on results:
- PRECIPITATION:
Precipitation of the test item was observed in experiment I at 520.0 μg/mL without metabolic activation and at 520.0 μg/mL and above with metabolic activation. In experiment II precipitation was noted at 594.3 μg/mL and above with and without metabolic activation.
TOXICITY:
No relevant toxic effects occurred up to the maximum concentration.
MUTAGENICITY:
No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration. The mutant frequency remained well within the historical range of solvent controls. The induction factor reached or exceeded the threshold of three times the corresponding solvent control in the first culture of the first experiment at 65.0 and 520μg/mL without metabolic activation. In the second culture of the first experiment with metabolic activation the threshold was exceeded at 1040.0 μg/mL. These effects however, were based upon rather low solvent controls of 4.3 and 6.9 mutant colonies/106 cells and thus, biologically irrelevant.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in the second culture of the first experiment with metabolic activation. As the mutation frequency did not exceed the historical range of solvent controls and actually remained within the range of the solvent controls of this study, the statistical result was judged as biologically irrelevant.
In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 4.3 up to 35.6 mutants per 106 cells; the range of the groups treated with the test item was from 4.5 up to 32.3 mutants per 106 cells.
EMS (150 μg/mL) and DMBA (1.1 μg/mL in experiment I, and 2.2 μg/mL in expeirment II) were used as positive controls and showed a distinct increase in induced mutant colonies. - Conclusions:
- Interpretation of results (migrated information):
negative
NM01 is considered to be non-mutagenic in this HPRT assay. - Executive summary:
The study was performed to investigate the potential of NM01 to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The main experiments were performed with and without liver microsomal activation and a treatment period of 4 hours. The highest concentration of 2100 μg/mL in the pre-experiment was equal to approximately 10 mM. Dose calculation was adjusted to preliminary purity data at study start (>95 %). The concentration range of the main experiments was limited by precipitation. The test item was suspended in deionised water. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.
Referenceopen allclose all
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Justification for classification or non-classification
Since a genotoxic potential was not detected in three in vitro tests according to OECD TGs 471, 473 and 476, respectively, the classification criteria of the CLP Regulation are not met.
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