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EC number: 939-579-8 | CAS number: -
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
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- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
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- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
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- Endpoint summary
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- 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
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- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
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- Carcinogenicity
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- Specific investigations
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- Additional toxicological data
Genetic toxicity: in vitro
Administrative data
- 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
- Study period:
- 24 May - 17 July 2006
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP study conducted in compliance with OECD Guideline 476 without any deviation.
Cross-referenceopen allclose all
- Reason / purpose for cross-reference:
- reference to same study
- Reason / purpose for cross-reference:
- reference to other study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 006
- Report date:
- 2006
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
Test material
- Reference substance name:
- Reaction mass of sodium 3-{[3-(diethylamino)propyl]carbamoyl}-2-(dodecylamino)propanoate and sodium 3-{[3-(diethylamino)propyl]carbamoyl}-3-(dodecylamino)propanoate
- EC Number:
- 939-579-8
- Molecular formula:
- Empirical Formula : C11H21N3O3Na C2nH4n+1 (n=4 – 9), C11H21N3O3NaC18H35, C11H21N3O3NaC18H33 Molecular formula of the two main constituents : C23H46N3O3Na
- IUPAC Name:
- Reaction mass of sodium 3-{[3-(diethylamino)propyl]carbamoyl}-2-(dodecylamino)propanoate and sodium 3-{[3-(diethylamino)propyl]carbamoyl}-3-(dodecylamino)propanoate
- Details on test material:
- - Name of test material (as cited in study report): Chimexane HB
- Physical state: Colourless to pale yellow, translucent to slightly opaque gel
- Analytical purity: 55.9 % active content
- Lot/batch No.: 0126344
- Date received: 22 May 2006
- Expiration date of the lot/batch: March 2007
- Storage condition of test material: Room temperature in the dark
Constituent 1
Method
- Target gene:
- hprt gene
Species / strain
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Source of cells: Dr Donald Clive, Burroughs Wellcome Co, North Carolina, USA.
- Type and identity of media: Growth media, RPMI 1640
RPMI A: Penicillin (100 units/mL), streptomycin (100 μg/mL), amphotericin B (2.5 μg/mL) and pluronic (0.5 mg/mL)
RPMI 10: Horse serum (heat inactivated, 10 % v/v), penicillin (100 units/mL), streptomycin (100 μg/mL), amphotericin B (2.5 μg/mL) and pluronic (0.5 mg/mL)
RPMI 20: Horse serum (heat inactivated, 20 % v/v), penicillin (100 units/mL), streptomycin (100 μg/mL) and amphotericin B (2.5 μg/mL)
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for spontaneous mutant frequency: Yes
- Other details: For each experiment, one vial was thawed and the cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5 % v/v CO2 in air. The cells were allowed to grow well and subcultures were established in an appropriate number of flasks. - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- 5 % S9 mix; S9 fraction was prepared from liver homogenates of Sprague Dawley male rats treated with Aroclor 1254
- Test concentrations with justification for top dose:
- Cytotoxicity study (range-finder experiment):
- Without S9 mix: 4.688, 9.375, 18.75, 37.5, 75 and 150 μg/mL
- With S9 mix: 156.3, 312.5, 625, 1250, 2500 and 5000 μg/mL
Main study:
- Experiment 1 (-S9): 20, 40, 60, 80, 100, 110, 120, 130, 140 and 150 μg/mL
- Experiment 1 (+S9): 15, 30, 60, 80, 100, 120, 140, 150, 160 and 175 μg/mL
- Experiment 2 (-S9): 20, 40, 60, 80, 90, 100, 110, 120 and 140 μg/mL
- Experiment 2 (+S9): 20, 40, 60, 80, 100, 120, 130, 140, 150, 160 and 175 μg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Purified water
- Test item was dissolved in purified water with the aid of vortex mixing, warming at 37 or 60 °C and sonication as required to give the maximum required treatment solution concentration. This solution was filter-sterilised (Pall Acrodisc 32 filter, 0.2 μm pore size) and further dilutions were made using purified water. The test article solutions were protected from light and used within 1.25 h of initial formulation of the test article
Controlsopen allclose all
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- purified water diluted 10-fold in the treatment medium
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- with metabolic activation
Migrated to IUCLID6: 2 and 3 µg/mL
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- purified water diluted 10-fold in the treatment medium
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Remarks:
- without metabolic activation
Migrated to IUCLID6: 0.10 and 0.15 µg/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: In RPMI 1640 medium
DURATION
- Exposure duration: 3 h, 37 ± 1 ºC
- Expression time (cells in growth medium): 7 days, 37 ± 1 ºC in a humidified incubator gassed with 5 % v/v CO2 in air
- Viability scoring: 7 days, 37 ± 1 ºC in a humidified incubator gassed with 5 % v/v CO2 in air
- Selection time (if incubation with a selection agent): 11-12 days, 37 ± 1 ºC in a humidified incubator gassed with 5 % v/v CO2 in air
SELECTION AGENT (mutation assays): 6-thioguanine (6-TG) at 15 µg/mL (final concentration)
NUMBER OF REPLICATIONS:
- Vehicle and test substance groups (single cultures for cytotoxicity study and duplicate cultures for experiment 1 and 2).
- Single culture for positive control groups for experiments 1 and 2.
NUMBER OF CELLS EVALUATED: 1.6, 1.6 and 20000 cells per well plated for survival, viability and 6-TG resistance respectively.
DETERMINATION OF CYTOTOXICITY
- Method: Percentage Relative Survival (%RS)
OTHER:
- Wells containing viable clones were identified by eye using background illumination and counted.
- Cell concentrations in the selected cultures were determined using a Coulter counter. - Evaluation criteria:
- For valid data, the test article was considered to induce forward mutation at the hypoxanthine phosphoribosyl transferase (hprt) locus in mouse lymphoma L5178Y cells if:
- the assay was valid
- the mutant frequency at one or more concentrations was significantly greater than that of the negative control (p≤0.05)
- there was a significant concentration-relationship as indicated by the linear trend analysis (p≤0.05)
- the effects described above were reproducible.
- Test article was considered positive in this assay if all of the above criteria were met.
- Test article was considered negative in this assay if none of the above criteria are met.
- Results that only partially satisfy the assessment criteria described above were to be considered on a case-by-case basis. Positive responses seen only at high levels of cytotoxicity may require careful interpretation when assessing their biological significance.
- Extreme caution should be exercised with positive results obtained at levels of RS lower than 10 %. - Statistics:
- - Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines. Thus the control log mutant frequency (LMF) was compared with the LMF from each treatment concentration, and secondly the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.
Results and discussion
Test results
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility: Test item was soluble in purified water at concentrations up to at least 89.44 mg/mL (equivalent to 50 mg/mL, allowing for 55.9 % active content of test article).
- Effects of osmolality: No marked effect on osmolality (greater than a shift of 50 mOsm/kg) as compared to concurrent vehicle controls.
- Effects of pH: Increases of greater than 1 pH unit were observed in the absence of S9 at 2500 mg/mL and above. No further pH measurements were considered necessary as no increases of 1 pH unit were observed at 1250 mg/mL and below and the highest concentration tested in the main experiments was limited by toxicity to a maximum of 150 mg/mL. No increases of greater than 1 pH unit were observed in the presence of S9.
Precipitation:
- Cytotoxicity study (range-finder experiment): Upon addition of the test article to the cultures, no precipitate was observed. However, after the 3 h treatment incubation period, precipitate was observed at 312.5-2500 μg/mL in the presence of S9.
- Experiment 1: Upon addition of the test article to the cultures, no precipitate was observed. However, after the 3 h treatment incubation period, precipitate was observed at the highest two concentrations tested in the presence of S9 (160 and 175 μg/mL).
- Experiment 2: Upon addition of the test article to the cultures, no precipitate was observed. However, after the 3 h treatment incubation period, precipitate was observed at the highest five concentrations tested in the presence of S9 (130-175 μg/mL).
RANGE-FINDING/SCREENING STUDIES:
- Six concentrations were tested, in the absence and presence of S9, ranging from 4.688 to 150 μg/mL.
- Highest concentrations that survived treatment (75 μg/mL in the absence of S9 and 156.3 μg/mL in the presence of S9) yielded 84 and 16 % relative survival, respectively.
- See table 7.6.1/1 for more details.
MUTAGENICITY TESTS:
- When tested up to toxic concentrations, no statistically significant increases in mutant frequency were observed following treatment with Chimexane HB at any concentration tested in Experiments 1 and 2. A weak linear trend was observed in the presence of S9 in Experiment 2 but, as no significant increases in mutant frequency were observed and the effect was not reproduced between experiments, this was not considered biologically relevant.
- It may be noted that cultures treated at 130 μg/mL in the presence of S9 in Experiment 2 were highly toxic, yielding 5 % relative survival. These data were included in the statistical analysis as there was a steep concentration-related response between 100 and 130 μg/mL but there were no statistically significant increases in mutant frequency at the latter, highly toxic concentration.
- See table 7.6.1/2 and 7.6.1/3.
COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were compared with the historical data of the laboratory. - Remarks on result:
- other: other: TK+/- cells
- Remarks:
- Migrated from field 'Test system'.
Any other information on results incl. tables
Table 7.6.1/1: Range-finder experiment
Treatment (μg/mL) |
-S9 % RS |
+S9 % RS |
0 |
100 |
100 |
4.688 |
98 |
NT |
9.375 |
65 |
NT |
18.75 |
80 |
NT |
37.5 |
72 |
NT |
75 |
84 |
NT |
150 |
0 |
NT |
156.3 |
NT |
16 |
%RS: Percentage Relative Survival; NT: Not tested
Note: Concentrations of 312.5-5000 mg/mL treated in the presence of S9 were not plated, due to excessive toxicity
Table 7.6.1/2: Summary of mutation data -Experiment 1 (3 hour treatment in the absence and presence of S9)
Treatment (µg/mL) |
-S9 |
Treatment (µg/mL) |
+S9 |
||
%RS |
MF§ |
%RS |
MF§ |
||
0 |
100 |
5.26 |
0 |
100 |
4.41 |
20 |
92.87 |
5.19 NS |
15 |
102.30 |
4.36 NS |
40 |
83.20 |
5.23 NS |
30 |
103.00 |
3.19 NS |
60 |
88.23 |
3.96 NS |
60 |
94.84 |
4.54 NS |
80 |
74.26 |
4.12 NS |
80 |
89.05 |
4.80 NS |
100 |
27.70 |
4.33 NS |
100 |
94.88 |
4.69 NS |
110 |
17.82 |
2.30 NS |
120 |
87.57 |
3.90 NS |
- |
- |
- |
140 |
53.95 |
6.46 NS |
- |
- |
- |
150 |
13.97 |
4.68 NS |
Linear trend NS |
Linear trend NS |
||||
NQO 0.1 |
60.64 |
24.25 |
B[a]P 2 |
63.59 |
42.40 |
NQO 0.15 |
65.39 |
45.77 |
B[a]P 3 |
51.63 |
102.29 |
Table 7.6.1/3: Summary of mutation data -Experiment 2 (3 hour treatment in the absence and presence of S9)
Treatment (µg/mL) |
-S9 |
Treatment (µg/mL) |
+S9 |
||
%RS |
MF§ |
%RS |
MF§ |
||
0 |
100 |
5.16 |
0 |
100 |
4.73 |
20 |
105.89 |
5.19 NS |
20 |
96.17 |
4.25 NS |
40 |
86.65 |
6.15 NS |
40 |
95.32 |
3.41 NS |
60 |
58.80 |
7.64 NS |
60 |
97.73 |
4.86 NS |
80 |
11.70 |
4.75 NS |
80 |
100.67 |
4.26 NS |
- |
- |
- |
100 |
69.92 |
7.26 NS |
- |
- |
- |
120 |
24.50 |
7.36 NS |
- |
- |
- |
130 PP |
5.46 |
7.13 NS |
Linear trend NS |
Linear trend * |
||||
NQO 0.1 |
78.79 |
35.61 |
B[a]P 2 |
83.80 |
80.00 |
NQO 0.15 |
62.72 |
95.81 |
B[a]P 3 |
47.65 |
124.46 |
§ : 6-TG resistant mutants/10^6 viable cells 7 days after treatment ; %RS : Percent relative survival adjusted by post treatment cell counts ; NS: Not significant ; *, **, *** Test for linear trend: x^2 (one-sided), significant at 5, 1 and 0.1 % level respectively ; PP : Precipitation observed by eye following the treatment incubation period
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation
Under the test conditions, Chimexane HB did not induce any mutations in mammalian cells (strain: L5178Y mouse lymphoma cells) with and without metabolic activation. - Executive summary:
In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, mouse lymphoma L5178Y tk+/- cells were exposed to Chimexane HB dissolved in purified water in RPMI 1640 medium for 3 h, with and without metabolic activation (5 % S9 fraction of male Sprague Dawley rats liver induced with Aroclor 1254), at the following concentrations:
Cytotoxicity study (range-finder experiment):
- Without S9 mix: 4.688, 9.375, 18.75, 37.5, 75 and 150 μg/mL
- With S9 mix: 156.3, 312.5, 625, 1250, 2500 and 5000 μg/mL
Main study:
- Experiment 1 (-S9): 20, 40, 60, 80, 100, 110, 120, 130, 140 and 150 μg/mL
- Experiment 1 (+S9): 15, 30, 60, 80, 100, 120, 140, 150, 160 and 175 μg/mL
- Experiment 2 (-S9): 20, 40, 60, 80, 90, 100, 110, 120 and 140 μg/mL
- Experiment 2 (+S9): 20, 40, 60, 80, 100, 120, 130, 140, 150, 160 and 175 μg/mL
In the cytotoxicity study, the highest concentrations that survived treatment (75 μg/mL in the absence of S9 and 156.3 μg/mL in the presence of S9) yielded 84 and 16 % relative survival, respectively. In experiment 1, the highest concentrations analysed were 110 μg/mL in the absence of S9 and 150 μg/mL in the presence of S9, which yielded 18 and 14 % relative survival, respectively. In experiment 2, the highest concentrations analysed were 80 μg/mL in the absence of S9 and 130 μg/mL in the presence of S9, which yielded 12 and 5 % relative survival, respectively. When tested up to toxic concentrations, no statistically significant increases in mutant frequency were observed following treatment with Chimexane HB at any concentration tested in experiments 1 and 2. A weak linear trend was observed in the presence of S9 in experiment 2 but, as no significant increases in mutant frequency were observed and the effect was not reproduced between experiments, this was not considered biologically relevant. Mutant frequencies in negative control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals [4-nitroquinoline 1-oxide at 0.10 or 0.15 µg/mL (without S9 mix) and benzo(a)pyrene at 2 or 3 µg/mL (with S9 mix)] indicating the validity of the study.
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