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EC number: 939-017-1 | CAS number: 1469982-94-2
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- The experimental phase of this study was performed between 26 June 2012 and 20 December 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 013
- Report date:
- 2013
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- see Appendix 2
- Type of assay:
- in vitro mammalian chromosome aberration test
Test material
- Reference substance name:
- Isostearamide DEA
- IUPAC Name:
- Isostearamide DEA
- Reference substance name:
- HiTec 6457 Fuel Additive
- IUPAC Name:
- HiTec 6457 Fuel Additive
- Test material form:
- other: liquid
- Details on test material:
- Sponsor's identification: Isostearamide DEA
Description: Amber coloured liquid
Purity: UVCB - 100%
Batch number: OE11124(6/202593/00)
Date received: 14 May 2012
Expiry date: Not supplied
Storage conditions: Room temperature in the dark
Constituent 1
Constituent 2
Method
- Target gene:
- Not applicable.
Species / strain
- Species / strain / cell type:
- lymphocytes: human
- Details on mammalian cell type (if applicable):
- For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for
suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a
viral infection - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- phenobarbitone and beta-naphthoflavone induced rat liver, S9
- Test concentrations with justification for top dose:
- Cell Growth Inhibition Test:
4(20)-hour without S9: 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/ml.
4(20)-hour with S9: 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/ml.
20-hour without S9: 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/ml.
Experiment 1
4(20)-hour without S9: 0*, 25, 50*, 100*, 200*, 400* and 800 µg/ml.
4(20)-hour with S9: 0*, 25, 50*, 100*, 200*, 400* and 800 µg/ml.
Experiment 2
24-hour without S9: 0*, 12.5, 25, 50*, 100*, 200*, 400 µg/ml.
4(20)-hour with S9: 0*, 25, 50, 100*, 200*, 400*, 800 µg/ml.
* Dose levels selected for metaphase analysis - Vehicle / solvent:
- Justification for choice of solvent/vehicle:
The test item was immiscible in in Eagle's minimal essential medium with HEPES buffer (MEM) but was fully miscible in dimethyl sulphoxide in solubility checks performed in house therefore, the test item was accurately weighed, dissolved and serial dilutions prepared in dimethyl sulphoxide prior to
each experiment.
Controlsopen allclose all
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- In the presence of S9
Migrated to IUCLID6: (CP)EXAMPLE:
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- In the absence of S9
Migrated to IUCLID6: (MMC)
- Details on test system and experimental conditions:
- METHODS OF APPLICATION:
In medium
DURATION
- Pre-incubation period:
48 hours
- Exposure duration:
Experiment 1 – 4 hours with and without S9. Experiment 2 – 24 hours without S9, 4 hours with S9.
- Expression time (cells in growth medium):
20 hours for 4 hours exposure
- Selection time (in incubation with a selective agent):
Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells):
24 hours
SELECTION AGENT (MUTATION ASSAYS):
No selection agent selected
SPINDLE INHIBITOR (Cytogenetic assays):
Demecolcine
STAIN (for cytogenetic assays):
When slides were dry they were stained in 5% giemsa for 5 minutes, rinsed, dried and coverslipped using mounting medium.
NUMBER OF REPLICATIONS:
Duplicate cultures
NUMBER OF CELLS EVALUATED:
100/culture
DETERMINATION OF CYTOTOXICITY
-Method:
Mitotic index – A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic
index as a percentage of the vehicle control value.
-Scoring of Chromosome Damage:
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted
according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing (Appendix 1). Cells with
chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.
OTHER EXAMINATIONS:
- Determination of polyploidy:
Frequency of polyploid cells
OTHER:
none - Evaluation criteria:
- A positive response is recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the
concurrent control, either with or without a clear dose-response relationship. For modest increases in aberration frequency, a dose response
relationship is generally
required and appropriate statistical tests may be applied in order to record a positive response. - Statistics:
- The frequency of cells with aberrations excluding gaps and the frequency of polyploidy cells were compared, where necessary, with the concurrent
vehicle control value using Fisher’s Exact test.
Results and discussion
Test results
- Species / strain:
- lymphocytes: Human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Refer to information on results and attached tables.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- The molecular weight of the test item was assumed to be greater than 500 (because the test item is deemed to be a complex mixture), therefore, the maximum dose level was 5000 µg/ml. The purity of the test item was not required and was not accounted for in the formulations. There was no significant change in pH (more than 1 pH unit) when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm at the dose levels investigated (Scott et al 1991)
- Remarks on result:
- other: strain/cell type:
- Remarks:
- Migrated from field 'Test system'.
Any other information on results incl. tables
Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 19.53 to 5000 µg/ml. The maximum dose was based on the maximum recommended dose level, 5000 µg/ml. Precipitate observations were made from the blood-free cultures. In the 4(20)-hour exposure group in the absence of S9, precipitate was noted at the end of exposure at and above 78.13 µg/ml which became aggregated at and above 2500 µg/ml. However, in the 4(20)-hour exposure group with S9 precipitate was observed at and above 156.25 µg/ml but no aggregated precipitate was observed. In the 24‑hour continuous exposure group, precipitate was observed at the end of exposure at and above 78.13 µg/ml which became aggregated at and above 1250 µg/ml. Haemolysis was also observed at and above 312.5 µg/ml in the blood cultures of the 4(20)-hour exposures only.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 5000 µg/ml in all three exposure groups, however, the toxic limit of the test item was concluded to be 312.5µg/ml due to the precipitate preventing adequate exposure of the test item to the cells. The mitotic index data are presented in Table 1.
The selection of the maximum dose level was based on toxicity rather than the onset of the precipitate in all exposure groups tested.
Chromosome Aberration Test – Experiment 1
The dose levels of the controls and the test item are given in the table below:
Group |
Final concentration ofIsostearamideDEA(µg/ml) |
4(20)-hour without S9 |
0*, 25, 50*, 100*, 200*, 400*, 800, MMC0.4* |
4(20)-hour with S9 (2%) |
0*, 25, 50*, 100*, 200*, 400*, 800, CP5* |
*: dose levels selected for metaphase analysis
MMC: Mitomycin C
CP: Cyclophosphamide
The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to 400 µg/ml in the absence of metabolic activation (S9) and up to 800 µg/ml in the presence of metabolic activation (S9).
In the absence of S9, precipitate of the test item was observed in the blood cultures at 100 µg/ml at the end of the exposure period. However, precipitate was observed in the blood cultures in the presence of S9 at and above 400 µg/ml.
The mitotic index data are given in Table 2. They confirm the qualitative observations in that a marked inhibition of mitotic index was observed, and that 64% and 58% mitotic inhibition was achieved at 400 µg/ml in both the absence and presence of S9, respectively. The maximum dose level selected for metaphase analysis was, therefore, 400 µg/ml in both the absence and presence of S9.
The chromosome aberration data are given in Table 4 and Table 5. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test item did not induce any statistically significant increases in the frequency of cells with aberrations in either the absence or presence of metabolic activation.
The polyploid cell frequency data are given in Tables 4 and 5. The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.
Chromosome Aberration Test – Experiment 2
The dose levels of the controls and the test item are given in the table below:
Group |
Final concentration ofIsostearamideDEA(µg/ml) |
24-hour without S9 |
0*, 12.5, 25, 50*, 100*, 200*, 400, MMC0.2* |
4(20)-hour with S9 (1%) |
0*, 25, 50, 100*, 200*, 400*, 800, CP5* |
*: dose levels selected for metaphase analysis
MMC: Mitomycin C
CP: Cyclophosphamide
The qualitative assessment of the slides determined that there were metaphases suitable for scoring present up to 200 µg/ml in the absence of S9 and at 400 µg/ml in the presence of S9. Precipitate of the test item was observed at the end of exposure at and above 100 µg/ml in both the absence and presence of S9.
The mitotic index data are given in Table 3. They confirm the qualitative observations in that inhibition of mitotic index was observed, and that 56% mitotic inhibition was achieved at 200 µg/ml in the absence of S9 and 65% mitotic inhibition was achieved at 400 µg/ml in the presence of S9.
The maximum dose level selected for metaphase analysis was therefore, 200 µg/ml and 400 µg/ml in the absence and presence of S9, respectively.
The chromosome aberration data are given in Table 6 and Table 7. All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations in either the absence or presence of metabolic activation.
The polyploid cell frequency data are given in Tables 6 and 7. The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.
The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments using a dose range that included a dose level that was generally just outside the optimum 50% mitotic inhibition.
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results (migrated information):
negative
The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments using a dose range that included a dose level that was generally just outside the optimum 50% mitotic inhibition. - Executive summary:
Introduction. This report describes the results of anin vitrostudy for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al, 1990). The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Regulation (EC) No. 440/2008 of 30 May 2008. The study design was also compatible with the UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity.
Methods. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, i.e. In Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration); whilst in the absence of metabolic activation the exposure time was increased to 24 hours.
The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows:
Group
Final concentration of test item (µg/ml)
4(20)-hour without S9
25, 50, 100, 200, 400, 800
4(20)-hour with S9 (2%)
25, 50, 100, 200, 400, 800
24-hour without S9
12.5, 25, 50, 100, 200, 400
4(20)-hour with S9 (1%)
25, 50, 100, 200, 400, 800
Results. All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.
All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments using a dose range that included a dose level that was just outside the optimal 50% mitotic inhibition.
Conclusion. The test item, Isostearamide DEA, was considered to be non-clastogenic to human lymphocytes in vitro.
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