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EC number: 203-338-9 | CAS number: 105-85-1
- 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
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- Nanomaterial aspect ratio / shape
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- Nanomaterial surface chemistry
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- Nanomaterial pour density
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- Nanomaterial radical formation potential
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- Endpoint summary
- Stability
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- 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
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Based on the results of an Ames test (OECD 471) it is concluded that the test item is non-mutagenic in the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia colistrain WP2uvrA in the absence and presence of S9-mix.
Based on the results of an micronucleus test (OECD 487) it is concluded that the test item is not clastogenic or aneugenic in human lymphocytes.
Based on the results of a HPRT test (OECD 476) it is concluded that the test item does not induce gene mutations at the HPRT locus
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:
- 10 May 2017 - 10 August 2017
- 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:
- 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 2008
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial forward mutation assay
- 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:
- Phenobarbital/β-naphthoflavone induced rat liver S9
- Test concentrations with justification for top dose:
- Pre-Experiment/Experiment I:
3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II:
Strains TA 1535 and WP2 uvrA: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
The remaining strains: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Justification for top dose: according to OECD guideline 471 - Vehicle / solvent:
- - Vehicle/solvent used: DMSO
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- methylmethanesulfonate
- other: 4-nitro-o-phenylene-diamine: TA 1537, TA 98 without metabolic activation, 2-aminoanthracene: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA with metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: experiment I: in agar (plate incorporation); experiment II: preincubation
DURATION
- Preincubation period: 60 minutes.
- Exposure duration: ≥ 48 hours
NUMBER OF REPLICATIONS: 3 (two independent experiments)
DETERMINATION OF CYTOTOXICITY
- Method: counting numbers of revertants and inspection of the bacterial background lawn - Evaluation criteria:
- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant. - Statistics:
- no statistical analysis
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at concentrations ≥ 333 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at concentrations ≥ 100 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at concentrations ≥ 333 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at concentrations ≥ 100 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at concentrations ≥ 333 µg/plate
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item occurred up to the highest investigated dose. - Conclusions:
- Based on the results of this study it is concluded that the test item is non-mutagenic in the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2uvrA in the absence and presence of S9-mix.
- Executive summary:
The genetic toxicity of the test item was assessed using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2uvrA, in accordance with OECD 471 guideline and GLP principles in two independent experiments (direct plate assay and a preincubation assay). No precipitation of the test item occurred up to the highest investigated dose. The negative and strain-specific positive control values were within the laboratory background historical control data ranges.
The plates incubated with the test item showed reduced background growth up in all strains with metabolic activation and in all strains, except of strain WP2uvrA without metabolic activation.
Toxic effects, evident as a reduction in the number of revertants, were observed in all strains with metabolic activation and in strains TA 1537, TA 98, and TA 100 without metabolic activation.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Based on the results of this study it is concluded that the test item is non-mutagenic in the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2uvrA in the absence and presence of S9-mix.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 24 April 2017 - 06 September 2017
- 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:
- 2016
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Version / remarks:
- 1998
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: mammalian cell gene mutation assay
- Target gene:
- HPRT locus
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany
- Suitability of cells: established cell line for in vitro experiments
- Doubling time: 12 - 16 h
- Methods for maintenance in cell culture: stock cultures were propagated at 37 °C in 75 cm2 plastic flasks. About 2-3Χ10exp6 cells were seeded into each flask with 15 mL culture medium, cells were sub-cultured once or twice weekly.
- Modal number of chromosomes: 22
MEDIA USED
- Type and identity of media including CO2 concentration: MEM (minimal essential medium) containing Hank’s salts, neomycin (5 μg/mL), 10% FBS, and amphotericin B (1%), 1,5 % CO2
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/β-naphthoflavone induced rat liver S9
- Test concentrations with justification for top dose:
- pre-experiemnt (+/- S9): 15.1; 30.2; 60.3; 120.6; 241.3; 482.5; 965; and 1930 μg/mL (= 10 mM)
experiment I (-S9): 5.85; 8.78; 13.17; 19.75; 29.63; 44.44; 66.67; and 100.0 μg/mL
experiment I (+S9): 12.5; 25.0; 50.0; 100.0; 200.0; 300.0; 400.0; and 600.0 µg/mL
justification for top dose (main experiment): based on cytotoxicity of the test item and phase separation (observed in the pre-experiment) - Vehicle / solvent:
- - Vehicle/solvent used: DMSO (final concentration in nutrient medium 0.5%)
- Justification for choice of solvent/vehicle: due to solubility properties and its relative non-toxicity to the cell cultures - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 hours
- Expression time: 7 days
- Selection time: 8 days
SELECTION AGENT: 6-thioguanine
STAIN: 10% methylene blue in 0.01% KOH solution
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: all colonies with more than 50 cells were counted
DETERMINATION OF CYTOTOXICITY
- Method: relative survival (cloning efficiency) - Evaluation criteria:
- The gene mutation assay is considered acceptable if:
- The mean values of the numbers of mutant colonies per 10exp6 cells found in the solvent controls of both parallel cultures remain within the 95% confidence interval of the laboratory historical control data range.
- Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistical significant increase compared with the concurrent solvent control.
- Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in positive results.
- An adequate number of cells and concentrations (at least four test item concentrations) are analysable even for the cultures treated at concentrations that cause 90% cytotoxicity during treatment.
- The criteria for the selection of the top concentration are fulfilled. - Statistics:
- A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated 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.
A t-Test was performed using a validated test script of “R”, a language and environment for statistical computing and graphics, to evaluate an isolated increase of the mutation frequency at a test point exceeding the 95% confidence interval. Again a t-test is judged as significant if the p-value (probability value) is below 0.05. - Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at concentrations ≥ 44.44 μg/mL
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at concentrations ≥ 100 μg/mL
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Water solubility: phase separation at concentrations ≥ 44.4 µg/mL (without metabolic activation) and at concentrations ≥ 100 µg/mL (with metabolic activation)
- Precipitation: none
RANGE-FINDING/SCREENING STUDIES: yes, pre-experiment
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: relative survival (cloning efficiency) - Conclusions:
- An in vitro gene mutation study (HPRT) was conducted in V79 cells of the Chinese hamster. It was concluded that the test substance did not induce gene mutations at the HPRT locus and therefore was considered to be non-mutagenic.
- Executive summary:
An in vitro gene mutation study (HPRT) according to OECD Guideline 476 was conducted in V79 cells of the Chinese hamster. A pre-experiment was performed with and without metabolic activation for the selection of the test substance concentration. Concentrations between 15.1 and 1930 μg/mL (=10 mM) were tested. The analysed concentration range of the main experiment was limited by cytotoxicity of the test item and phase separation. For the main experiment without metabolic activation a concentration range between 5.85 and 100 μg/mL was used and for the main experiment with metabolic activation a concentration range between 12.5 and 600 μg/mL. The main assay was performed in duplicates. Cells were exposed to the test item for 4 hours. No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment. The controls confirmed the validity of the test. It was concluded that the test substance did not induce gene mutations at the HPRT locus and therefore was considered to be non-mutagenic.
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 10 December 2015 - 25 March 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
- Species / strain / cell type:
- lymphocytes:
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: healthy adult, non-smoking volunteers (aged 18 to 35 years) - Cytokinesis block (if used):
- Cytochalasin B
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix
- Vehicle / solvent:
- - Vehicle/solvent used: Dimethyl sulfoxide
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- Cultured peripheral human lymphocytes were used as test system. Peripheral human lymphocytes are recommended in the international OECD guideline.
Blood was collected from healthy adult, non-smoking volunteers (aged 18 to 35 years). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2015) are presented below:
Dose range finding study: age 28, AGT = 13.4 h
First cytogenetic assay: age 26, AGT = 12.8 h
Second cytogenetic assay: age 28, AGT = 13.4 h - Evaluation criteria:
- A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with an Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with an Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.
In case the Chi-square test shows that there are statistically significant differences between one or more of the test item groups and the vehicle control group a Cochran Armitage trend test (p < 0.05) was performed to test whether there is a significant trend in the induction. - Statistics:
- GraphPad PRISM version 4.03 (Graphpad Software, San Diego, USA) and ToxRat Professional
v 3.0.0 (ToxRat Solutions® GmbH, Germany) were used for statistical analysis of the data. - Key result
- Species / strain:
- lymphocytes:
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no mutagenic potential (based on QSAR/QSPR prediction)
- Conclusions:
- The test item is not clastogenic or aneugenic in human lymphocytes.
- Executive summary:
An in vitro micronucleus assay according to OECD 487 was performed in cultured peripheral human lymphocytes. The number of micronuclei formed was assessed in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation. The possible clastogenicity and aneugenicity of the test item was tested in two independent experiments.
The test item was dissolved in dimethyl sulfoxide. In the first cytogenetic assay, the test item was tested up to 200 μg/mL for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction. Appropriate toxicity was reached at this dose level. In the second cytogenetic assay, the test item was tested up to 50 μg/mL for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. Appropriate toxicity was reached at this dose level. The controls confirmed the validity of the results.
The test item did not induce a statistically significant and biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments. It is concluded that the test item is not clastogenic or aneugenic in human lymphocytes.
Referenceopen allclose all
Table 1: Summary of Experiment I
Metabolic Activation |
Test Group |
Dose Level (per plate) |
Revertant Colony Counts (Mean ± SD) |
||||
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
WP2 uvrA |
|||
Without Activation |
DMSO |
|
10 ± 4 |
12 ± 3 |
22 ± 5 |
162 ± 12 |
36 ± 6 |
Untreated |
|
11 ± 4 |
9 ± 3 |
25 ± 2 |
175 ± 24 |
37 ± 3 |
|
Test item |
3 µg |
12 ± 2 |
12 ± 2 |
26 ± 9 |
151 ± 4 |
34 ± 7 |
|
10 µg |
13 ± 3 |
13 ± 3 |
25 ± 5 |
142 ± 13 |
35 ± 10 |
||
33 µg |
10 ± 1 |
11 ± 4 |
26 ± 5 |
137 ± 7 |
40 ± 2 |
||
100 µg |
9 ± 5 |
7 ± 4 |
16 ± 5 |
110 ± 15 |
40 ± 9 |
||
333 µg |
10 ± 4 |
8 ± 3R |
20 ± 6R |
54 ± 2R |
41 ± 1 |
||
1000 µg |
7 ± 3 |
10 ± 2R |
17 ± 5R |
51 ± 4R |
37 ± 6 |
||
2500 µg |
8 ± 3 |
8 ± 5R |
16 ± 3R |
57 ± 10R |
40 ± 4 |
||
5000 µg |
8 ± 2 |
10 ± 2R |
13 ± 1R |
35 ± 3M R |
41 ± 8 |
||
NaN3 |
10 µg |
1284 ± 13 |
|
|
2059 ± 79 |
|
|
4-NOPD |
10 µg |
|
|
346 ± 17 |
|
|
|
4-NOPD |
50 µg |
|
62 ± 5 |
|
|
|
|
MMS |
2.0 µL |
|
|
|
|
934 ± 30 |
|
With Activation |
DMSO |
|
14 ± 3 |
18 ± 6 |
26 ± 7 |
158 ± 7 |
47 ± 2 |
Untreated |
|
14 ± 6 |
16 ± 5 |
33 ± 4 |
166 ± 8 |
54 ± 8 |
|
Test item |
3 µg |
12 ± 3 |
16 ± 3 |
32 ± 5 |
130 ± 26 |
47 ± 5 |
|
10 µg |
13 ± 3 |
18 ± 8 |
31 ± 4 |
134 ± 8 |
39 ± 9 |
||
33 µg |
13 ± 4 |
12 ± 1 |
28 ± 9 |
143 ± 16 |
51 ± 8 |
||
100 µg |
12 ± 5 |
15 ± 2 |
32 ± 8 |
148 ± 9 |
53 ± 11 |
||
333 µg |
9 ± 3 |
10 ± 4R |
31 ± 4R |
94 ± 24R |
40 ± 11 |
||
1000 µg |
11 ± 3R |
4 ± 1M R |
16 ± 2R M |
25 ± 2R |
29 ± 8 |
||
2500 µg |
6 ± 2R M |
3 ± 1M R |
1 ± 1M R |
1 ± 1R |
21 ± 6 |
||
5000 µg |
7 ± 3M R |
3 ± 1M R |
0 ± 0R |
0 ± 0R |
25 ± 2 |
||
2-AA |
2.5 µg |
533 ± 60 |
154 ± 14 |
3890 ± 433 |
4101 ± 294 |
|
|
2-AA |
10.0 µg |
|
|
|
|
444 ± 24 |
NaN3: sodium azide, 2-AA: 2-aminoanthracene, 4-NOPD: 4-nitro-o-phenylene-diamine, MMS:methyl methane sulfonate, R: Reduced background growth, M: Manual count
Table 2: Summary of Experiment II
Metabolic Activation |
Test Group |
Dose Level (per plate) |
Revertant Colony Counts (Mean ± SD) |
||||
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
WP2 uvrA |
|||
Without Activation |
DMSO |
|
11 ± 4 |
8 ± 3 |
18 ± 4 |
182 ± 12 |
41 ± 9 |
Untreated |
|
10 ± 2 |
10 ± 3 |
22 ± 1 |
213 ± 3 |
44 ± 7 |
|
Test item |
3 µg |
|
9 ± 3 |
21 ± 5 |
180 ± 3 |
|
|
10 µg |
11 ± 5 |
8 ± 4 |
18 ± 8 |
172 ± 4 |
44 ± 5 |
||
33 µg |
9 ± 2 |
10 ± 3 |
18 ± 3 |
153 ± 18 |
46 ± 9 |
||
100 µg |
10 ± 2 |
6 ± 1R |
19 ± 6 |
71 ± 25R |
35 ± 3 |
||
333 µg |
9 ± 3 |
9 ± 3R |
18 ± 5R |
47 ± 9R |
36 ± 8 |
||
1000 µg |
8 ± 2 |
8 ± 1R |
19 ± 3R |
46 ± 6R |
31 ± 7 |
||
2500 µg |
11 ± 3R |
10 ± 4R |
7 ± 3R M |
26 ± 5M R |
41 ± 9 |
||
5000 µg |
10 ± 5R |
2 ± 1M R |
6 ± 1M R |
8 ± 2M R |
40 ± 7 |
||
NaN3 |
10 µg |
1218 ± 30 |
|
|
1468 ± 71 |
|
|
4-NOPD |
10 µg |
|
|
290 ± 7 |
|
|
|
4-NOPD |
50 µg |
|
81 ± 6 |
|
|
|
|
MMS |
2.0 µL |
|
|
|
|
537 ± 47 |
|
With Activation |
DMSO |
|
11 ± 2 |
10 ± 1 |
34 ± 6 |
181 ± 13 |
52 ± 9 |
Untreated |
|
12 ± 1 |
12 ± 4 |
38 ± 3 |
201 ± 3 |
71 ± 14 |
|
Test item |
3 µg |
|
13 ± 4 |
30 ± 8 |
166 ± 14 |
|
|
10 µg |
14 ± 2 |
12 ± 4 |
38 ± 7 |
171 ± 21 |
58 ± 11 |
||
33 µg |
10 ± 2 |
10 ± 4 |
38 ± 3 |
158 ± 5 |
57 ± 8 |
||
100 µg |
13 ± 2 |
11 ± 3 |
36 ± 9 |
133 ± 18 |
62 ± 9 |
||
333 µg |
7 ± 2M R |
3 ± 1R M |
6 ± 1R M |
71 ± 7M R |
36 ± 8R |
||
1000 µg |
5 ± 1M R |
2 ± 1M R |
1 ± 1R |
28 ± 9M R |
27 ± 4R |
||
2500 µg |
3 ± 1M R |
0 ± 0R |
0 ± 0R |
7 ± 2R M |
9 ± 2M R |
||
5000 µg |
2 ± 1M R |
0 ± 0R |
0 ± 0R |
0 ± 0R |
11 ± 1M R |
||
2-AA |
2.5 µg |
376 ± 1 |
119 ± 20 |
3852 ± 420 |
4094 ± 290 |
|
|
2-AA |
10.0 µg |
|
|
|
|
492 ± 60 |
NaN3: sodium azide, 2-AA: 2-aminoanthracene, 4-NOPD: 4-nitro-o-phenylene-diamine, MMS:methyl methane sulfonate, R: Reduced background growth, M: Manual count
Table 2: Historicla data
Strain |
|
without S9 mix |
with S9 mix |
||||||
Mean |
SD |
Min |
Max |
Mean |
SD |
Min |
Max |
||
TA 1535 |
Solvent control Untreated control Positive control |
12 12 1130 |
2.5 3.1 143.1 |
6 6 334 |
25 28 1816 |
12 12 388 |
2.5 2.9 58.2 |
7 7 176 |
26 26 668 |
TA 1537 |
Solvent control Untreated control Positive control |
10 11 82 |
2.2 2.7 12.7 |
6 5 43 |
19 21 157 |
13 14 191 |
3.5 4.0 60.8 |
7 7 83 |
30 31 434 |
TA 98 |
Solvent control Untreated control Positive control |
25 27 378 |
4.4 4.9 73.7 |
13 12 211 |
43 43 627 |
34 37 3949 |
6.2 6.5 771.8 |
15 11 360 |
58 57 6586 |
TA 100 |
Solvent control Untreated control Positive control |
156 176 1966 |
26.0 23.6 293.2 |
78 79 498 |
209 217 2767 |
148 172 3798 |
32.3 25.4 830.4 |
73 85 536 |
208 218 6076 |
WP2uvrA |
Solvent control Untreated control Positive control |
41 42 798 |
5.6 5.8 362.7 |
27 30 319 |
63 63 4732 |
50 52 378 |
6.8 6.8 112.6 |
28 36 167 |
72 88 1265 |
Table 1: Summary of Results
|
conc. µg/mL |
PS |
S9 mix |
Relative cloning efficiency I % |
Relative cell density % |
Rel. adjusted cloning efficiency I % |
Mutant colonies/ 106cells |
95% confidence interval |
Relative cloning efficiency I % |
Relative cell density % |
Rel. adjusted cloning efficiency I % |
Mutant colonies/ 106cells |
95% confidence interval |
Column |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
13 |
Experiment I / 4h treatment |
|
|
|
culture I |
culture II |
||||||||
Solvent control with DMSO |
|
- |
- |
100.0 |
100.0 |
100.0 |
30.1 |
1.7 - 30.2 |
100.0 |
100.0 |
100.0 |
15.7 |
1.7 - 30.2 |
Positive control (EMS) |
300.00 |
- |
- |
108.6 |
76.5 |
83.1 |
320.3 |
13.4 - 367.1 |
108.1 |
52.9 |
57.2 |
267.1 |
13.4 - 367.1 |
Test item |
5.85 |
- |
- |
109.9 |
75.1 |
82.6 |
23.4 |
1.7 - 30.2 |
105.0 |
82.9 |
87.0 |
25.5 |
1.7 - 30.2 |
Test item |
8.78 |
- |
- |
103.5 |
88.0 |
91.0 |
15.4 |
1.7 - 30.2 |
104.9 |
69.2 |
72.6 |
15.3 |
1.7 - 30.2 |
Test item |
13.17 |
- |
- |
115.1 |
71.4 |
82.1 |
13.9 |
1.7 - 30.2 |
104.7 |
90.5 |
94.8 |
13.0 |
1.7 - 30.2 |
Test item |
19.75 |
- |
- |
113.4 |
90.2 |
102.3 |
31.9 |
1.7 - 30.2 |
107.0 |
74.0 |
79.2 |
19.9 |
1.7 - 30.2 |
Test item |
29.63 |
- |
- |
109.2 |
81.0 |
88.4 |
18.8 |
1.7 - 30.2 |
105.9 |
65.2 |
69.1 |
22.8 |
1.7 - 30.2 |
Test item |
44.44 |
PS |
- |
19.5 |
57.0 |
11.1 |
17.6 |
1.7 - 30.2 |
10.2 |
69.7 |
7.1 |
16.1 |
1.7 - 30.2 |
Test item |
66.67 |
PS |
- |
16.4 |
31.1 |
5.1 |
# |
8.6 |
51.7 |
4.5 |
# |
||
Test item |
100.00 |
PS |
- |
19.0 |
32.7 |
6.2 |
# |
10.4 |
52.1 |
5.4 |
# |
||
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Solvent control with DMSO |
|
- |
+ |
100.0 |
100.0 |
100.0 |
13.9 |
2.0 - 29.4 |
100.0 |
100.0 |
100.0 |
21.7 |
2.0 - 29.4 |
Positive control (DMBA) |
2.3 |
- |
+ |
77.7 |
91.2 |
70.8 |
117.6 |
0.0 - 437.7 |
97.1 |
51.5 |
50.0 |
140.0 |
0.0 - 437.7 |
Test item |
12.5 |
- |
+ |
61.7 |
94.3 |
58.2 |
23.2 |
2.0 - 29.4 |
69.0 |
123.4 |
85.1 |
13.8 |
2.0 - 29.4 |
Test item |
25.0 |
- |
+ |
67.7 |
94.7 |
64.1 |
12.0 |
2.0 - 29.4 |
60.9 |
74.3 |
45.2 |
27.3 |
2.0 - 29.4 |
Test item |
50.0 |
- |
+ |
79.7 |
115.8 |
92.3 |
21.5 |
2.0 - 29.4 |
64.4 |
81.4 |
52.5 |
18.5 |
2.0 - 29.4 |
Test item |
100.0 |
PS |
+ |
40.6 |
79.3 |
32.2 |
19.4 |
2.0 - 29.4 |
63.4 |
61.2 |
38.8 |
46.4 |
2.0 - 29.4 |
Test item |
200.0 |
PS |
+ |
## |
6.7 |
culture was not continued## |
## |
1.5 |
culture was not continued## |
||||
Test item |
300.0 |
PS |
+ |
culture was not continued## |
culture was not continued## |
||||||||
Test item |
400.0 |
PS |
+ |
culture was not continued## |
culture was not continued## |
||||||||
Test item |
600.0 |
PS |
+ |
culture was not continued## |
culture was not continued## |
PS = phase separation visible at the end of treatment, # culture was not continued to avoid analysis of too many phase separating concentrations, ## culture was not continued due to exceedingly severe cytotoxic effects
Table 2: Historical data
2014 – 2016 |
||
Number of mutant colonies per 106cells |
||
without metabolic activation (4 hours treatment time) |
||
|
Positive control |
Solvent control |
Range: |
53.9 – 872.3 |
3.4 – 41.0 |
Mean value: |
190.3 |
15.9 |
Standard deviation: |
88.4 |
7.1 |
95% confidence limit |
-- |
1.7 – 30.2 |
Number of studies: |
111 |
111 |
with metabolic activation (4 hours treatment time) |
||
|
Positive control |
Solvent control |
Range: |
56.7 – 739.9 |
2.4 – 39.2 |
Mean value: |
215.8 |
15.7 |
Standard deviation: |
110.9 |
6.8 |
95% confidence: |
-- |
2.0 – 29.4 |
Number of studies: |
105 |
105 |
The 95% confidence interval is derived from the mean value plus/minus 2 times the standard deviation.
Table 1 Cytokinesis-block proliferation index of human lymphocytes cultures treated with Citronellyl Formate in the first cytogenetic assay
Without metabolic activation (-S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration µg/ml |
CBPI |
Mean CBPI |
% cytostasis |
0 10 50 100 150 175 2001 2501 3001 0.25 MMC-C 0.38 MMC-C 0.1 Colch |
1.75 - 1.78 1.75 - 1.77 1.78 - 1.78 1.74 - 1.74 1.61 - 1.64 1.48 - 1.49 1.40 - 1.41 1.03 - 1.27 1.04 -2 1.55 - 1.59 1.47 - 1.48 1.22 - 1.24 |
1.76 1.76 1.78 1.74 1.63 1.49 1.41 1.15 ND 1.57 1.48 1.23 |
0 0 -2 3 18 36 47 80 ND 25 38 70 |
With metabolic activation (+S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration µg/ml |
CBPI |
Mean CBPI |
% cytostasis |
0 10 50 100 150 2001 2501 3001 15 CP 17.5 CP |
1.78 - 1.79 1.73 - 1.73 1.76 - 1.79 1.70 - 1.74 1.53 - 1.58 1.31 - 1.34 1.21 -2 2-2 1.39 - 1.40 1.34 - 1.34 |
1.79 1.73 1.78 1.72 1.55 1.32 ND 2 1.40 1.34 |
0 7 1 8 30 59 ND 2 50 57 |
1Citronellyl Formate precipitated in the culture medium
2Cell lysis
ND = Not determined
Note: All calculations were performed without rounding off.
Table 2 Number of mononucleated or binucleated cells with micronuclei of human lymphocyte cultures treated with Citronellyl Formate in the first cytogenetic assay
Without metabolic activation (-S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration (µg/ml) |
Cytostasis (%) |
Number of mononucleated cells with micronuclei1) |
Number of binucleated cells with micronuclei1) |
||||
1000 |
1000 |
2000 |
1000 |
1000 |
2000 |
||
A |
B |
A+B |
A |
B |
A+B |
||
0 50 150 2002) 0.25 MMC-C 0.1 Colch |
0 -2 18 47 25 70 |
0 0 1 2 0 28 |
1 1 2 3 3 52 |
1 1 3 5 3 80*** |
1 5 11 5 42 64 |
5 6 9 43) 38 444) |
6 11 20** 9 80*** 108*** |
With metabolic activation (+S9-mix)
3 hours exposure time, 27 hours harvest time
Concentration (µg/ml) |
Cytostasis (%) |
Number of mononucleated cells with micronuclei1) |
Number of binucleated cells with micronuclei1) |
||||
1000 |
1000 |
2000 |
1000 |
1000 |
2000 |
||
A |
B |
A+B |
A |
B |
A+B |
||
0 50 150 2002) 15 CP |
0 1 30 59 50 |
2 4 2 6 1 |
1 26) 0 1 27) |
3 6 2 7 3 |
5 5 3 6 32 |
7 10 4 3 155) |
12 15 7 9 47*** |
*) Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.
1) 1000 bi- and mononucleated cells were scored for the presence of micronuclei.
Duplicate cultures are indicated by A and B.
2) Citronellyl Formate precipitated in the culture medium.
3) 695 binucleated cells were scored for the presence of micronuclei (see study plan deviation 1).
4) 668 binucleated cells were scored for the presence of micronuclei.
5) 432 binucleated cells were scored for the presence of micronuclei.
6) 784 mononucleated cells were scored for the presence of micronuclei (see study plan deviation 1).
7) 841 mononucleated cells were scored for the presence of micronuclei.
Table 3 Cytokinesis-block proliferation index of human lymphocyte cultures treated with Citronellyl Formate in the second cytogenetic assay
Without metabolic activation (-S9-mix)
24 hours exposure time, 24 hours harvest time
Concentration µg/ml |
CBPI |
Mean CBPI |
% Cytostasis |
0 5 10 25 50 75 100 125 0.15 MMC-C 0.23 MMC-C 0.05 Colch. |
1.86 – 1.86 1.75 – 1.78 1.73 – 1.76 1.61 – 1.62 1.44 – 1.45 1.27 – 1.28 1.07 – 1.09 1.01 – 1.01 1.42 – 1.45 1.35 – 1.36 1.02 – 1.03 |
1.86 1.77 1.74 1.61 1.44 1.28 1.08 1.01 1.44 1.36 1.03 |
0 11 14 29 49 68 91 99 50 59 97 |
Note: All calculations were performed without rounding off.
Table 4 Number of mononucleated or binucleated cells with micronuclei of human lymphocyte cultures treated with Citronellyl Formate in the second cytogenetic assay
Without metabolic activation (-S9-mix)
24 hours exposure time, 24 hours harvest time
Concentration (µg/ml) |
Cytostasis (%) |
Number of mononucleated cells with micronuclei1) |
Number of binucleated cells with micronuclei1) |
||||
1000 |
1000 |
2000 |
1000 |
1000 |
2000 |
||
A |
B |
A+B |
A |
B |
A+B |
||
0 5 25 50 0.15 MMC-C 0.05 Colch |
0 11 29 49 50 97 |
0 0 2 1 2 32 |
1 0 1 1 4 44 |
1 0 3 2 6* 76*** |
2 3 7 4 29 62) |
3 4 6 5 36 53) |
5 7 13* 9 65*** 11** |
*) Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.
1) 1000 bi- and mononucleated cells were scored for the presence of micronuclei.
Duplicate cultures are indicated by A and B.
2) 619 binucleated cells were scored for the presence of micronuclei.
3) 567 binucleated cells were scored for the presence of micronuclei.
Table 5 Historical control data forin vitromicronucleus studies of the solvent control
|
Mononucleated |
Binucleated |
||||
+ S9-mix |
- S9-mix |
+ S9-mix |
- S9-mix |
|||
3 hour exposure |
3 hour exposure |
24 hour exposure |
3 hour exposure |
3 hour exposure |
24 hour exposure |
|
Mean number of micronucleated cells |
0.85 |
0.93 |
0.85 |
3.26 |
3.80 |
4.06 |
SD |
0.88 |
0.97 |
1.13 |
2.17 |
2.63 |
2.47 |
n |
54 |
56 |
55 |
54 |
56 |
55 |
Upper control limit (95% control limits) |
3.36 |
3.35 |
3.17 |
8.58 |
10.53 |
10.85 |
Lower control limit (95% control limits) |
-1.66 |
-1.49 |
-1.46 |
-2.06 |
-2.92 |
-2.74 |
SD = Standard deviation
n = Number of observations
Distribution historical negative control data from experiments performed between January 2012 and June 2015.
Table 6 Historical control data for in vitro micronucleus studies of the positive control substances
|
Mononucleated |
Binucleated |
|||
- S9-mix |
+ S9-mix |
- S9-mix |
|||
3 hour |
24 hour exposure |
3 hour exposure |
3 hour exposure |
24 hour exposure |
|
Mean number of micronucleated cells |
46.2 |
52.1 |
29.3 |
30.4 |
30.0 |
SD |
28.1 |
25.0 |
14.9 |
16.4 |
10.1 |
n |
54 |
56 |
58 |
60 |
60 |
Upper control limit (95% control limits) |
93.1 |
92.7 |
57.8 |
56.48 |
54.7 |
Lower control limit (95% control limits) |
-0.58 |
11.5 |
0.70 |
4.28 |
5.31 |
SD = Standard deviation
n = Number of observations
Distribution historical positive control data from experiments performed between January 2012 and June 2015.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Ames test (2017):
The genetic toxicity of the test item was assessed using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2uvrA, in accordance with OECD 471 guideline and GLP principles in two independent experiments (direct plate assay and a preincubation assay). No precipitation of the test item occurred up to the highest investigated dose. The negative and strain-specific positive control values were within the laboratory background historical control data ranges.
The plates incubated with the test item showed reduced background growth in all strains with metabolic activation and in all strains, except of strain WP2uvrA, without metabolic activation.
Toxic effects, evident as a reduction in the number of revertants, were observed in all strains with metabolic activation and in strains TA 1537, TA 98, and TA 100 without metabolic activation.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Based on the results of this study it is concluded that the test item is non-mutagenic in the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2uvrA in the absence and presence of S9-mix.
HPRT (2017):
An in vitro gene mutation study (HPRT) according to OECD Guideline 476 was conducted in V79 cells of the Chinese hamster. A pre-experiment was performed with and without metabolic activation for the selection of the test substance concentration. Concentrations between 15.1 and 1930 μg/mL (=10 mM) were tested. The analysed concentration range of the main experiment was limited by cytotoxicity of the test item and phase separation. For the main experiment without metabolic activation a concentration range between 5.85 and 100 μg/mL was used and for the main experiment with metabolic activation a concentration range between 12.5 and 600 μg/mL. The main assay was performed in duplicates. Cells were exposed to the test item for 4 hours. No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment. The controls confirmed the validity of the test. It was concluded that the test substance did not induce gene mutations at the HPRT locus and therefore was considered to be non-mutagenic.
Micronucleus test (2016):
The genetic toxicity of the test item was assessed in a micronucleus test according to OECD 487. The test was valid and
the test item is not clastogenic or aneugenic in human lymphocytes under the experimental conditions.
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 (EC) No 1272/2008. Based on
available data on genetic toxicity, the
test item is not classified according
to Regulation (EC) No 1272/2008 (CLP), as amended for the thenth time in
Regulation (EU) No 2017/776.
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