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EC number: 284-634-5 | CAS number: 84961-45-5 Extractives and their physically modified derivatives such as tinctures, concretes, absolutes, essential oils, oleoresins, terpenes, terpene-free fractions, distillates, residues, etc., obtained from Ceratonia siliqua. Leguminosae.
- 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 / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 22 Sep 2017 - 28 Dec 2017
- Reliability:
- 1 (reliable without restriction)
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline 487 (In Vitro Mammalian Cell Micronucleus Test)
- Version / remarks:
- 29 July 2016
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell micronucleus test
Test material
- Test material form:
- liquid
- Details on test material:
- - Physical appearance: Dark brown to black liquid
- Storage of test material: At room temperature
Constituent 1
Method
Species / strain
- Species / strain / cell type:
- lymphocytes: human peripheral blood
- Details on mammalian cell type (if applicable):
- Type and identity of media:
- Sex, age and number of blood donors if applicable:
Blood was collected from healthy adult, non-smoking volunteers
The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2016) are presented below:
Dose-range finding study: age 22, AGT = 13.2 h
First cytogenetic assay (presence of S9-mix): age 31, AGT = 13.2 h
First cytogenetic assay (absence of S9-mix): age 25, AGT = 13.2 h
Second cytogenetic assay: age 29, AGT = 14.2 h
Blood samples
Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.
- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin was added.
- Cytokinesis block (if used):
- Cytochalasine B (5 μg/mL) for 24 hours (1.5 times normal cell cycle)
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
- Test concentrations with justification for top dose:
- Dose range finding test:
Without S9-mix, 3 hr exposure; 27 hr harvest: 0, 156, 313, 625, 1250, 2500 and 5000 μg/mL
Without S9-mix, 24 hr exposure, 24 hour harvest 0, 156, 313, 625, 1250, 2500 and 5000 μg/mL
With S9-mix, 3 exposure; 27 hr harvest: 0, 156, 313, 625, 1250, 2500 and 5000 μg/mL
First cytogenetic test:
Without S9-mix, 3hr exposure; 27 hr harvest: 0, 1250, 2500 and 5000 μg/mL
With S9-mix, 3hr exposure; 27 hr harvest: 0, 1250, 2500 and 5000 μg/mL
Second cytogenetic test:
Without S9-mix, 24 hr exposure: 0, 1250, 2500 and 5000 μg/mL
A concentration of 5000 µg/mL showed no precipitation in the culture medium and was used as the highest concentration of Carob Bean Extract. - Vehicle / solvent:
- - Vehicle: dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle:
DMSO is recommended in international OECD guidelines to be used as solvent.
Controlsopen allclose all
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Hanks’ Balanced Salt Solution without calcium and magnesium.
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- Without Metabolic Activation
- Positive control substance:
- mitomycin C
- Remarks:
- Mitomycin C was used as a direct acting clastogen at a final concentration of 0.25 and 0.38 µg/mL for a 3 hour exposure period and 0.15 and 0.23 µg/mL for a 24 hour exposure period.
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Hanks’ Balanced Salt Solution without calcium and magnesium.
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- Without Metabolic Activation
- Positive control substance:
- other: Colchicine
- Remarks:
- Colchicine was used as a direct acting aneugen at a final concentration of 0.1 µg/mL for a 3 hour exposure period and 0.05 µg/mL for a 24 hour exposure period
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Hanks’ Balanced Salt Solution without calcium and magnesium.
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- With Metabolic Activation
- Positive control substance:
- cyclophosphamide
- Remarks:
- Cyclophosphamide was used as an indirect acting clastogen, requiring metabolic activation, at a final concentration of 10, 12.5, 15 and 17,5 µg/mL for a 3 hour exposure period.
- Details on test system and experimental conditions:
- Environmental conditions:
Humidity: 37 - 89%
CO2: 5.0 ± 0.5%
Temperature: 34.5 - 37.1°C
METHOD OF APPLICATION:
in medium
DURATION
- Preincubation period: 3 hour
- Exposure duration:
Without and with S9-mix: 3 hour treatment, 27 hour harvest time
The exposure period was followed by centrifugation, rinsing, a second centrifugation and incubation for another 24 hours with cytokinase block.
Without S9-mix: 24 hour treatment, 24 hour harvest time.
The exposure period was followed by immediate fixation without rinsing.
ARREST OF CELL DIVISION: 5 µg/mL Cytochalasine B
STAIN: 5% (v/v) Giemsa
NUMBER OF REPLICATIONS: 2
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
To harvest the cells, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cells in the remaining cell pellet were re-suspended in 1% Pluronic F68. After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloridesolution. Immediately after, ethanol: acetic acid (Merck) fixative (3:1 v/v) wasadded. Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet werefixated carefully with 3 changes of ethanol: acetic acid fixative (3:1 v/v). Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96%(v/v) ethanol/ether and cleaned with a tissue. The slides were marked with the Charles River Den Bosch study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa solution in Sörensen buffer pH 6.8. Thereafterslides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper
NUMBER OF CELLS EVALUATED: A minimum of 500 cells per culture was counted, scoring cells with one, two or more nuclei (multinucleated cells).
- Evaluation criteria:
- A test was considered acceptable if it met the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item colchicine induces a statistically significant increase in the number of mononucleated cells with micronuclei and the positive control items MMC-C and CP induces a statistically significant increase in the number of binucleated cells with micronuclei. The positive control data will be analyzed by the Chi-square test (one-sided, p < 0.05).
A test substance was considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if:
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 a Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test substance was 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, onesided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range. - Statistics:
- To evaluate the statistical significane of induction, the Chi-square test is performed ( Chi-square test, onesided, p < 0.05).
Results and discussion
Test results
- Key result
- Species / strain:
- lymphocytes: human peripheral blood
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolarity: no, the pH and osmolarity at a concentration of 5000 μg/ml were 7.627 and 374 mOsm/kg respectively (compared to 7.806 and 432 mOsm/kg in the solvent control).
- Definition of acceptable cells for analysis:
Cells could be scored when they had seperate main nuclei of approximately equal size, when the nuclear boundaries were able to be distinguished or when the main nuclei were linked by nucleoplasmic bridges. Trinucleated, quadranucleated, or multinucleated cells or cells where main nuclei were undergoing apoptosis were not scored.
RANGE-FINDING/SCREENING STUDIES:
In the absence of S9-mix, at the 3 hours exposure time, Carob Bean Extract induced a
statistically significant increase in the number of binucleated cells with micronuclei at dose
levels 1250 and 5000 µg/mL. However, the number of binucleated cells with micronuclei
was within the 95% control limits of the distribution of the historical negative control
database. In addition a negative Cochran Armitage trend test was obtained (p = 0.101).
Therefore, this increase is considered not biologically significant
FIRST CYTOGENETIC ASSAY
All dose levels were selected for scoring:
Without S9-mix:
In the presence of S9-mix, Carob Bean Extract did not induce a statistically significant or
biologically relevant increase in the number of mono- and binucleated cells with micronuclei.
With S9-mix:
In the absence of S9-mix, at the 3 hours exposure time, Carob Bean Extract induced a
statistically significant increase in the number of binucleated cells with micronuclei at dose
levels 1250 and 5000 µg/mL. However, the number of binucleated cells with micronuclei
was within the 95% control limits of the distribution of the historical negative control
database. In addition a negative Cochran Armitage trend test was obtained (p = 0.101).
Therefore, this increase is considered not biologically significant
SECOND CYTOGENETIC ASSAY
All dose levels were selected for scoring:
Without S9-mix:
The test item did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei.
ACCEPTABILITY:
The number of mono- and binucleated cells with micronuclei found in the solvent control was within the 95% control limits of the distribution of the historical negative control
The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei.
The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition colchicine also showed a statistically significant increase in the number of binucleated cells with micronuclei and cyclophosphamide also showed a statistically significant increase in the number of mononucleated cells
In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database.
There can be concluded that the test conditions were adequate and that the metabolic activation system functioned properly.
Any other information on results incl. tables
Tables: See Attached Background material
Applicant's summary and conclusion
- Conclusions:
- An in vitro micronucleus assay in cultured peripheral human lymphocytes, performed according to OECD 487 and GLP principles, showed that Carob Bean Extract is not clastogenic or aneugenic in human lymphocytes
- Executive summary:
An in vitro micronucleus assay in cultured peripheral human lymphocytes with Carob Bean Extract was performed according to OECD 487 and GLP principles. The solvent and positive controls, both with and without metabolic activation, functioned properly within the acceptabillity range. Based on the results it can be concluded that the test conditions were adequate and that the metabolic activation system functioned properly. The dose levels at which the test substance was exposed were 0, 1250, 2500 and 5000 μg/mL. No significant genetoxicity or cytotxicity was observed at all dose levels and therefore it can be concluced that Carob Bean Extract is not clastogenic or aneugenic in human lymphocytes under the experimental conditions.
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