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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

AMES assay:

Test chemical failed to induce mutation in bacterial cells both in the presence and absence of metabolic activationa.Hence is negative for mutation in vitro.

In vitro mammalian chromosomal aberrations assay:

In the study test chemical was assessed for its possible mutagenic potential. For this purpose in vitro mammalian chromosome aberration test was performed on Mammalian cells. The test chemical was exposed to Chinese hamster Ovary (CHO). No mutagenic effects were observed in the study. Therefore the test chemical was considered to be non mutagenic in Chinese hamster Ovary (CHO) by purpose in vitro mammalian chromosome aberration test. Hence the substance can be classified as non mutagenic in vitro.

In vitro mammalian cell gene mutation assay:

The test chemical did not induce gene mutation in mammalian cell gene mutation assay in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
data from handbook or collection of data
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
other: Refer below principle
Principles of method if other than guideline:
Weight of evidence prepared from various below mention studies
1. Bacterial reverse mutation assay was performed to evaluate the mutagenic nature of the test compound Dibutyltin dilaurate
2. To evaluate the mutagenic potential of Docosanoic acid in Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA by AMES test.
GLP compliance:
not specified
Type of assay:
bacterial gene mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium, other: TA98, TA100, TA1535, and TA97
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
bacteria, other: Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
not specified
Metabolic activation:
with and without
Metabolic activation system:
The S-9 fractions of Aroclor 1254-induced, male Sprague-Dawley rat and male Syrian hamster livers were prepared
Test concentrations with justification for top dose:
1. 0, 1, 3, 10, 33, 100, 333, 166 µg/plate
2. 0, 156, 313, 625, 1250, 2500, 5000µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Distilled Water
- Justification for choice of solvent/vehicle: The chemical was soluble and stable in water
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
H2O
True negative controls:
not specified
Positive controls:
yes
Remarks:
For all strains
Positive control substance:
other: 2-Aminoanthracene (2-AA)
Remarks:
With S9
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
H2O
True negative controls:
not specified
Positive controls:
yes
Remarks:
TA 100/TA 1535
Positive control substance:
sodium azide
Remarks:
Without S9
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
H2O
True negative controls:
not specified
Positive controls:
yes
Remarks:
TA 1537/ TA97
Positive control substance:
9-aminoacridine
Remarks:
Without S9
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Distilled water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: -S9 mix; 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide(TA100, TA98, WP2uvrA) , Sodium azide(TA1535) and 9-Aminoacridine(TA1537) +S9 mix; 2-Aminoanthracene(all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: Triplicate

NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data available

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other:

OTHER: No data available
Rationale for test conditions:
No data
Evaluation criteria:
An individual trial was judged mutagenic (+) if a dose-related increase over the corresponding solvent control was seen, and it was judged weakly mutagenic C+W) if a low-level dose response was seen. A trial was considered questionable (?) if a dose-related increase was judged insufficiently high to justify a call of "+W," if only a single dose was elevated over the control, or if a non-dose-related increase was seen.

The chemical was judged to be mutagenic (+), or weakly mutagenic (+W), if it produced a reproducible, dose-related increase in his+ revertants over the corresponding solvent controls in replicate trials.

It chemical was considered to be questionable (?) if a reproducible increase of his+ revertants did not meet the criteria for either a " + " or " + W," or if only single doses produced an increase in his+ revertants in repeat trials.
Statistics:
No data available
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, and TA97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
bacteria, other: Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data available
- Effects of osmolality: No data available
- Evaporation from medium: No data available
- Water solubility: No data available
- Precipitation: No data available
- Other confounding effects: No data available

RANGE-FINDING/SCREENING STUDIES: The chemical was tested initially in a toxicity assay to determine the appropriate dose range. The toxicity assay was performed by using TA100 or the system developed by Waleh et al. Toxic concentrations were those at which a decrease in the number of his+ colonies was seen or at which there was a clearing in the density of the background lawn

COMPARISON WITH HISTORICAL CONTROL DATA: No data available

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data available
Remarks on result:
other: No mutagenic potential
Conclusions:
Test chemical did not induce gene mutation in bacterial cells and hence is negative for mutation in vitro.
Executive summary:

Data for the various test chemicals was reviewed to determine the mutagenic nature of Castor oil, ethoxylated (61791-12-6) .The studies are as mentioned below:

Salmonella/microsome test in the absence of exogenous metabolic activation and in the presence of liver S-9 from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters was performed to evaluate the mutagenic nature of test chemical using S. typhimurium tester strains TA1535, TA97, TA98 and TA100. The study was performed as per the preincubation assay. The test compound was used at a dosage level of 0, 1, 3, 10, 33, 100, 333, 166 µg/plate in the preincubation assay of 48 hrs. Test chemical failed to induce mutation in the S. typhimurium tester strains TA1535, TA97, TA98 and TA100 and hence is negative for mutation in vitro.

Supported by other study in bacterial cell. Gene mutation toxicity study was performed to determine the mutagenic nature of test chemical. Pre-incubation method was performed to determine its mutagenic nature. Test chemical did not induce gene mutations in the S. typhimurium TA100, TA1535, TA98, TA1537, E. coli WP2 uvrA in a bacterial reverse mutation assay. The assay was performed in the presence and absence of S9 metabolic activation system at dose levels of 0, 156, 313, 625, 1250, 2500, 5000 µg /plate. Test chemical did not induce gene mutations in the S. typhimurium strains TA100, TA1535, TA98, TA1537 and E. coli WP2 uvrA in a bacterial reverse mutation assay in the presence and absence of S9 metabolic activation system up to a concentration of 5000 µg/plate and hence is not likely to classify for gene mutation in vitro.

Based on the data available for the test chemicals, the test chemical did not induce gene mutation in bacterial cells in the presence and absence of S9 metabolic activation system and hence is negative for mutation in vitro.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from publication.
Qualifier:
according to guideline
Guideline:
other: Refer below principle
Principles of method if other than guideline:
To evaluate the mutagenic potential of test substance in mammalian cell by in vitro mammalian chromosome aberration test.
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
No data
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
No data
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
not specified
Metabolic activation system:
no data
Test concentrations with justification for top dose:
Not specified
Vehicle / solvent:
No data
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
Not specified
Rationale for test conditions:
No data
Evaluation criteria:
No data
Statistics:
No data
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
not specified
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
No data
Remarks on result:
other: No mutagenic effects were observed
Conclusions:
The Chromosomal aberration test for the test chemical in Chinese hamster Ovary (CHO) shows negative results.
Executive summary:

In the study test chemical was assessed for its possible mutagenic potential. For this purpose in vitro mammalian chromosome aberration test was performed on Mammalian cells. The test chemical was exposed to Chinese hamster Ovary (CHO). No mutagenic effects were observed in the study. Therefore the test chemical was considered to be non mutagenic in Chinese hamster Ovary (CHO) by purpose in vitro mammalian chromosome aberration test. Hence the substance can be classified as non mutagenic in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Experimental data from various test chemicals
Justification for type of information:
Data for the target chemical is from various test chemicals
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1
Principles of method if other than guideline:
WoE derived based on the experimental data from various test chemicals
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
1. HPRT
2. TK
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
1
Details on mammalian cell type (if applicable):
- Cell line used: Chinese Hamster Ovary (CHO) cells
- Type and identity of media: Ham's F-12K (Kaighn's) Medium containing 2 mM L-Glutamine supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (10,000 U/mL).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Not applicable
- Periodically checked for karyotype stability: Not applicable
Additional strain / cell type characteristics:
other: Hypodiploid, modal No. 20
Species / strain / cell type:
mouse lymphoma L5178Y cells
Remarks:
clone 3.7.2C / 2
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 or Fischer’s medium. Growth medium consisted of Fischer’s medium supplemented with heat-treated horse serum (10% viv), 110 µg/ml sodium pyruvate, 2 mM L-glutamine, 0.05% Pluronic F68
(nonionic surfactant), and pen-strep (95 U/ml, 95 µg/ml). Treatment medium was always Fischer’s growth medium with the serum content reduced to 5% by volume
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes, the cell line was free from mycoplasma as determined every 2-3 months by cultivating techniques
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: No data
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
1. 0, 1, 2.5, 5 or 10 mM
2. Without S9: 25-30 µg/mL
With S9: 30-40 µg/mL
Vehicle / solvent:
1. - Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/ vehicle: Test chemical was dissolved in ethanol.

2. - Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: Tes chemical solubility in water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
other: N-ethyl-N-nitrosourea (ENU) (Without S9)
Remarks:
1
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
other: MCA (With S9)
Details on test system and experimental conditions:
1. METHOD OF APPLICATION: In medium with pre-incubation

DURATION
- Preincubation period: One week involving 3 days of incubation with Hypoxanthine-aminopterin-thymidine (HAT) in medium as a mutant cleansing stage, followed by overnight incubation with hypoxanthine-thymidine (HT) in medium prior to a 3-4 days incubation in regular cell medium. After seeding and prior to treatment, the mutant-free cells were incubated for an additional of 24 hours.
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 7 days (harvest of cells)

SELECTION AGENT (mutation assays): 6-thioguanine (TG)
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): Crystal violet

NUMBER OF REPLICATIONS: A minimum of 2 replicates per dose concentration including negative and positive control.

NUMBER OF CELLS EVALUATED: 5 x 10 E5 cells were plated 7 days after treatment and whatever cells left, after 14 days of incubation with the selection medium, were evaluated.

DETERMINATION OF CYTOTOXICITY
- Cytotoxicity test
After being exposed to the test chemical for 3 hours, in the absence or presence of S9, cells were trypsinized and 0.5 x 10 E5 cells per well was seeded in duplicates from two parallel duplicate cultures into 6-well plates in fresh medium. The relative total growth and cytotoxicity was evaluated 24 and 48 hours after seeding.

OTHER EXAMINATIONS: Not applicable

2. METHOD OF APPLICATION: in medium
Cells at start of experiment: 6000000 cells

DURATION
- Preincubation period: No data
- Exposure duration: 4 hrs
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): 11-12 days

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Triplicate

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Relative total growth was determined (RTG)

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data
Rationale for test conditions:
No data
Evaluation criteria:
1. Dose dependent increase in the gene mutation activity
2. The cell line was observed for gene mutation
Statistics:
No data
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
1
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
1
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
5 mM or less
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not valid
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
1
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
At 10 mM
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
No data
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical did not induce gene mutation in mammalian cell gene mutation assay in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.
Executive summary:

Data available for the various test chemicals was reviewed to determine the mutagenic nature of the test chemical. The tsudies are as mentioned below:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of test chemical when administered to Chinese Hamster Ovary (CHO) cells. In the genotoxicity test, zinc distearate was administered to CHO cells for 3 hrs at the dose levels of 0, 1.0, 2.5, 5.0 or 10.0 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such as N-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. The positive control ENU gave a clear indication of gene mutations occurring while no other treatment gave rise to gene toxicity. Two very diffuse colonies were seen in one well out of four for the negative control in the presence with 4% S9 liver microsomal fraction. These diffuse colonies are not regarded to be relevant since the two spots were only mildly colored by crystal violet, thus indicating that it were small clusters of apoptotic cells taking their last breath instead of cells surviving the TG-selection. The presence of several colonies were observed in cells treated with 10.0 mM in the presence with S9 liver microsomal fraction. When the mutation frequency was determined, a frequency of 3.43 x 10-4was shown after a 3 hour exposure of ENU as the positive control and in the absence of S9 liver microsomal fraction, while exposure to 10.0 mM zinc stearate in the presence of S9 liver microsomal fraction showed a frequency of 5.13 x 10-4. The results indicate that the test chemical does not give rise to gene mutations when used at concentrations of ≤ 5.0 mM, but when exposed to concentrations above 5.0 mM for 3 hrs, and in organisms with a fully functioning metabolic activation, the test chemical may induce gene mutations. When treated with test chemical and in the absence with S9 liver microsomal fraction, the results show no evidence of genotoxicity when CHO cells are exposed to concentrations of 1.0, 2.5, 5.0 or 10.0 mM, thus indicating that the test chemical does not give rise to gene mutations in organisms who have no or non-functional metabolic activation at the above mentioned concentrations. With S9 metabolic activation

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to test chemical in the concentration of 0, 1, 2.5, 5 or 10 mM and S9-induced metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity after treatment with test chemical. The results showed evidence of gene toxicity when cells were exposed to test chemical at 10 mM, while treatment with 5 mM or less did not cause gene toxicity. Therefore, it is considered that test chemical in the concentrations of 5 mM or below does not cause genetic mutation(s), while concentrations at 10 mM may, when CHO cells are exposed to the test chemical in the presence of metabolic activation. In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to test chemical in the concentration of 0, 1, 2.5, 5 or 10 mM and without metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity after treatment with the test chemical. Independently of treatment concentration of test chemical, the results showed no evidence of potential gene toxicity. Therefore, it is considered that test chemical in the concentration of 0, 1, 2.5, 5 or 10 mM does not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the absence of metabolic activation.

In vitro mammalian cell gene mutation test was performed to determine the mutagenic nature of the test chemical. The study was performed using L5178Y mouse lymphoma cells, clone 3.7.2C in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in water and used at dose level of 25-30µg/mL without S9 and 30-40µg/mL with S9. Cells from THMG-treated stock cultures were seeded at 6000000 cells into 50-ml centrifuge tubes and were resuspended in Fischer's treatment medium. The final volumes were 10 ml after adding S9 mix in activation experiments (or additional medium in nonactivation experiments) and the test chemical solution. The tubes were closed tightly and placed on a roller drum for 4 hr at 1&15 rpm at 37°C for the treatment period. After treatment, the cells were centrifuged at low speed (approximately 1508 for 8-10 min), and the supernatant treatment medium was removed. Each culture was washed twice by resuspension and centrifugation in fresh growth medium. The cells were then resuspended in 20 ml of Fischer's or RPMI 1640 growth medium to obtain densities of 300000 cells/ml, and the cultures were returned to the roller drum for a 2-day expression and growth period. Viable cell densities were determined by hemacytometer each day using trypan blue dye exclusion. If the density exceeded 400000 cells/ml after day 1, the culture was diluted to 300000 cells/ml with fresh growth medium (maintaining the 20 ml volume). On day 2, the cultures showing an increase in cell density were diluted to 300000 cells/ml, and samples were cloned in soft agar medium. A 10 ml sample containing 300000 cells was added to a flask containing 90 ml cloning medium and 3 Fg/ml TFT. The contents were mixed by swirling and were poured into three 100-mm dishes such that each dish contained approximately 1000000 cells in 33 ml medium. The CE was determined by serially diluting a 1 ml sample from each culture and seeding 600 cells into a flask containing 100 ml of cloning medium. This flask was emptied into three 100-mm dishes to obtain approximately 200 cells/dish in 33 ml of medium. The dishes were incubated for 11-12 days at 37°C with 5% CO2/humidified air to allow colony development. Based on the observations made, thetest chemical did not induce gene mutation inL5178Y mouse lymphoma cells, clone 3.7.2C in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Based on the data available for the test chemical, the test chemical did not induce gene mutation in mammalian cell gene mutation assay in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Data for the various test chemicals was reviewed to determine the mutagenic nature of Castor oil, ethoxylated (61791-12-6). The studies are as mentioned below:

 

AMES assay:

Salmonella/microsome test in the absence of exogenous metabolic activation and in the presence of liver S-9 from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters was performed to evaluate the mutagenic nature of test chemical using S. typhimurium tester strains TA1535, TA97, TA98 and TA100. The study was performed as per the preincubation assay. The test compound was used at a dosage level of 0, 1, 3, 10, 33, 100, 333, 166 µg/plate in the preincubation assay of 48 hrs. Test chemical failed to induce mutation in the S. typhimurium tester strains TA1535, TA97, TA98 and TA100 and hence is negative for mutation in vitro.

 

Supported by other study in bacterial system, gene mutation toxicity study was performed to determine the mutagenic nature of test chemical. Pre-incubation method was performed to determine its mutagenic nature. Test chemical did not induce gene mutations in the S. typhimurium TA100, TA1535, TA98, TA1537, E. coli WP2 uvrA in a bacterial reverse mutation assay. The assay was performed in the presence and absence of S9 metabolic activation system at dose levels of 0, 156, 313, 625, 1250, 2500, 5000 µg /plate. Test chemical did not induce gene mutations in the S. typhimurium strains TA100, TA1535, TA98, TA1537 and E. coli WP2 uvrA in a bacterial reverse mutation assay in the presence and absence of S9 metabolic activation system up to a concentration of 5000 µg/plate and hence is not likely to classify for gene mutation in vitro.

 

In vitro mammalian chromosomal aberrations assay:

In the study test chemical was assessed for its possible mutagenic potential. For this purpose in vitro mammalian chromosome aberration test was performed on Mammalian cells. The test chemical was exposed to Chinese hamster Ovary (CHO). No mutagenic effects were observed in the study. Therefore the test chemical was considered to be non mutagenic in Chinese hamster Ovary (CHO) by purpose in vitro mammalian chromosome aberration test. Hence the substance can be classified as non mutagenic in vitro.

In another study, genetic toxicity in vitro was assessed for test chemical. For this purpose in vitro mammalian chromosome aberration test was performed according to OECD 473 and Guidelines for Screening Mutagenicity Testing of Chemicals (Japan).The test material was exposed to Chinese hamster lung (CHL)cells in the presence and absence of metabolic activation S9. The concentration of test material used in the presence and absence of metabolic activation were mention below: -S9 mix(24hr continuous exposure): 0, 350, 700, 1400, 2800 µg/mL, -S9 mix(48hr continuous exposure): 0, 288, 575, 1150, 2300 µg/mL, -S9 mix(short-term exposure): 0, 875, 1750, 3500 µg/mL, +S9 mix(short-term exposure): 0, 875, 1750, 3500 µg/mL. No chromosomal abbreviation, gaps were observed in cells, in the presence and absence of metabolic activation. Therefore test chemical was considered to be non-mutagenic in Chinese hamster lung (CHL)cells by in vitro mammalian chromosome aberration test. Hence the substance cannot be classified as gene mutant in vitro.

Supported by another study, chromosome aberration study was performed to evaluate the mutagenic nature of the test chemical. The study was performed using cultured Chinese hamster lung fibroblast (CHL cells) at seven dose levels, four soluble concentrations 0.13, 0.25, 0.5, 1.0 ug/mL and three insoluble concentrations 5, 10, 20 ug/mL in the presence and absence of a metabolic activation system (rat liver S9 mix).CHL cells were treated with the test substance for 6 hours, changed to fresh culture medium, and cultured for total 24 hours. In the presence of S9 mix, the frequency of structural chromosome aberrations was not significantly induced at the three concentrations, 0.5, 1.0, 2.0 ug/mL, and similarly, in the absence of S9 mix, the frequency of aberrant cells was not induced at the three concentrations, 0.5, 1.0, 2.0 ug/mL (p>0.05, each). In addition, treating with the test substance at 0.5, 1.0, 2.0 ug/mL for 24 hours in the absence of S9 mix, a significant induction was not detected in regard to the frequency of aberrant cells (p>0.05). The frequency of chromosome aberration by the vehicle control was found to be within a normal range for the acceptability of the test, the positive control substances, cyclophosphamide (with S9 mix) and mitomycin C (without S9 mix) induced statistically significant chromosomal aberrations under each condition (p<0.001 each) and hence is not likely to classify as a gene mutant in vitro. Based on the observations made, the test chemical did not showe any evidence of clastogenic activity in mammalian cell culture system in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

In vitro mammalian cell gene mutation assay:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of test chemical when administered to Chinese Hamster Ovary (CHO) cells. In the genotoxicity test, zinc distearate was administered to CHO cells for 3 hrs at the dose levels of 0, 1.0, 2.5, 5.0 or 10.0 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such as N-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test. The positive control ENU gave a clear indication of gene mutations occurring while no other treatment gave rise to gene toxicity. Two very diffuse colonies were seen in one well out of four for the negative control in the presence with 4% S9 liver microsomal fraction. These diffuse colonies are not regarded to be relevant since the two spots were only mildly colored by crystal violet, thus indicating that it were small clusters of apoptotic cells taking their last breath instead of cells surviving the TG-selection. The presence of several colonies were observed in cells treated with 10.0 mM in the presence with S9 liver microsomal fraction. When the mutation frequency was determined, a frequency of 3.43 x 10-4was shown after a 3 hour exposure of ENU as the positive control and in the absence of S9 liver microsomal fraction, while exposure to 10.0 mM zinc stearate in the presence of S9 liver microsomal fraction showed a frequency of 5.13 x 10-4. The results indicate that the test chemical does not give rise to gene mutations when used at concentrations of ≤ 5.0 mM, but when exposed to concentrations above 5.0 mM for 3 hrs, and in organisms with a fully functioning metabolic activation, the test chemical may induce gene mutations. When treated with test chemical and in the absence with S9 liver microsomal fraction, the results show no evidence of genotoxicity when CHO cells are exposed to concentrations of 1.0, 2.5, 5.0 or 10.0 mM, thus indicating that the test chemical does not give rise to gene mutations in organisms who have no or non-functional metabolic activation at the above mentioned concentrations. With S9 metabolic activation

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to test chemical in the concentration of 0, 1, 2.5, 5 or 10 mM and S9-induced metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity after treatment with test chemical. The results showed evidence of gene toxicity when cells were exposed to test chemical at 10 mM, while treatment with 5 mM or less did not cause gene toxicity. Therefore, it is considered that test chemical in the concentrations of 5 mM or below does not cause genetic mutation(s), while concentrations at 10 mM may, when CHO cells are exposed to the test chemical in the presence of metabolic activation. In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to test chemical in the concentration of 0, 1, 2.5, 5 or 10 mM and without metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity after treatment with the test chemical. Independently of treatment concentration of test chemical, the results showed no evidence of potential gene toxicity. Therefore, it is considered that test chemical in the concentration of 0, 1, 2.5, 5 or 10 mM does not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the absence of metabolic activation.

In vitro mammalian cell gene mutation test was performed to determine the mutagenic nature of the test chemical. The study was performed using L5178Y mouse lymphoma cells, clone 3.7.2C in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in water and used at dose level of 25-30µg/mL without S9 and 30-40µg/mL with S9. Cells from THMG-treated stock cultures were seeded at 6000000 cells into 50-ml centrifuge tubes and were resuspended in Fischer's treatment medium. The final volumes were 10 ml after adding S9 mix in activation experiments (or additional medium in nonactivation experiments) and the test chemical solution. The tubes were closed tightly and placed on a roller drum for 4 hr at 1&15 rpm at 37°C for the treatment period. After treatment, the cells were centrifuged at low speed (approximately 1508 for 8-10 min), and the supernatant treatment medium was removed. Each culture was washed twice by resuspension and centrifugation in fresh growth medium. The cells were then resuspended in 20 ml of Fischer's or RPMI 1640 growth medium to obtain densities of 300000 cells/ml, and the cultures were returned to the roller drum for a 2-day expression and growth period. Viable cell densities were determined by hemacytometer each day using trypan blue dye exclusion. If the density exceeded 400000 cells/ml after day 1, the culture was diluted to 300000 cells/ml with fresh growth medium (maintaining the 20 ml volume). On day 2, the cultures showing an increase in cell density were diluted to 300000 cells/ml, and samples were cloned in soft agar medium. A 10 ml sample containing 300000 cells was added to a flask containing 90 ml cloning medium and 3 Fg/ml TFT. The contents were mixed by swirling and were poured into three 100-mm dishes such that each dish contained approximately 1000000 cells in 33 ml medium. The CE was determined by serially diluting a 1 ml sample from each culture and seeding 600 cells into a flask containing 100 ml of cloning medium. This flask was emptied into three 100-mm dishes to obtain approximately 200 cells/dish in 33 ml of medium. The dishes were incubated for 11-12 days at 37°C with 5% CO2/humidified air to allow colony development. Based on the observations made, thetest chemical did not induce gene mutation inL5178Y mouse lymphoma cells, clone 3.7.2C in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Based on the data summarized and applying the weight of evidence approach, Castor oil, ethoxylated (61791-12-6) did not induce gene mutation in vitro.Hence it is not likely to be mutagenic in vitro.

Justification for classification or non-classification

Based on the data summarized and applying the weight of evidence approach, Castor oil, ethoxylated (61791-12-6) did not induce gene mutation in vitro.Hence it is not likely to be mutagenic in vitro.