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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro mammalian cell gene mutation assay:

The given test chemical does not give rise to gene mutations when exposed at ≤ 5.0 mM for 3 hrs or more, and it does not give rise to cytotoxicity in CHO cells at concentrations of ≤ 5.0 mM.

Ames assay:

The test chemical did not induce mutation in the Salmonella typhimurium strains both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.

In vitro mammalian chromosome aberration study:

The test chemical did not induce chromosome aberrations in the mammalian cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study report
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
The purpose of this study was to assess toxic and genotoxic effects of test chemical on Chinese Hamster Ovary (CHO) cells by using several different in vitro-based assays, including genotoxicity tests based on the OECD Guideline No. 476 “In Vitro Mammalian Cell Gene Mutation Test”.
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
Cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HPRT) due to the mutation HPRT+/- to HPRT-/- are resistant to cytotoxic effects of 6-thioguanine (TG). HPRT proficient cells are sensitive to TG (which causes inhibition of cellular metabolism and halts further cell division since HPRT enzyme activity is important for DNA synthesis), so mutant cells can proliferate in the presence of TG, while normal cells, containing hypoxanthine-guanine phosphoribosyl transferase cannot. This in vitro test is an assay for the detection of forward gene mutations at the in hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosomes of hypodiploid, modal No. 20, CHO cells. Gene and chromosome mutations are considered as an initial step in the carcinogenic process.The hypodiploid CHO cells are exposed to the test item with and without exogenous metabolic activation. Following an expression time the descendants of the treated cell population are monitored for the loss of functional HPRT enzyme.HPRT catalyses the transformation of the purine analogue 6-thioguanine (TG) and thus rendering the analogue cytotoxic to normal cells. Hence, cells with mutations in the HPRT gene cannot phosphoribosylate the analogue and survive treatment with TG.Therefore, mutated cells are able to proliferate in the presence of TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 days.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Cell line used: Chinese Hamster Ovary (CHO) cells
- Type and identity of media: Ham’s F12K (Kaighn’s) Medium including 2 mM Glutamine supplemented with 10% Foetal Bovine Serum (FBS; qualified, EU-approved, South America origin), penicillin (50 U/ml) and streptomycin (50 µg/ml).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: No detection for mycoplasma was performed
- Periodically checked for karyotype stability: No detection for karyotype stability was performed
Additional strain / cell type characteristics:
other: Hypodiploid, modal No. 20
Cytokinesis block (if used):
Not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats (Supplier: Molecular Toxicology Inc. via Trinova Biochem GmbH, Giessen, Germany)
Test concentrations with justification for top dose:
0, 0.5, 1.0, 2.5 or 5.0 mM
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle:Phenethyl phenylacetate was easily dissolved in ethanol.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
other: N-ethyl-N-nitrosourea (ENU)
Details on test system and experimental conditions:
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- Method: mitotic index; cloning efficiency; relative total growth; other: 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:
Rationale for test conditions:
No data
Evaluation criteria:
The plates were scored for total number of colonies by manual counting. As a result, the mutation frequency could be calculated
Statistics:
Mean were observed.
Species / strain:
Chinese hamster Ovary (CHO)
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
Additional information on results:
No data
Remarks on result:
other: No mutagenic effect were observed.

GENE MUTATION TEST

 

Table 1A.Effect of 5-Decanolide exposure on gene toxicity in CHO cells. After being exposed to the test chemical for 3 hrs, cells was washed with sterile PBS and then incubated for 7 days at 37°C, 5% CO2. After 7 days, cells were re-seeded in new 6-well plates in the absence or presence of 10mM TG as a selection agent and returned to the incubator for 14 days at 37°C, 5% CO2. On day 15, all 6-well plates were stained with crystal violet and the number of colonies were counted manually. The results are presented as the total number of colonies found in the number of independent wells analyzed (e.g. 0 colonies in 4 wells will give 0/4) (n = 2 samples from 2 independent cultures).

 

 

With S9

Without S9

 

with TG

without TG

with TG

without TG

Neg. control

0/4

773/4

0/4

856/4

Pos. control

0/4

788/4

36/4

769/4

0.5 mM

0/4

773/4

0/4

720/4

1.0 mM

0/4

603/4

0/4

747/4

2.5 mM

0/4

666/4

0/4

725/4

5.0 mM

0/4

611/4

0/4

403/4

 

 

 

Table 1B.Mutation frequency in CHO cells after 3 hrs of exposure to 5-Decanolide in the absence or presence of 4% S9 liver microsomal fraction. N/A, no colonies present in the samples selected with TG, i.e. no mutation frequency could be determined.

 

 

With S9

Without S9

Neg. control

N/A

N/A

Pos. control

N/A

4.18x10-4

0.5 mM

N/A

N/A

1.0 mM

N/A

N/A

2.5 mM

N/A

N/A

5.0 mM

N/A

N/A

Conclusions:
Based on the results of the current study, it can be concluded that the given test chemical does not give rise to gene mutations when exposed at ≤ 5.0 mM for 3 hrs or more, and it does not give rise to cytotoxicity in CHO cells at concentrations of ≤ 5.0 mM.
Executive summary:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the given test chemical as per OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test) when administered to Chinese Hamster Ovary (CHO) cells.

A preliminary dose-finding study was conducted prior to the main study. A range of different test chemical concentrations were tested in 96-well plates and analyzed by two commonly used assays, i.e. the colorimetric assay of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the bicinchoninic acid (BCA) assay to assess cell viability and protein concentration, respectively.

From the basis of the results from the MTT and BCA assays, test concentrations of the test chemical was chosen to be included in the gene toxicity test.

In the genotoxicity test, the given test chemical was administered to CHO cells for 3 hrs at the dose levels of 0, 0.5, 1.0, 2.5 or 5.0 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 results showed indication of gene mutations occurring only in the positive control ENU while no other treatment gave rise to gene toxicity. No cytotoxic effects was observed in CHO cells when exposed towards test chemical for 3 hrs in the absence or presence of S9 liver microsomal fraction.

Based on the results of the current study, it can be concluded that the given test chemical does not give rise to gene mutations when exposed at ≤ 5.0 mM for 3 hrs or more, and it does not give rise to cytotoxicity in CHO cells at concentrations of ≤ 5.0 mM.

Endpoint:
in vitro gene mutation study in bacteria
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 summarized based on data from various test chemicals
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: As mentioned below
Principles of method if other than guideline:
WoE for the target CAS is summarized based on data from various test chemicals.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
3. Histidine
4. Histidine
Species / strain / cell type:
other: TA92, TA1535,TA100, TA1537, TA94 and TA98
Remarks:
3
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Remarks:
4
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
3. No data
4. No data
Metabolic activation:
with and without
Metabolic activation system:
3. The liver microsome fraction (S-9) was prepared from the liver of Fischer rats
4. Liver homogenates (S-9 fraction) from Aroclor 1254-induced male Sprague-Dawley rats (RLI) or male Syrian hamsters (HLI)
Test concentrations with justification for top dose:
3. Six different concentrations were used with 10 mg/plate as the maximum concentration.
4. Lab 1: 0, 100, 333, 1000, 3333 or 10000 µg/plate
Lab 2: 0, 100, 333, 1000, 3333 or 10000 µg/plate
Vehicle / solvent:
3. - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The chemical was soluble in DMSO
4. - Vehicle(s)/solvent(s) used: Lab 1: ET95 and Lab 2: DMSO
- Justification for choice of solvent/vehicle: Chemical solubility in respective solvents mentioned
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Remarks:
3
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Lab 1: ET95 and Lab 2: DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 4-nitro-o-phenylenediamine (for TA98; without S9) and 2-amino-anthracene (2 AA; with S9)
Remarks:
4
Details on test system and experimental conditions:
3. METHOD OF APPLICATION: Preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs

NUMBER OF REPLICATIONS: Duplicate
4. METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs

NUMBER OF REPLICATIONS: All tests were repeated atleast once
Rationale for test conditions:
3. No data
4. No data
Evaluation criteria:
3. The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated).
4. A mutagenic response was defined as a reproducible, dose-related increase in the number of histidine-independent colonies over the spontaneous incidence; there was no requirement for a specific magnitude of increase.
Statistics:
3. No data
4. No data
Species / strain:
other: TA92, TA1535,TA100, TA1537, TA94 and TA98
Remarks:
3
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
other: TA100, TA1535, TA98, TA1537
Remarks:
4
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
3. The maximum dose for negative results represents the highest non-cytotoxic dose used in the experiment.
4. RANGE-FINDING/SCREENING STUDIES: The final dose level selection was based on the results of a preliminary range-finding study conducted with TA100 in the presence and absence of S-9.
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical did not induce mutation in the Salmonella typhimurium strains both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.
Executive summary:

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned below:

 

A bacterial reverse mutation test was performed to determine the mutagenic nature of the test substance. The study was performed using Salmonella typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 with and without S9 metabolic activation system. The test was performed as a preincubation assay at six different concentrations with 10 mg/plate being the maximum concentration. Cells were pre-incubated with both the test chemical and S9-mix for 20 min. The number of revertant (His+) colonies was scored after 48 hours of incubation. The result was considered positive if the number of revertant colonies was found twice the number of the controls (exposed to the appropriate solvent or untreated). The test chemical did not induce a doubling of revertant colonies over the control using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 in the presence and absence of S9 metabolic activation system. Hence, it is considered as Non-mutagenic (negative) in vitro in the test system used under the certain experimental conditions.

 

In another study, the bacterial reverse mutation assay was performed for the test chemical using Salmonella typhimurium strains TA100, TA1535, TA98, TA1537 with and without rat and hamster liver S9 mix. The test chemical was dissolved in ET95 (lab 1) and DMSO (lab 2). The study was performed using the preincubation protocol at five dose levels of 0, 100, 333, 1000, 3333 or 10000 µg/plate (lab 1 and 2) with incubation period of 48 hrs in the presence and absence of S9 mix. The final dose level selection was based on the results of a preliminary range-finding study conducted with TA100 in the presence and absence of S-9. No mutagenic response was noted for the test compound in the preliminary dose range finding study and the main study performed. The given test chemical did not induce mutation in the Salmonella typhimurium strain TA100, TA1535, TA98, TA1537 with and without rat and hamster liver S9 mix in both lab 1 and lab 2 studies and hence is negative for gene mutation in vitro.

 

Thus, based on the above summarized studies on test chemical, it can be concluded that the given test chemical did not induce mutation in the Salmonella typhimurium strains both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.

Endpoint:
in vitro cytogenicity / chromosome aberration 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 summarized based on the various test chemicals.
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: As mentioned below
Principles of method if other than guideline:
WoE for the target CAS is summarized based on data from various test chemicals.
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
6. No data
7. No data
Species / strain / cell type:
other: Chinese hamster fibroblast cell line
Remarks:
6
Details on mammalian cell type (if applicable):
- Type and identity of media: Minimum Essential Medium (MEM; GIBCO) supplemented by 10% calf serum.
- Properly maintained: Yes, by 4 day passages
- Periodically checked for Mycoplasma contamination: No data available
- Periodically checked for karyotype stability: No data available
- Periodically "cleansed" against high spontaneous background: No data available
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
other: Chinese hamster fibroblast cell line CHL
Remarks:
7
Details on mammalian cell type (if applicable):
- Type and identity of media: Minimum
Essential Medium (MEM; GIBCO) supplemented by 10% calf serum
- Properly maintained: yes by 4 day passages
- Periodically checked for Mycoplasma contamination: No data available
- Periodically checked for karyotype stability: No data available
- Periodically "cleansed" against high spontaneous background: No data available
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
6. No data
7. No data
Metabolic activation:
without
Metabolic activation system:
6. not specified
7. No metabolic activation system was used
Test concentrations with justification for top dose:
6. At three different doses with 2 mg/mL being the maximum dose concentration.
7. At three different doses with 0.125 mg/mL being the maximum dose concentration
Vehicle / solvent:
6. - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The chemical was soluble in DMSO
7. - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The chemical was soluble in DMSO
Untreated negative controls:
yes
Remarks:
Untreated cells served as negative controls.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Remarks:
6
Untreated negative controls:
yes
Remarks:
Untreated cells served as negative control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Remarks:
7
Details on test system and experimental conditions:
6. METHOD OF APPLICATION: In medium

DURATION
- Preincubation period: No data available
- 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): Giemsa solution (1.5%, pH 6.8)
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: No data available

NUMBER OF CELLS EVALUATED: 100 well spread metaphases

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

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: No data available
- Other: No data available
7. METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data
SELECTION AGENT (mutation assays): Giemsa solution (1.5%, pH 6.8)
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: 100 well spread metaphases

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

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

OTHER: No data
Rationale for test conditions:
6. No data
7. No data
Evaluation criteria:
6. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. In which the incidence of aberrations was usually less than 3.0%. The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%,and positive if it was more than 10.0%.
7. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%.
Statistics:
6. No data
7. No data
Species / strain:
other: Chinese hamster fibroblast cell line
Remarks:
6
Metabolic activation:
not applicable
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
other: Chinese hamster fibroblast cell line CHL
Remarks:
7
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
6. RANGE-FINDING/SCREENING STUDIES: The maximum dose of each sample was selected by a preliminary test in which the dose needed for 50% cell-growth inhibition was estimated using a cell densitometer.
7. RANGE-FINDING/SCREENING STUDIES: The maximum dose of each sample was selected by a preliminary test in which the dose needed or 50% cell-growth inhibition was estimated using a cell densitometer
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical did not induce chromosome aberrations in the mammalian cell line in the absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.
Executive summary:

In different studies, the given test chemical has been investigated for the mutagenic nature. The studies are as mentioned below:

 

An in vitro mammalian chromosomal aberration study was performed to determine the mutagenic nature of test chemical. The cells were exposed to the test material at three different doses with 2 mg/ml being the maximum concentration for 48 hr. Colcemid (0.2 µg/ml in final concentration) was added to the culture 2 hrs before cell harvesting. The slides were stained with Giemsa solution for 12-15 min. A hundred well-spread metaphases were observed under the microscope. In the present studies, no metabolic activation (liver microsome solution, S9 mix) systems were applied. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others were recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%. The results were considered to be negative if the incidence was less than 4.9 %, equivocal if it was between 5.0 and 9.9 %, and positive if it was more than 10.0 %. The results showed that the given test chemical did not give a rise of the number of cells with chromosome aberrations and/or induce polyploid cells when CHL cells were exposed to 2 mg/ml of test chemical in the absence of S9-mix. Thus, it is considered as Non-mutagenic (negative) when CHL cells are exposed to the test chemical without metabolic activation under the presented experimental conditions.

 

In another study, chromosomal aberration study was performed to determine the mutagenic nature of the test chemical. The cells were exposed to the test material at three different doses with 0.125 mg/mL being the maximum concentration for 48hr. Colcemid (final concn 0.2 µg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution for 12-15 min. A hundred well-spread metaphases were observed under the microscope. In the present studies, no metabolic activation systems were applied. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%. The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%. The test chemical did not induce chromosomal aberration in Chinese hamster fibroblast cell line CHL and hence it is not likely to classify as a gene mutant in vitro.

 

Thus, based on the above summarized studies on test chemical, it can be concluded that the given test chemical did not induce chromosome aberrations in the mammalian cell line in the presence and absence of S9 metabolic activation system and hence it is not mutagenic in the chromosome aberration study performed.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Gene mutation in vitro:

Data available from various sources was reviewed to determine the mutagenic nature of the given test chemical. The studies are as mentioned below:

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 the given test chemical as per OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test) when administered to Chinese Hamster Ovary (CHO) cells.

A preliminary dose-finding study was conducted prior to the main study. A range of different test chemical concentrations were tested in 96-well plates and analyzed by two commonly used assays, i.e. the colorimetric assay of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the bicinchoninic acid (BCA) assay to assess cell viability and protein concentration, respectively.

From the basis of the results from the MTT and BCA assays, test concentrations of the test chemical was chosen to be included in the gene toxicity test.

In the genotoxicity test, the given test chemical was administered to CHO cells for 3 hrs at the dose levels of 0, 0.5, 1.0, 2.5 or 5.0 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 results showed indication of gene mutations occurring only in the positive control ENU while no other treatment gave rise to gene toxicity. No cytotoxic effects was observed in CHO cells when exposed towards test chemical for 3 hrs in the absence or presence of S9 liver microsomal fraction.

Based on the results of the current study, it can be concluded that the given test chemical does not give rise to gene mutations when exposed at ≤ 5.0 mM for 3 hrs or more, and it does not give rise to cytotoxicity in CHO cells at concentrations of ≤ 5.0 mM.

 

Ames assay:

A bacterial reverse mutation test was performed to determine the mutagenic nature of the test substance. The study was performed using Salmonella typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 with and without S9 metabolic activation system. The test was performed as a preincubation assay at six different concentrations with 10 mg/plate being the maximum concentration. Cells were pre-incubated with both the test chemical and S9-mix for 20 min. The number of revertant (His+) colonies was scored after 48 hours of incubation. The result was considered positive if the number of revertant colonies was found twice the number of the controls (exposed to the appropriate solvent or untreated). The test chemical did not induce a doubling of revertant colonies over the control using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 in the presence and absence of S9 metabolic activation system. Hence, it is considered as Non-mutagenic (negative) in vitro in the test system used under the certain experimental conditions.

 

In another study, the bacterial reverse mutation assay was performed for the test chemical using Salmonella typhimurium strains TA100, TA1535, TA98, TA1537 with and without rat and hamster liver S9 mix. The test chemical was dissolved in ET95 (lab 1) and DMSO (lab 2). The study was performed using the preincubation protocol at five dose levels of 0, 100, 333, 1000, 3333 or 10000 µg/plate (lab 1 and 2) with incubation period of 48 hrs in the presence and absence of S9 mix. The final dose level selection was based on the results of a preliminary range-finding study conducted with TA100 in the presence and absence of S-9. No mutagenic response was noted for the test compound in the preliminary dose range finding study and the main study performed. The given test chemical did not induce mutation in the Salmonella typhimurium strain TA100, TA1535, TA98, TA1537 with and without rat and hamster liver S9 mix in both lab 1 and lab 2 studies and hence is negative for gene mutation in vitro.

 

 

In vitro mammalian chromosome aberration study:

An in vitro mammalian chromosomal aberration study was performed to determine the mutagenic nature of test chemical. The cells were exposed to the test material at three different doses with 2 mg/ml being the maximum concentration for 48 hr. Colcemid (0.2 µg/ml in final concentration) was added to the culture 2 hrs before cell harvesting. The slides were stained with Giemsa solution for 12-15 min. A hundred well-spread metaphases were observed under the microscope. In the present studies, no metabolic activation (liver microsome solution, S9 mix) systems were applied. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others were recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%. The results were considered to be negative if the incidence was less than 4.9 %, equivocal if it was between 5.0 and 9.9 %, and positive if it was more than 10.0 %. The results showed that the given test chemical did not give a rise of the number of cells with chromosome aberrations and/or induce polyploid cells when CHL cells were exposed to 2 mg/ml of test chemical in the absence of S9-mix. Thus, it is considered as Non-mutagenic (negative) when CHL cells are exposed to the test chemical without metabolic activation under the presented experimental conditions.

 

In another study, chromosomal aberration study was performed to determine the mutagenic nature of the test chemical. The cells were exposed to the test material at three different doses with 0.125 mg/mL being the maximum concentration for 48hr. Colcemid (final concn 0.2 µg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution for 12-15 min. A hundred well-spread metaphases were observed under the microscope. In the present studies, no metabolic activation systems were applied. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%. The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%. The test chemical did not induce chromosomal aberration in Chinese hamster fibroblast cell line CHL and hence it is not likely to classify as a gene mutant in vitro.

 

Justification for classification or non-classification

Based on the data available and applying weight of evidence approach, the given test chemical does not exhibit gene mutation in vitro by Ames assay, In vitro mammalian chromosome aberration study and In vitro mammalian cell gene mutation assay. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.