Dodecan-5-olide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

The test chemical did not induce mutation in the Salmonella typhimurium and Escherichia coli 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.

In vitro mammalian cell gene mutation study:

The test chemical did not induce mutation in the mouse lymphoma L5178 and Chinese hamster Ovary (CHO) Cells 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.

Link to relevant study records

Reference

Reference 1

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 for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

2. Histidine
3. Histidine for Salmonella typhimurium and tryptophan Escherichia coli

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Remarks:

2

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

bacteria, other: S. typhimurium TA100, TA1535, TA98, TA1537, E. coli WP2 uvrA

Remarks:

3

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

2. No data
3. not specified

Metabolic activation:

with and without

Metabolic activation system:

2. rat liver S9
3. Rat liver, induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

2. 0, 33, 100, 1000, 2500, or 5000 µg/plate
3.-S9 mix; 0, 156, 313, 625, 1250, 2500 and 5000 ug/plate
+S9 mix; 0, 156, 313, 625, 1250, 2500 and 5000 ug/plate

Vehicle / solvent:

2. Ethanol
3. - Vehicle(s)/solvent(s) used: [none; DMSO
- Justification for choice of solvent/vehicle: The test substance is soluble in DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

not specified

Remarks:

2

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

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 and WP2 uvrA), Sodium azide(TA1535), 9-Aminoacridine hydrochloride (TA1537) +S9 mix; 2-Aminoanthracene(all strains)

Remarks:

3

Details on test system and experimental conditions:

2. METHOD OF APPLICATION: Plate incorporation assay
3. Details on test system and conditions
METHOD OF APPLICATION: Pre incubation method
NUMBER OF REPLICATIONS: Duplicate
OTHER EXAMINATIONS: 3 plates per test were observed.

Rationale for test conditions:

2. No data
3. not specified

Evaluation criteria:

2. Increase in revertants/plate was observed
3. Evaluation was done considering a dose dependent increase in the number of revertants/plate.

Statistics:

2. No data available
3. Yes, SD ± Mean was observed.

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102

Remarks:

2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

bacteria, other: Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA

Remarks:

3

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

2. No data available
3. ADDITIONAL INFORMATION ON CYTOTOXICITY: No cytotoxicity was observed up to a concentration of 5000 µg/plate with or without metabolic activation.

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not induce mutation in the Salmonella typhimurium and Escherichia coli 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:

The Ames salmonella typhimurium mutagenicity test was conducted for the given test chemical to evaluate its gene toxic effects when exposed to Salmonella typhimurium TA1535, TA1537, TA98, TA100, and TA102 both with and without metabolic activation system extracted from rat liver using the test material at a dose level of 33, 100, 1000, 2500, or 5000 µg/plate using ethanol as solvents. Plate incorporation protocol was followed. The given test chemical is considered to be non mutagenic to S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 with and without metabolic activation in the Plate incorporation assay.

 

In another study, the given test chemical was investigated for its ability to induce mutagenic activity when tested in an in vitro reverse mutagenicity test as per OECD 471, 472 and Guidelines for Screening Mutagenicity Testing of Chemicals (Japan). The test material was exposed to Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA in the presence and absence of metabolic activation S9. The concentration of test material used in the presence and absence of metabolic activation were 156, 313, 625, 1250, 2500 and 5000 µg/plate. No mutagenic effects were observed in all strains, in the presence and absence of metabolic activation. Therefore, test chemical was considered to be non-mutagenic in Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA by AMES test. Hence the substance cannot be classified as 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 mutation in the Salmonella typhimurium and Escherichia coli 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.

Reference 2

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 for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

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:

5. No data
6. Not specified

Species / strain / cell type:

mammalian cell line, other: Chinese hamster fibroblast cell line CHL

Remarks:

5

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 lung(CHL)cells

Remarks:

6

Details on mammalian cell type (if applicable):

No data

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

5. No data
6. not specified

Metabolic activation:

with and without

Metabolic activation system:

5. No metabolic activation system was used
6. Rat liver, induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

5. At three different doses with 0.125 mg/mL being the maximum dose concentration
6. -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

Vehicle / solvent:

5. - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The chemical was soluble in DMSO
6.- Vehicle(s)/solvent(s) used: 1% Carboxymethylcellulose sodium
- Justification for choice of solvent/vehicle: The test chemical is soluble in Carboxymethylcellulose sodium.

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:

5

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

- Vehicle(s)/solvent(s) used: 1% Carboxymethylcellulose sodium

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: -S9 mix, Mitomycin C +S9 mix, Cyclophosphamide

Remarks:

6

Details on test system and experimental conditions:

5. 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
6. Details on test system and conditions
Chinese hamster lung(CHL)cells were used .

Rationale for test conditions:

5. No data
6. not specified

Evaluation criteria:

5. 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%.
6. The cells were observed for chromosomal abbreviation, gaps.

Statistics:

5. No data
6. not specified

Species / strain:

mammalian cell line, other: Chinese hamster fibroblast cell line CHL

Remarks:

5

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

Species / strain:

other: CHL/IU cells

Remarks:

6

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:

valid

Additional information on results:

5. 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
6. Cytotoxicity conc.: With metabolic activation: None
Without metabolic activation: >= 2,703 ug/mL (24 hr), 2,242 ug/mL (48 hr)

Remarks on result:

other: No mutagenic potential

Conclusions:

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.

Executive summary:

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

In vitro mammalian chromosome aberration test was performed to evaluate the mutagenic nature of the test chemical as per OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test). 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.

 

In another study, the gene mutation study was conducted according to in vitro mammalian chromosome aberration test to determine the mutagenic nature of the given test chemical as per 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 given as: -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; and +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.

 

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.

Reference 3

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 summarized based on data 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 using the Hprt and xprt genes)

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 cell gene mutation tests using the thymidine kinase gene

Target gene:

8. 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.


9. Thymidine Kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

9

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

8

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

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:

8). 0, 0.5, 1.0, 2.5 or 5.0 mM
9) 0,5,19,15,20,30,30,40,50 µg/mL

Vehicle / solvent:

Vehicle and solvent used:
8) Ethanol
- Justification for choice of solvent/vehicle:Phenethyl phenylacetate was easily dissolved in ethanol.

9) Water
- Justification for choice of solvent/vehicle: The test substance is soluble in water.

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)

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

methylmethanesulfonate

Details on test system and experimental conditions:

8). 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)
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.


9). NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 hours

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 48 hours
- Selection time (if incubation with a selective agent): 10 to 12 days
- Fixation time (start of exposure up to fixation or harvest of cells): After the 48-hour expression period, 3 x 10^6 cells were plated in medium and soft agar supplemented with trifluorothymidine (TFT) for selection of TFT-resistant cells (TK-/-) and in nonselective medium and soft agar to determine cloning efficiency. Plates were incubated at 37 C. in 5% CO2 for 10 to 12 days.
- Method used: Microwell plates for the mouse lymphoma assay.
- If a selective agent is used: trifluorothymidine was used
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 3 x 10^6 cells

Rationale for test conditions:

8). No data
9). No Data Available

Evaluation criteria:

8). The plates were scored for total number of colonies by manual counting. As a result, the mutation frequency could be calculated.
9). The mammalian cells were observed for mutagenic frequency in cells.

Statistics:

8). Mean were observed.
9). Yes, SD ± Mean was 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

Species / strain:

mouse lymphoma L5178Y cells

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

Remarks on result:

other: Non-mutagenic

Conclusions:

The test chemical did not induce mutation in the mouse lymphoma L5178 and Chinese hamster Ovary (CHO) Cells 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 read across to test chemical has been investigated for the mammalian cell mutagenic nature. The studies are as mentioned below:

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.

In another study, the in vitro mammalian cell gene mutation assay using the mouse lymphoma L5178Y cells was performed to observe the genotoxic potential of the test chemical. In this study, the average mutant frequency was observed on the Thymidine Kinase gene locus. The study was performed using with and without metabolic activation. Freshly prepared S9 from the livers of either Aroclor 1254-induced or non-induced male Fischer 344 rats. The concentration of test material used in two experiment were 0,5,19,15,20,30,30,40,50 µg/mL. The test chemical was dissolved in Water which was consider as a vehicle control. Methyl methanesulphonate was used a positive control. The average mutant frequency was considered as a measure for mutagenicity. However, the test chemical did not induce mutagenicity at the gene locus and therefore was considered to be non-mutagenic in mouse lymphoma cells by in vitro mammalian cell gene mutation assay.

 

Thus, based on above summarized studies for read-across to test chemical, it can be concluded that the given test chemical did not induce mutation in the Mouse lymphoma L5178 and Chinese hamster Ovary (CHO) Cells both in the presence and absence of S9 metabolic activation system and hence is not likely to be mammalian cell mutagenic under the conditions of this study.

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:

Ames assay:

The Ames salmonella typhimurium mutagenicity test was conducted for the given test chemical to evaluate its gene toxic effects when exposed to Salmonella typhimurium TA1535, TA1537, TA98, TA100, and TA102 both with and without metabolic activation system extracted from rat liver using the test material at a dose level of 33, 100, 1000, 2500, or 5000 µg/plate using ethanol as solvents. Plate incorporation protocol was followed. The given test chemical is considered to be non mutagenic to S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 with and without metabolic activation in the Plate incorporation assay.

 

In another study, the given test chemical was investigated for its ability to induce mutagenic activity when tested in an in vitro reverse mutagenicity test as per OECD 471, 472 and Guidelines for Screening Mutagenicity Testing of Chemicals (Japan). The test material was exposed to Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA in the presence and absence of metabolic activation S9. The concentration of test material used in the presence and absence of metabolic activation were 156, 313, 625, 1250, 2500 and 5000 µg/plate. No mutagenic effects were observed in all strains, in the presence and absence of metabolic activation. Therefore, test chemical was considered to be non-mutagenic in Salmonella typhimurium TA100, TA1535, TA98, TA1537, Escherichia coli WP2 uvrA by AMES test. Hence the substance cannot be classified as gene mutant in vitro.

   

In vitro mammalian chromosome aberration study:

In vitro mammalian chromosome aberration test was performed to evaluate the mutagenic nature of the test chemical as per OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test). 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.

 

In another study, the gene mutation study was conducted according to in vitro mammalian chromosome aberration test to determine the mutagenic nature of the given test chemical as per 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 given as: -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; and +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.

In vitro mammalian cell gene mutation study:

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

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.

In another study, thein vitro mammalian cell gene mutation assay using the mouse lymphoma L5178Y cells was performed to observe the genotoxic potential of the test chemical. In this study, the average mutant frequency was observed on the Thymidine Kinase gene locus. The study was performed using with and without metabolic activation. Freshly prepared S9 from the livers of either Aroclor 1254-induced or non-induced male Fischer 344 rats. The concentration of test material used in two experiment were 0,5,19,15,20,30,30,40,50 µg/mL. The test chemical was dissolved in Water which was consider as a vehicle control. Methyl methanesulphonate was used a positive control. The average mutant frequency was considered as a measure for mutagenicity. However, the test chemical did not induce mutagenicity at the gene locus and therefore was considered to be non-mutagenic in mouse lymphoma cells by in vitro mammalian cell gene mutation assay.

 

Thus, based on above summarized studies for read-across to test chemical, it can be concluded that the given test chemical did not induce mutation in the Mouse lymphoma L5178 and Chinese hamster Ovary (CHO) Cells both in the presence and absence of S9 metabolic activation system and hence is not likely to be mammalian cell mutagenic under the conditions of this study.

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 study. Hence, the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.