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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical using Salmonella typhimurium both in the presence and absence of metabolic activation system. The test chemical did not induce gene mutation in the Salmonella typhimurium 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 chromosome aberration study:

In vitro chromosome aberration study was performed to determine the mutagenic nature of the test chemical using Chinese hamster cell line both in the presence and absence of metabolic activation system. The test compound did not induce chromosome aberration in the Chinese hamster cell line in the presence and absence of metabolic activation system and hence 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:
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 auhtortative database
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
other: Salmonella typhimurium
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
No data
Test concentrations with justification for top dose:
Concentrations up to growth inhibition and cell toxicity
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:
No data
Rationale for test conditions:
No data
Evaluation criteria:
Increase in the number of revertants/dose
Statistics:
No data
Species / strain:
S. typhimurium, other: Salmonella typhimurium
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
Positive controls validity:
not specified
Additional information on results:
No data
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical did not induce gene mutation in the Salmonella typhimurium in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.
Executive summary:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical using Salmonella typhimurium both in the presence and absence of metabolic activation system. The test chemical did not induce gene mutation in the Salmonella typhimurium in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant 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 authoritative database
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Principles of method if other than guideline:
In vitro mammalian chromosome aberration test study was performed to determine the mutagenic nature of the test chemical
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
No data
Species / strain / cell type:
mammalian cell line, other: Chinese hamster
Details on mammalian cell type (if applicable):
No data
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
No data
Test concentrations with justification for top dose:
Concentrations up to growth inhibition and cell toxicity
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:
No data
Rationale for test conditions:
No data
Evaluation criteria:
No data
Statistics:
No data
Species / strain:
mammalian cell line, other: Chinese hamster
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
Positive controls validity:
not specified
Additional information on results:
No data
Remarks on result:
other: No mutagenic potential
Conclusions:
The test compound did not induce chromosome aberration in the Chinese hamster cell line in the presence and absence of metabolic activation system and hence is not likely to classify as a gene mutant in vitro.
Executive summary:

In vitro chromosome aberration study was performed to determine the mutagenic nature of the test chemical using Chinese hamster cell line both in the presence and absence of metabolic activation system. The test compound did not induce chromosome aberration in the Chinese hamster cell line in the presence and absence of metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

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

Additional information

Gene mutation toxicity in vitro:

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

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical using Salmonella typhimurium both in the presence and absence of metabolic activation system. The test chemical did not induce gene mutation in the Salmonella typhimurium 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 chromosome aberration study was performed to determine the mutagenic nature of the test chemical using Chinese hamster cell line both in the presence and absence of metabolic activation system. The test compound did not induce chromosome aberration in the Chinese hamster cell line in the presence and absence of metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Gene mutation toxicity study was also performed to determine the mutagenic nature of the test chemical. The study was performed by the preincubation protocol using Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100 both in the presence and absence of S9 metabolic activation system. Preincubation was carried at 37°C for 20 mins followed by exposure period of 48 hrs at dose levels of 0.0, 3.3, 10.0, 33.0, 100.0, 333.0 µg/plate. Concurrent solvent and positive control chemicals were also included in the study. The test chemical did not induce gene mutation in the Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100 both in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

In another study, gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium G46, TA1535, TA100, C3076, TA1537, D3052, TA1538, TA98 and E. coli WP2 and WP2 uvrA- with and without liver enzymes activation system. Ten ml of minimal agar medium (not containing test compound) was poured into a square Petri dish (9 x 9 cm) which is tilted at a slight angle. The agar was then allowed to solidify into a wedge-shaped layer by standing at room temperature. Meanwhile, a 1000-µg/ml mixture of test compound in agar was prepared by adding 10 ml of minimal agar to 0.1 ml of a 100-mg/mI solution of test compound in dimethyl sulfoxide. When appropriate, water or dimethoxyethane was used instead of dimethyl sulfoxide. The cooled agar plates were then placed on a level surface, and an overlay of the 10 ml of agar containing the test compound was poured onto the plate to form a reversed wedge of agar on top of the first wedge. A concentration gradient of compound was produced by allowing the compound in the upper wedge to diffuse into the lower layer for 2 hr at room temperature. The concentration range in this plate is approximately 100 to 1000µg/ml. Three additional plates with concentration ranges of 10 to 100µg/ml, 1 to 10µg/ml, and 0.1 to 1µg/ml were prepared. A streaking device consisting of 10 sterile 50-µL pipets was dipped into suspensions of the 10 test strains and allowed to fill by capillary action. The pipets were then touched to the upper edge of the gradient and drawn across the plate. The study was performed in the presence and absence of liver enzyle activating system and the plates were incubated for 48 hrs at 37°C. The test chemical did not induce gene mutation in Salmonella typhimurium G46, TA1535, TA100, C3076, TA1537, D3052, TA1538, TA98 and E. coli WP2 and WP2 uvrA- in the presence and absence of liver enzymes activation systemand hence the chemical is not likely to classify as a gene mutant in vitro.

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. The test chemical was studied at a dose level of 50-150µg/mL (in the absence of S9) and 25- 100µg/mL (in the presence of S9) using Chinese hamster ovary cells (CHO-W-B1).Cells were exposed to the test chemical for 2 hr in the presence of S9 or throughout the incubation period without S9. 100 cells were scored from each of the three highest dose groups having sufficient metaphases for analysis.All types of aberrations were recorded separately, but for data analysis they were grouped into categories of “simple” (breaks and terminal deletions), “complex” (exchanges and rearrangements), “other” (includes pulverized chromosomes), and “total”. Gaps and endo-reduplications were recorded but were not included in the totals. Polyploid cells were not scored but used metaphases with 19-23 chromosomes (the modal number being 21). Based on the results noted, the test chemical did not induce chromosome aberrations in the Chinese hamster ovary cells (CHO-W-B1) in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

In the same study, sister chromatid exchange test was performed to determine the mutagenic nature of the test chemical. The test chemical was studied at a dose level of 75-150µg/mL using Chinese hamster ovary cells (CHO-W-B1) both in the presence and absence of S9 metabolic activation system.            

5-Bromodeoxyuridine (BrdUrd; 10 pM) was added 2 hr after addition of the test chemical (without S9) or immediately after the S9 mix plus chemical had been removed. The chemical treatment periods were appoximately 25 hr without S9 and 2 hr with S9. The total incubation time with BrdUrd was 25-26 hr, with colcemid (0.1µg/ml) present during the final 2-3 hr. Immediately before the cells were harvested, the cell monolayers were examined, and the degree of confluence and availability of mitotic cells were noted. Cells were collected by mitotic shake-off at doses up to the maximum considered likely to yield sufficient metaphase cells for analysis; supernatant medium was returned to appropriate flasks so that subsequent harvests could bemade from the same cultures if necessary. Because all mitotic cells were removed in the initial harvest, cells collected during subsequent harvests had come into mitosis during the period between harvests and thus had been exposed to colcemid for an average of 4 hr. After 1-3 min treatment with hypotonic solution (75 mM KCl), cells were fixed in 3: 1 methano1: glacial acetic acid (V/V). For a preliminary assessment of cell cycle delay, test slides were prepared from cells treated at the highest dose levels to see if later harvests were necessary. These test slides were stained with “dilute” Hoeschst 33258 (0.5µg/ml in Sorensen’s buffer, pH 6.8) and examined by fluorescence microscopy to assess cell cycle kinetics. In control cultures, almost all cells completed two cycles in BrdUrd (M2 cells) in 25-26 hr, whereas, in treated cultures, cell cycle delay was common. In cases of severe delay, additional harvests were made from the same cultures at a later time to obtain sufficient second metaphase (M2) cells for SCE analysis. After staining for 10 min in “concentrated” Hoechst 33258 (5µg/ml in pH 6.8 buffer) and exposure to “black light” at 55 to 60°C for about 5 min, slides were stained in Giemsa. All slides were coded, and 50 cells per dose were scored from the three highest doses at which sufficient M2 cells were available. When cell cycle delay was noted, cell kinetics were recorded by classifying each of 100 metaphases as M1, M1+, or M2, i.e., having completed one (M1), two (M2), or between one and two (M1 +) cell cycles in BrdUrd. The test chemical did not induce an increase in the number of Sister chromatid exchanges in the Chinese hamster ovary cells (CHO-W-B1) in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Based on the data available and applying the weight of evidence appoach, the test chemical does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data available and applying the weight of evidence appoach, the test chemical does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.