Registration Dossier

Administrative data

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 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 or 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 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:
Histidine
Species / strain / cell type:
S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537, and TA 1538
Remarks:
2
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 100, TA 1537, TA1538 and TA98
Remarks:
3
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:
2. S9 liver homogenate metabolic activation
3. S-9 liver fractions were prepared from Aroclor-pretreated rats (Aroclor 1254, 500 mg/kg ip) and adjusted to 25 mg protein/ml
Test concentrations with justification for top dose:
2. 0, 50, 150, 500, 1500, and 5000 μg/plate
3. 5 doses of the test substance were used upto 3600 µg/plate
Vehicle / solvent:
2. - Vehicle(s)/solvent(s) used: A vehicle control was used.
3. - Vehicle(s)/solvent(s) used: DMSO/water
- Justification for choice of solvent/vehicle: DMSO was used as solvent for the test materials that were poorly soluble in water
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
other: 4-nitro-o-phenylenediamine for TA1538
Remarks:
2
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO / water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
Remarks:
3
Details on test system and experimental conditions:
2. NUMBER OF REPLICATIONS:
- Number of cultures per concentration - triplicate
3. METHOD OF APPLICATION: Plate incorporation method

DURATION
- Preincubation period: No data available
- Exposure duration: 48 hr
- Expression time (cells in growth medium): 48 hr
Rationale for test conditions:
No data
Evaluation criteria:
2. If there were increase in the mutation frequency in any of the strains tested at any of the concentrations tested.
3. A reproducible, dose-related and at least two-fold elevation of the spontaneous revertant frequency. Agents producing reproducible, dose-related and significant (P≤ 0.01) but less than two-fold elevations were classified as marginally mutagenic under the experimental conditions.
Statistics:
2. No data
3. Statistical significance was determined according to the methods of Kastenbaum & Bowman (1970).
Species / strain:
S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537, and TA 1538
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
True negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA 1535, TA 100, TA 1537, TA1538 and TA98
Remarks:
3
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:
No data
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:

 

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical by Ames assay in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537, and TA 1538. Five dose levels (50, 150, 500, 1500, and 5000 μg/plate) were studied, in triplicate, with and without addition of S9 liver homogenate metabolic activation. Positive controls included N-ethyl-N-nitro-N-nitroguanidine for TA100 and TA1535; 9-aminoacridine for TA1537; 4-nitro-o-phenylenediamine for TA1538 and 4-nitroquinoline-1-oxide for TA98. A vehicle control was used, as well as a positive control with 2-aminoanthracene, which is mutagenic only with metabolic activation. The increase in the mutation frequency in any of the strains tested at any of the concentrations tested considered for the mutagenic potential of the test chemical. Test chemical was not toxic to any of the strains tested at any of the concentrations tested. It produced no significant increase in the mutation frequency in any of the strains tested at any of the concentrations tested. All of the positive controls produced marked increases in the mutation frequency, and the S9 liver homogenate metabolic activation was confirmed active with the additional positive control. Therefore, the given test chemical did not induce mutation in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, TA1538 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, mutagenic nature of the test chemical was determined by using gene mutation toxicity study. The test was performed by plate incorporation method using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 with and without S9 metabolic activation system at 5 different doses upto 3600 µg/plate and the plates were incubated for 48 hrs. Agents producing reproducible, dose-related and significant (P≤ 0.01) but less than two-fold elevations were classified as marginally mutagenic under the experimental conditions. Statistical significance was determined according to the methods of Kastenbaum & Bowman (1970). The test chemical did not induce mutation in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, TA1538 in the presence and absence of S9 metabolic activation system and hence 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 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:
No data
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
5
Details on mammalian cell type (if applicable):
Details on mammalian cell line
- Type and identity of media: Mc-Coy’s 5a medium with 10% fetal calf serum, L-glutamine, and antibiotics
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):
- Type and identity of media: Chinese hamster lung derived fibroblast cell line (CHL).
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
5. The S9 mix consisted of 15 µl/ml liverhomogenate (from male Sprague-Dawley rats, induced with Aroclor 1254), 2.4 mg/ml NADP, and 4.5 mg/ml isocitric acid in serum-free medium.
6. Rat liver, induced with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
5. Without S9: 300-3000 µg/ml
With S9: 300-3000 µg/ml
6. -S9 mix(24hr continuous exposure): 0, 375, 750, 1500 µg/mL
-S9 mix(48hr continuous exposure): 0, 375, 750, 1500 µg/mL
-S9 mix(short-term exposure): 0, 375, 750, 1500µg/mL
+S9 mix(short-term exposure): 0, 375, 750, 1500 µg/mL
Vehicle / solvent:
5. - Vehicle(s)/solvent(s) used: Water, dimethyl sulfoxide (DMSO), ethanol, or Acetone (in the order of preference).
6. - Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Water, dimethyl sulfoxide (DMSO), ethanol, or Acetone (in the order of preference)
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
triethylenemelamine
cyclophosphamide
mitomycin C
Remarks:
5
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
6
Details on test system and experimental conditions:
5. Details on test system and conditions
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period:
- Exposure duration: With S9: 2 hrs
Without S9: Apprx. 8.5-9hrs
- Expression time (cells in growth medium):8.5-9hrs
- Selection time (if incubation with a selection agent):after 18-26 hrs
- Fixation time (start of exposure up to fixation or harvest of cells):8-12 hrafter the beginning of treatment.

SELECTION AGENT (mutation assays):Geimsa stain

NUMBER OF CELLS EVALUATED:100 cells

OTHER EXAMINATIONS:
- Determination of polyploidy:Yes
6. Details on test system and conditions
Cells were seeded on a multi-plate for cell culture, and the test substance solution was treated 3 days after the culture. In the case of the continuous treatment method, the treatment is carried out continuously for 24 or 48 hours, and in the short treatment method, after treatment for 6 hours in the presence of S9 mix (+ S 9 mix) or absence (-S 9 mix), fresh The medium was replaced with a culture medium, and the culture was continued for 18 hours.
After fixation of the cells with 10% neutral buffered formalin solution (Wako Pure Chemical Industries, Ltd.), the cells were stained with 0.1% crystal violet (Kanto Kagaku Co., Ltd.) aqueous solution for 10 minutes. An appropriate amount of pigment eluate (30% ethanol, 1% acetic acid aqueous solution) was added and allowed to stand for about 5 minutes to elute the dye, and the absorbance at 580 nm was measured. For each dose group, the ratio to the absorbance in the solvent control group, ie the cell viability was calculated.

SPINDLE INHIBITOR (cytogenetic assays): colcemid
NUMBER OF CELLS EVALUATED: 100 cells were evaluated.

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
Rationale for test conditions:
No data
Evaluation criteria:
5. Chromosomal aberrations were noted; Cells were selected for scoring on the basis of good morphology and completeness of karyotype (21 ± 2 chromosomes).
Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized chromosomes).Gaps and endo-reduplications were recorded but were not included in the totals. Aberrations were not scored in polyploidy cells but metaphases with 19-23 chromosomes were used (the modal number being 21).
6. The cells were observed for chromosomal gaps, chromatid breaks (ctb), chromosome breaks (csb), chromosome segments (csb), chromosome segments They were classified as structural anomalies of exchange (cte), chromosome exchange (cse) and others (oth). At the same time, the incidence of ploidy cells was recorded.
Statistics:
5. For chromosome aberrations, linear regression analysis of the percentage of cells with aberrations vs the log-dose was used as the test for trend. Toexamine absolute increases over control levels at each dose, a binomial sampling assumption was used. The P values were adjusted to take into account the multiple dose comparisons. For data analysis, we used the“total” aberration category, and the criterion for a positive response was that theadjusted P value be < 0.05.
6. No data
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
5
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
Species / strain:
other: Chinese hamster lung (CHL) 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: doses were chosen for the aberration test based on a preliminary testof cell survival 24 hr after treatment. Doses were based on observations of cell confluence and mitotic cell availability in the SCE test.
6. No data
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical did not induce chromosome aberrations in the mammalian cell line in the presence or 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. Cloned Chinese hamster ovary cells (CHO-W-B1) were cultured in Mc-Coy’s 5a medium with 10% fetal calf serum, L-glutamine, and antibiotics. Test was carried out with and without an in vitro metabolic activation system (S9 mix). The S9 mix consisted of 15µl/ml liver homogenate (from male Sprague-Dawley rats, induced with Aroclor 1254), 2.4 mg/ml NADP, and 4.5 mg/ml isocitric acid in serum-free medium. In without metabolic activation, the test chemical was left in culture until colcemid addition, whereas with activation the test chemical was added along with S9 mix for only 2 hr at the beginning of the test period. The doses used for the study were 300-3000 µg/ml with and without S9.

The test chemical did not induce chromosome aberrations in the Chinese hamster ovary 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 another genetic toxicity in vitro study, the given test chemical was assessed for mutagenic effects. For this purpose in vitro mammalian chromosome aberration test was performed according to 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, 375, 750, 1500 µg/mL; -S9 mix(48hr continuous exposure): 0, 375, 750, 1500 µg/mL; -S9 mix(short-term exposure): 0, 375, 750, 1500µg/mL; +S9 mix(short-term exposure): 0, 375, 750, 1500 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.

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:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical by Ames assay in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537, and TA 1538. Five dose levels (50, 150, 500, 1500, and 5000 μg/plate) were studied, in triplicate, with and without addition of S9 liver homogenate metabolic activation. Positive controls included N-ethyl-N-nitro-N-nitroguanidine for TA100 and TA1535; 9-aminoacridine for TA1537; 4-nitro-o-phenylenediamine for TA1538 and 4-nitroquinoline-1-oxide for TA98. A vehicle control was used, as well as a positive control with 2-aminoanthracene, which is mutagenic only with metabolic activation. The increase in the mutation frequency in any of the strains tested at any of the concentrations tested considered for the mutagenic potential of the test chemical. Test chemical was not toxic to any of the strains tested at any of the concentrations tested. It produced no significant increase in the mutation frequency in any of the strains tested at any of the concentrations tested. All of the positive controls produced marked increases in the mutation frequency, and the S9 liver homogenate metabolic activation was confirmed active with the additional positive control. Therefore, the given test chemical did not induce mutation in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, TA1538 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, mutagenic nature of the test chemical was determined by using gene mutation toxicity study. The test was performed by plate incorporation method using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538 with and without S9 metabolic activation system at 5 different doses upto 3600 µg/plate and the plates were incubated for 48 hrs. Agents producing reproducible, dose-related and significant (P≤ 0.01) but less than two-fold elevations were classified as marginally mutagenic under the experimental conditions. Statistical significance was determined according to the methods of Kastenbaum & Bowman (1970). The test chemical did not induce mutation in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, TA1538 in the presence and absence of S9 metabolic activation system and hence 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 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:

In-vitro mammalian chromosome aberration test was performed to evaluate the mutagenic nature of the test chemical. Cloned Chinese hamster ovary cells (CHO-W-B1) were cultured in Mc-Coy’s 5a medium with 10% fetal calf serum, L-glutamine, and antibiotics. Test was carried out with and without an in vitro metabolic activation system (S9 mix). The S9 mix consisted of 15µl/ml liver homogenate (from male Sprague-Dawley rats, induced with Aroclor 1254), 2.4 mg/ml NADP, and 4.5 mg/ml isocitric acid in serum-free medium. In without metabolic activation, the test chemical was left in culture until colcemid addition, whereas with activation the test chemical was added along with S9 mix for only 2 hr at the beginning of the test period. The doses used for the study were 300-3000 µg/ml with and without S9.

The test chemical did not induce chromosome aberrations in the Chinese hamster ovary 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 another genetic toxicity in vitro study, the given test chemical was assessed for mutagenic effects. For this purpose in vitro mammalian chromosome aberration test was performed according to 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, 375, 750, 1500 µg/mL; -S9 mix(48hr continuous exposure): 0, 375, 750, 1500 µg/mL; -S9 mix(short-term exposure): 0, 375, 750, 1500µg/mL; +S9 mix(short-term exposure): 0, 375, 750, 1500 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.

 

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 and In vitro mammalian chromosome aberration study. Hence, the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.