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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in vitro:

Ames test:

Test substance  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 and hence the chemical is not likely to classify as a gene mutant in vitro.

Chromosome aberration study:

The incidence of chromosome aberration in Chinese hamster fibroblast cell line for the test chemical  falls between 5.0 and 9.9 %. Hence the Gene toxicity in vitro result for disodium succinate is ambiguous/ equivocal.

In vitro Mammalian gene mutation assay

The test chemical did not induce a doubling of the mutant frequency both in the presence and absence of S9 activation system and hence is not likely to be 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 peer reviewed journal
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Salmonella/ microsome assay was performed to evaluate the mutagenic potential of test substance.
GLP compliance:
not specified
Type of assay:
bacterial gene mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium, other: TA92, TA1535, TA100, TA1537, TA94 and TA98
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
The liver microsome fraction (S-9) was prepared from the liver of Fischer rats
Test concentrations with justification for top dose:
6 different concentrations were used; 5000 µg/plate (5 mg/plate) was the maximum concentration applied
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Phosphate buffer
- Justification for choice of solvent/vehicle: The test chemical was soluble in Phosphate buffer
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Phosphate buffer
True negative controls:
not specified
Positive controls:
not specified
Details on test system and experimental conditions:
Details on test system and conditions
METHOD OF APPLICATION: preincubation

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

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

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: No data

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

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

OTHER: No data
Rationale for test conditions:
No data
Evaluation criteria:
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).
Statistics:
No data
Species / strain:
S. typhimurium, other: TA92, TA1535, TA100, TA1537, TA94 and TA98
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:
The maximum dose for negative results represents the highest non-cytotoxic dose used in the experiment
Remarks on result:
other: No mutagenic potential
Conclusions:
Test substance 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 and hence the chemical 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 test substance. The study was performed using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 with and without S9 metabolic activation system. The test was performed as per the preincubation assay at six different concentrations with 5.0 mg/plate being the maximum concentration. The chemical was dissolved in phosphate buffer. Preincubation was performed for 20 mins and the exposure duration was for 48 hrs. 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). Test substance 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 and hence the chemical is not likely to classify as a gene mutant in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
data from handbook or collection of data.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
other: Refer below principle
Principles of method if other than guideline:
Weight of evidence prepared from various publication mention below
The gene mutation study was conducted according to L5178Y TK+/- Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature of the test chemical
GLP compliance:
not specified
Type of assay:
other: Mammalian cell gene mutation assay
Target gene:
Thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Remarks:
TK+/- 3.7.C
Details on mammalian cell type (if applicable):
- Type and identity of media: The cells were grown in Fischer’s medium for leukemic cells of mice supplemented with 10% horse serum and 0.02% pluronic F-68.
- Properly maintained: No data available
- Periodically checked for Mycoplasma contamination: Yes
- 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):
No data
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 fraction was prepared from Aroclor 1254-induced male Sprague- Dawley rats.
Test concentrations with justification for top dose:
1,718-2000µg/mL
2,250-2000µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
methylmethanesulfonate
other: 3-methylcholanthrene at 1.86 × 10-5 M (or dimethylbenz[a]- anthracene at 0.5-4 µg/mL) for the test with metabolic activation.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium;
- Cell density at seeding (if applicable): 6000000 cells

DURATION
- Preincubation period: No data available
- Exposure duration: 4 h
- Expression time (cells in growth medium):48 h
- 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): 1×106 cells/plate for mutant selection
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: 1 X 106 cells/plate for mutant selection and 200
cells/plate for viable count determinations

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: The rate of cell growth was determined for each of the treated cultures

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

OTHER: No data available
Rationale for test conditions:
No data
Evaluation criteria:
Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. Doubling of the mutant frequency was previously reported as representing a positive effect. Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response.
Statistics:
No data available
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
No data
Remarks on result:
other: No mutagenic potential was observed
Conclusions:
The test chemical did not induce a doubling of the mutant frequency both in the presence and absence of S9 activation system and hence is not likely to be gene mutant in vitro.
Executive summary:

The gene mutation study was conducted according to L5178Y TK+/-Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature of the test chemical. The Cells at a concentration of 6 X 105/mL (6 X106cells total) were exposed for 4 h to a range of concentrations from 250 -2000 µg/mL of the test chemical. The cells were then washed, resuspended in growth medium, and incubated at 37°C for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls. Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase The test chemical did not induce a doubling of the mutant frequency both in the presence and absence of S9 activation system and hence is not likely to be gene mutant in vitro.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Data for the various test chemicals was reviewed to determine the mutagenic nature of Disodium succinate (150-90-3). The studies are as mentioned below:

AMES test

Gene mutation toxicity study was performed to determine the mutagenic nature of test substance. The study was performed using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 with and without S9 metabolic activation system. The test was performed as per the preincubation assay at six different concentrations with 5.0 mg/plate being the maximum concentration. The chemical was dissolved in phosphate buffer. Preincubation was performed for 20 mins and the exposure duration was for 48 hrs. 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). Test substance 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 and hence the chemical is not likely to classify as a gene mutant in vitro.

 Supported by other study. Bacterial Reverse Mutation Assay was performed to evaluate the mutagenic potential of test substance. The study was performed using Salmonella typhimuriuim strains TA97 and TA102 in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in distilled water and used at dose level of 100- 10000 µg/plate. The treated plates were observed for a dose dependent increase in the number of revertants/plate. Test substance did not induce gene mutation in the Salmonella typhimurium strains TA97 and TA102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In vitro chromosome abbreviation test

Chromosomal aberration study was performed to determine the mutagenic nature of test substance . The cells were exposed to the test material at three different doses with 15.0 mg/mL being the maximum concentration for 48hr. Colcemid (final concn 0.2µg/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 incidence of polyploid cells for 48hr after treatment was 0.0%. The incidence of cells with structural chromosomal aberrations at 0.4mg/ml for 48hr after treatment was 47.0%. Also positive at 0.3 mg/ml at 24hr (11.0 %) and at 48hr (30.0%). D20 was 0.25 mg/ml (the dose at which structural aberrations were detected in 20 % of theMetaphases observed). TR value (the frequency of cells with exchange-type aberrations per unit dose(mg/ml) ) was 0.35. The incidence of chromosome aberration in Chinese hamster fibroblast cell line for the test chemical falls between 5.0 and 9.9 %. Hence the Gene toxicity in vitro result for disodium succinate is ambiguous/ equivocal.

In vitro Mammalian gene mutation assay

The gene mutation study was conducted according to L5178Y TK+/-Mouse Lymphoma Mutagenicity Assay to determine the mutagenic nature of the test chemical. The Cells at a concentration of 6 X 105/mL (6 X106cells total) were exposed for 4 h to a range of concentrations from 250 -2000 µg/mL of the test chemical. The cells were then washed, resuspended in growth medium, and incubated at 37°C for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls. Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase The test chemical did not induce a doubling of the mutant frequency both in the presence and absence of S9 activation system and hence is not likely to be gene mutant in vitro.

Based on the data summarized, Disodium succinate (150-90-3) did not induce gene mutation .Hence it is not likely to be mutagenic in vitro.

Justification for classification or non-classification

Thus based on the above annotation and CLP criteria the test chemical Disodium succinate (150-90-3)not likly to induce gene mutation .Hence it is not likely to be mutagenic in vitro.