Dilithium glutarate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic Toxicity Bacterial Reverse Mutation Assay- Not Mutagenic to Salmonella Typhimurium bacterial Strains TA98, TA100, TA102, TA1535 and TA1537 (OECD TG 471)

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003/10/22 - 2003/11/7

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: ExxonMobil Research and Engineering (Paulsboro, New Jersey)
- Expiration date of the lot/batch: July 2008

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
-Physical State: white powder
- Storage condition of test material: Room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Test substance was soluble in distilled water at 250 mg/mL and was stable throughout duration of assay.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Adjustments were not made for the purity of the test substance. The test substance, as received, was considered "pure".
- Final dilution of a dissolved solid, stock liquid or gel: 100 uL of appropriate test substance dilution was added to test system containing 2 mL of molten top agar
- Final preparation of a solid: Test substance was diluted in distilled water to desired concentrations.

Target gene:

Each S. typhimurium tester strain contains, in addition to a mutation in the histidine operon, additional mutations that enhance sensitivity to some mutagens. The rfa mutation results in a cell wall deficiency that increases the permeability of the cell to certain classes of chemicals such as those containing large ring systems that would otherwise be excluded. The deletion in the uvrB gene results in a deficient DNA excision-repair system. Tester strains TA98, TA100, and TA 102 also contain the pKM101 plasmid (carrying the R-factor). It has been suggested that the plasmid increases sensitivity to mutagens by modifying an existing bacterial DNA repair polymerase complex involved with the mismatch-repair process.

TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. TA100 is reverted by both frameshift and base substitution mutagens and TA1535 and TA 102 are reverted only by mutagens that cause base substitutions.

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 Homogenate from liver of Arochlor 1254 pretreated Sprague Dawley Rats

Test concentrations with justification for top dose:

Rangefinding toxicity test (TA100) 0.5, 1, 5, 10, 50, 100, 500, 1000, 2000, 2500 microgram/plate (+S9 and -S9). No signs of toxicity were observed up to the highest dose tested.
Main tests: 25, 80, 250, 800, and 2500 microgram/plate (+S9 and -S9)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Lab supplied reagent grade water for the test substance and positive control Mitomycin C (initial assay); Distilled water for test substance and positive control Mitomycin C (repeat assay); Dimethylsulfoxide for all positive controls except Mitomycin C.
- Justification for choice of solvent/vehicle: Materials were soluble in respective vehicles throughout duration of study

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

other: 2-Aminoanthracene: 2.5 micrograms/plate with S9

Remarks:

Strain TA98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

other: 2-Aminoanthracene 2.5 micrograms per plate without S9; Danthron 30 micrograms per plate with S9

Remarks:

Strain TA100

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (+S9) and Water (-S9)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene 2.5 micrograms per plate with S9; N-Methyl-N-Nitro-N-Nitrosoguanidine 2.5 micrograms per plate without S9

Remarks:

Strain TA102

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene 2.5 micrograms per plate with S9; N-Methyl-N-Nitro-N-Nitrosoguanidine 2.5 micrograms per plate without S9

Remarks:

Strain TA1535

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene 2.5 micrograms per plate with S9; 9-Aminoanthracene 100 micrograms per plate without S9

Remarks:

Strain TA1537

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: Triplicate plating

DETERMINATION OF CYTOTOXICITY
- Method: The revertant colonies were counted manually or automatically with the Q Count Colony Counter. Evidence of test article precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.To determine the toxicity, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.

METABOLIC ACTIVATION SYSTEM
Rat liver microsomal enzymes (S9 homogenate) were obtained from Molecular Toxicology Inc., Boone, NC. and were prepared from male Sprague Dawley rats that had been pretreated with Aroclor 1254. The S9 used in this study was tested by the manufacturer for its ability to activate an S9 dependent mutagen. The S9 was found to be acceptable for use in mutation tests.  

PREPARATION OF S9
The S9 was stored at <-70oC until just prior to use. The rat S9 mix was prepared the day of dosing and was stored on ice until used. S9-mix contained per 80 mL: 3.2 mL of 0.1M NADP; 0.4 mL of 1.0M Glucose-6-phosphate; 40 mL 0.2 M sodium phosphate buffer pH 7.4; 1.6 mL 0.4 M MgCl2 and 1.65 M KCl solution; 26.8 mL reagent grade water; and 8.0 mL S9-fraction (10% (v/v) S9-fraction).
 
TEST SYSTEM
Test System: Salmonella typhimurium bacteria
Rationale: Recommended test system in international guidelines (e.g. OECD, EC).
Source: Molecular Toxicology Inc. (Moltox), Boone, NC.
Salmonella typhimuriumstrains were as follows:
Strain - Histidine mutation - Mutation type
TA1537 -hisC3076 - Frameshift mutations
TA98 -hisD3052/R-factor* - Frameshift mutations
TA1535 -hisG46 - Base-pair substitutions
TA100 -hisG46/R-factor* - Base-pair substitutions and Frameshift mutations
TA102- hisG428/R-factor* - Base-pair Substitution (Ochre mutation)
*: R-factor = plasmid pKM101 (increases error-prone DNA repair)
 
The strains were characterized by Moltox in accordance with published procedures of Maron and Ames, 1983.
 
PREPARATION OF BACTERIAL CULTURES
Frozen tester strains were thawed and inoculated into a nutrient broth culture one day prior to dosing and incubated at 37±20C for 10 hours. The number of cells per culture, following a 10 hour incubation, was determined to be 1.4 x109cells/mL in the initial assay and 1.0 x109cells/mL in the repeat assay.
 
ADMINISTRATION OF TEST SUBSTANCE VEHICLE AND POSITIVE CONTROLS
Samples of bacteria (0.1 mL), followed by vehicle (100 microL), appropriate test substance dilution (100 microL), appropriate positive control vehicle (100 microL) or appropriate positive control substance dilution (100 microL), and 0.5 mL of S9 mix (+S9) or saline (-S9), were added to sterile glass test tubes containing 2 mL of molten top agar. The mixture was vortexed and immediately poured on plates containing a layer of minimal agar medium. After the top agar solidified, the plates were inverted and incubated at 37±2 degrees C for 48 hours.

Rationale for test conditions:

The tester strains were exposed to the test article via the plate incorporation methodology originally described by Ames et al (1975) and Maron and Ames (1983). This methodology has been shown to detect a wide range of classes of chemical mutagens.

Evaluation criteria:

For tester strains TA98, TA1535, and TA1537, an individual dose was considered positive if the mean revertant colony count on the test plates was equal to or greater than three times the mean number of spontaneous revertants on the vehicle control plates. For tester strains TA100 and TA102, an individual dose was considered positive if the mean revertant colony count on the test plates was equal to or greater than two times the mean number of spontaneous revertants on the vehicle control plates. A positive result for the assay was defined as a dose-related increase in the mean number of revertant colonies over at least three concentrations of test substance, including at least one positive dose.
A lack of a positive response in the positive controls would render that portion of the test invalid. Toxicity was defined as a notable reduction in the background lawn and/or a greater than 50% reduction in the mean number of revertant colonies when compared to the vehicle control. When the mean revertant colony countfor a test substance concentration is greater than or equal to two or three times the vehicle control, toxicity may also be defined as a greater than 50% reduction in the mean number of revertant colonies at concentrations higher than the concentration that induced the largest increase in revertant colonies.

Statistics:

The mean revertant colony count and standard deviation were determined for each dose point.

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

both initial and repeat assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

both initial and repeat assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Remarks:

both initial and repeat assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

both initial and repeat assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

both initial and repeat assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Conclusions:

The in vitro Bacterial Reverse Mutation Assay to assess the genotoxicity of dilithium glutarate was negative. This finding does not warrant the classification of dilithium glutarate as a genotoxin under Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP).

Executive summary:

Dilithium glutarate was examined for its potential to induce mutations in Salmonella typhimurium bacterial cells, in both the presence and absence of an S9 metabolic activation system. Two separate bacterial reverse mutation assays were performed with identical doses for each test: 25, 80, 250, 800, or 2500 ug/plate. Dilithium glutarate did not induce a statistically significant increase in the number of revertant cells at any of the doses chosen with or without metabolic activation. Under the conditions in this study, dilithium glutarate was not mutagenic to Salmonella typhimurium bacterial cells. This finding does not warrant the classification of dilithium glutarate as a genotoxin under the Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Dilithium glutarate was examined for its potential to induce mutations in Salmonella typhimurium bacterial cells, in both the presence and absence of an S9 metabolic activation system. Mutagenic properties have not been demonstrated and classification is not warranted.

Justification for classification or non-classification

Dilithium glutarate was not mutagenic to Salmonella typhimurium bacterial cells in a reverse mutation assay. This finding does not warrant the classification of dilithium glutarate as a genotoxin under the Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP).