Dilithium salicylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 July 2017 - 26 Dec 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dilithium salicylate

EC Number:

254-228-2

EC Name:

Dilithium salicylate

Cas Number:

38970-76-2

Molecular formula:

C7H4Li2O3

IUPAC Name:

dilithium salicylate

Test material form:

solid: particulate/powder

Details on test material:

Lot # 17-24168
Expiration: 20 Mar 2019
White Powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: ExxonMobil Research & Engineering (Paulsboro, NJ) Lot # 17-24168
- Expiration date of the lot/batch: 20 March 2019
- Purity test date: 04 Aug 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Stable under conditions of assay
- Solubility and stability of the test substance in the solvent/vehicle: A preliminary solubility screen was conducted for Dilithium Salicylate in Tissue Culture Water; up to 50 mg/mL in tissue culture water, Dilithium Salicylate formed a clear colorless solution.

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.

Method

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, TA97a and TA100 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 TA97a are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. TA100 is reverted by both frameshift and base pair substitution mutagens and TA1535 is reverted only by mutagens that cause base pair substitutions.

The E. coli tester strain has an AT base pair at the critical mutation site within the trpE gene. Tester strain WP2uvrA (pKM101) has a deletion in the uvrA gene resulting in a deficient DNA excision-repair system. Tryptophan revertants can arise due to a base pair substitution at the originally mutated site or by a base pair substitution elsewhere in the chromosome causing the original mutation to be suppressed. Thus, the specificity of the reversion mechanism is sensitive to base pair substitution mutations.

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: hisG46

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: hisD3052/pKM101

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: hisG46/pKM101

Species / strain 4

Species / strain / cell type:

S. typhimurium, other: TA97a

Additional strain / cell type characteristics:

other: hisD6610/ pKM101

Species / strain 5

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: T/PE; pKM101

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 Supernatant (S9)

Test concentrations with justification for top dose:

A preliminary screen (5000, 1000, 500, 100, 50, 10, 5, and 1 ug/plate) produced cleared background lawn in bacterial strain S. typhirmurium TA100 at the three highest doses indicating cytotoxicity.

The following five doses were chosen for the main assay such that the highest dose produced evidence of cytotoxicity as per OECD TG 471:
5 µg/plate; 10 µg/plate; 50 µg/plate; 100 µg/plate; 500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Tissue cultue water
- Justification for choice of solvent/vehicle: A preliminary solubility screen was conducted for Dilithium Salicylate in DMSO and Tissue Culture Water. At 50 mg/mL in DMSO, an opaque white homogeneous suspension was formed. At 50 mg/mL in tissue culture water, Dilithium Salicylate formed a clear colorless solution.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA), CAS # 613-13-8

Remarks:

Positive control for all bacterial strains with S9

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

E. coli WP2 uvrA positive control without S9

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: acridine, 6-chloro-9-(3-((2-chloroethyl)amino) propyl)amino-2-methoxy, dihydrochloride (ICR- 191), CAS # 17070-45-0

Remarks:

S. typhimurium TA97a positive control without S9

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Daunomycin hydrochloride, CAS #23541-50-6

Remarks:

S. typhimurium TA98 positive control without S9

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

S. typhimurium TA100 and TA1535 positive control without S9

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 evaluated.

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 <-80oC until just prior to use.
NADPH REGENSYS B (used with S9) is stored at approximately -20ºC.
NADPH REGENSYS A buffer (also used with S9) and bacterial discs are stored at approximately 4ºC.
The rat S9 mix was prepared the day of dosing and was stored on ice until used. Lyophilized REGENESYS B is reconstituted in REGENESYS A buffer and Aroclor 1254 Supernatant (S9) is added to make a 10% S9 for use in the assay. The aliquots are refrigerated until use.
 
TEST SYSTEM
Test System: Salmonella typhimurium and Escherichia coli bacteria
Rationale: Recommended test system in international guidelines (e.g. OECD, EC).
Source: Molecular Toxicology Inc. (Moltox), Boone, NC.
Bacterial strains were as follows:
Strain - Histidine mutation - Mutation type
S. typhimurium TA98 -his D3052/uvrB/rfa/R-factor* - Frameshift mutations
S. typhimurium TA1535 -his G46/uvrB/rfa - Base-pair substitutions
S. typhimurium TA100 -his G46/uvrB/rfa/R-factor* - Base-pair substitutions and
Frameshift mutations
S. typhimurium TA97a -his D 6610/uvrB/rfa/R-factor* - Frameshift mutations
E. coli WP2 -trp/uvrA/R-factor*

*: R-factor = plasmid pKM101 (increases error-prone DNA repair)
 
 
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±2°C until an appropriate density is reached.

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:

TESTER STRAIN INTEGRITY: All Salmonella typhimurium tester strains must exhibit sensitivity to crystal violet and to ultraviolet light to demonstrate the presence of rfa mutation and uvrB mutation, respectively. The Escherichia coli tester strain must exhibit sensitivity to ultraviolet light to demonstrate the presence of uvrA mutation. Salmonella typhimurium strains TA98 and TA100 and Escherichia coli strain WP2uvrA (pKM101) must exhibit resistance to ampicillin to demonstrate the presence of the plasmid R-factor.The spontaneous reversion rates in the solvent/vehicle control must be in the range of in-house historical data.

TEST SUBSTANCE: For all tester strains, 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.

POSITIVE CONTROL: Positive control treatment for each tester strain of bacteria (see Section 4.2) must result in at least a 2-fold increase of revertants over the mean negative control value (vehicle). The effectiveness of the metabolic activation mix will be demonstrated by the positive response of the control.

VEHICLE CONTROL: The spontaneous reversion rate for each strain of bacteria will be calculated and compared to in-house historical ranges. Any deviations from those ranges will be taken into account when evaluating the data.

Statistics:

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

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium, other: TA97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Evaporation from medium: No evaporation from medium
- Water solubility: Test substance was soluble up to highest dose tested
- Precipitation: No precipitation was observed

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.

Remarks on result:

other: Main Assay

Applicant's summary and conclusion

Conclusions:

Under the conditions in this study, Dilithium Salicylate was not mutagenic in Salmonella typhimurium or Escherichia coli. These findings do not warrant classification of Dilithium Salicylate as a mutagen under the Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP).

Executive summary:

Dilithium Salicylate was examined for its potential to induce mutations in Salmonella typhimurium and Escherichia coli, in both the presence and absence of an S9 metabolic activation system. The doses were: 5, 10, 50, 100 and 500 ug/plate. In the mutagenicity assay, all data were acceptable and no significant increase in the number of revertants per plate were observed under any tester strain/activation condition combination. Under the conditions in this study, Dilithium Salicylate was not mutagenic to Salmonella typhimurium or Escherichia coli. These findings do not warrant classification of Dilithium Salicylate as a mutagen under the Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP).