Magnesium, bis[2-(hydroxy-.kappa.O)benzoato-.kappa.O]-, ar, ar'-di-C14-18-alkyl derivs.

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2009-08-31 to 2010-01-15

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP guideline study using a close structural analogue of the test material. Comparison of overall physico-chemical and toxicity profiles for target and source chemicals indicates it is appropriate to apply read-across data from the structural analogue when considering gene mutation in mouse lymphoma cells.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

dosing formulations were not analysed

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Benzoic acid, 2-hydroxy-, mono-C14-18-alkyl derivs., calcium salts (2:1)

EC Number:

931-276-9

Cas Number:

114959-46-5

Molecular formula:

See information in Section 1.2.

IUPAC Name:

Benzoic acid, 2-hydroxy-, mono-C14-18-alkyl derivs., calcium salts (2:1)

Details on test material:

- Name of test material (as cited in study report): [CAS Number 114959-46-5]
- Physical state: Purple flaky solid
- Analytical purity: 99 %
- Lot/batch No.: E00041-103
- Expiration date of the lot/batch: 2012-04-04
- Storage condition of test material: Room temperature protected from light

Method

Target gene:

Thymidine kinase locus of L5178Y mouse lymphoma cells

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

Preliminary toxicity test (4 hour exposure): zero, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 and 700 µg/mL
Preliminary toxicity test (24 hour exposure): zero, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 and 700 µg/mL
Initial assay without exogeneous metabolic activation (4 hour exposure): at least eight concentrations of test material including zero, 15, 25, 50, 60, 80 and 100 µg/mL
Initial assay with exogeneous metabolic activation (4 hour exposure): at least eight concentrations of test material including zero, 10, 15, 25, 50, 60 and 80 µg/mL
Extended assay without exogeneous metabolic activation (24 hour exposure): at least eight concentrations of test material including zero, 2.5, 5, 10, 15, 20 and 25 µg/mL

Vehicle / solvent:

Water

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

CHARACTERIZATION OF TEST AND CONTROL ARTICLES
The Sponsor determined the identity, strength, purity, and composition or other characteristics to define the test material.

The vehicle (solvent) used to deliver the substance to the test system was sterile distilled water (CAS 7732-18-5), lot #431944 expiration date March 2010, obtained from Gibco.

Methyl methanesulfonate (MMS), CAS 66-27-3, lot #76296KJ, expiration date 01 June 2012, supplied by Aldrich Chemical Company was diluted in water lot #431944 expiration date March 2010, from Gibco and used as the positive control for the non activated test system at stock concentrations of 1500 and 2000 µg/mL for the 4-hour exposure and 500 and 750 µg/mL for the 24- hour exposure.

7,12-Dimethyl-benz(a)anthracene (7,12-DMBA), CAS 57-97-6, lot #055K1360, expiration date 20 September 2010, supplied by Sigma Chemical Company was diluted in DMSO lot #48086822, expiration date 14 January 2011, from EMD Chemicals and used at stock concentrations of 100 and 125 µg/mL as the positive control for the S9-activated test system.

The stability of the negative and positive control materials and their respective mixtures was demonstrated by acceptable results that met the criteria for a valid test. Historical control data are presented in Appendix II.

TEST SYSTEM
L5178Y cells, clone 3.7.2C, were obtained from Patricia Poorman-Allen, Glaxo Wellcome Inc., Research Triangle Park, NC on 14 August 1995. Each lot of cryopreserved cells was tested using agar culture and Hoechst staining procedures and found to be free of mycoplasma contamination. Prior to use in the assay, L5178Y cells were cleansed of spontaneous TK-/- cells by culturing in a restrictive medium.

METABOLIC ACTIVATION SYSTEM
Aroclor 1254 induced rat liver S9 lot 2313, was purchased by BioReliance from Moltox (Boone, NC) and stored at = minus 60 degrees Centigrade until used. Each lot of S9 was assayed for sterility and its ability to metabolize at least two pro mutagens to forms mutagenic to Salmonella typhimurium TA100. The Record of Analysis is on file with the testing facility.

Immediately prior to use, the S9 was mixed with the cofactors and Fischer's Medium for Leukemic Cells of Mice with 0.1 % Pluronics (F0P) to contain 25 µL S9, 6.0 mg nicotinamide adenine dinucleotide phosphate (NADP), 11.25 mg DL-isocitric acid, and 975 µL F0P per mL S9-activation mixture and kept on ice until used. The cofactor/F0P mixture was adjusted to pH 7.0 and filter sterilized prior to the addition of S9.

SOLUBILITY TEST
A solubility test was conducted to select the vehicle. The test was conducted using purified water and dimethyl sulfoxide to determine the vehicle, selected in order of preference, which permitted preparation of the highest soluble or workable stock concentration, up to 50 mg/mL for aqueous vehicles or 500 mg/mL for organic vehicles.

PRELIMINARY TOXICITY ASSAY
The preliminary toxicity assay was used to establish the optimal concentrations for the mutagenesis assay. L5178Y cells were exposed to the solvent alone and nine concentrations of test material ranging from 0.15 to 700 µg/mL in both the absence and presence of S9 activation with a 4-hour exposure and without activation with a 24 hour exposure.

The osmolality of the solvent control and the highest soluble concentration in treatment medium were determined. For the 4-hour exposure, cell population density was determined 24 and 48 hours after the exposure to the test material; the cultures were adjusted to 3 x 10E5 cells/mL after 24 hours only. For the 24-hour exposure, cell population density was determined 24, 48, and 72 hours after the exposure to the test material. The cell population was adjusted to 3 x 10E5 cells/mL immediately after test material removal and 24 hours after test material removal. Cultures with less than 3 x10E5 cells/mL were not adjusted. Toxicity was measured as suspension growth of the treated cultures relative to the growth of the solvent control cultures after 48 hours.

MUTAGENESIS ASSAY
The initial mutagenesis assay (with and without S9 activation with a 4-hour exposure) and extended treatment assay (without activation with a 24-hour exposure) were used to evaluate the mutagenic potential of the test material. L5178Y mouse lymphoma cells were exposed to the solvent alone and at least eight concentrations of test material in duplicate in both the absence and presence of S9. Positive controls, with and without S9 activation, were tested concurrently.

TREATMENT OF THE TARGET CELLS
Treatment was carried out in conical tubes by combining 6 x 10E6 L5178Y/TK+/- cells, F0P medium or S9 activation mixture, and 1.0 mL dosing solution of test material in solvent or solvent alone in a total volume of 10 mL. The positive controls were treated with 100 µL MMS (at final concentrations in treatment medium of 15 and 20 µg/mL with a 4-hour exposure or 5.0 and 7.5 µg/mL with a 24-hour exposure) or 7,12 DMBA (at final concentrations in treatment medium of 1.0 and 1.25 µg/mL). Treatment tubes were gassed with 5 ± 1% carbon dioxide in air, capped tightly, and incubated with mechanical mixing for 4 or 24 hours at 37 ± 1 degrees Centigrade. The preparation and addition of the test material dosing solutions were carried out under amber lighting and the cells were incubated in the dark during the exposure period. After the treatment period, the cells were washed twice with F0P or F0P supplemented with 10 % horse serum, 2 mM L-glutamine, 100 U penicillin/mL and 100 µg streptomycin/mL (F10P). After the second wash, the cells were resuspended in 20 mL F10P, gassed with 5 ± 1 % carbon dioxide in air and placed on the roller drum apparatus at 37 ± 1 degree Centigrade.

EXPRESSION OF THE MUTANT PHENOTYPE
For expression of the mutant phenotype, the cultures were counted using an electronic cell counter and adjusted to 3x10E5 cells/mL at approximately 24 and 48 hours after treatment in 20 and 10 mL total volume, respectively. For the 24-hour exposure, cultures were adjusted to 3x10E5 cells/mL in 20 mL immediately after test article removal, then at 48 and 72 hours after treatment in 20 and 10 mL total volume, respectively. Cultures with less than 3x105 cells/mL were not adjusted.

For expression of the TK-/- cells, cells were placed in cloning medium (C.M.) containing 0.23% dissolved Noble agar in F0P plus 20% horse serum. Two flasks per culture to be cloned were labelled with the test article concentration, activation condition, and either TFT (trifluorothymidine, the selective agent) or VC (viable count). Each flask was prewarmed to 37 ± 1 degree Centigrade, filled with 100 mL
C.M., and placed in an incubator shaker at 37 ± 1 degree Centigrade until used. The cells were centrifuged at 1000 rpm for 10 minutes and the supernatant was decanted. The cells were then diluted in C.M. to concentrations of 3x10E6 cells/100 mL C.M. for the TFT flask and 600 cells/100 mL C.M. for the VC flask. After the dilution, 1.0 mL of stock solution of TFT was added to the TFT flask (final concentration of 3 µg/mL) and both this flask and the VC flask were placed on the shaker at 125 rpm and 37 ± 1 degrees Centigrade. After 15 minutes, the flasks were removed and the cell suspension was dispensed equally into each of three appropriately labelled Petri dishes. To accelerate the gelling process, the plates were placed in cold storage (approximately 4 degrees Centigrade) for approximately 30 minutes. The plates were then incubated at 37 ± 1 degrees Centigrade in a humidified 5 ± 1 % carbon dioxide atmosphere for 10-14 days.

SCORING PROCEDURES
After the incubation period, the VC plates were counted for the total number of colonies per plate and the total relative growth determined. The TFT-resistant colonies were then counted for each culture with = 20 % total relative growth (including at least one concentration with = 10% but = 20 % total growth). The diameters of the TFT-resistant colonies for the positive and solvent controls and, in the case of a positive response, the test article-treated cultures were determined over a range of approximately 0.2 to 1.1 mm. The rationale for this procedure is as follows: Mutant L5178Y TK-/- colonies exhibit a characteristic frequency distribution of colony sizes. The precise distribution of large and small TFT-resistant mutant colonies appears to be the characteristic mutagenic "fingerprint" of carcinogens in the L5178Y TK+/- system.

CRITERIA FOR A VALID TEST
The following criteria must be met for the mutagenesis assay to be considered valid:

Negative Controls
The average spontaneous mutant frequency of the solvent (or vehicle) control cultures must be within 35 to 140 TFT-resistant mutants per 10E6 surviving cells. Low spontaneous mutant frequencies, i.e., 20 to 34 mutants per 106 surviving cells, are considered acceptable if small colony recovery is demonstrated. The average cloning efficiency of the solvent (or vehicle) controls must be between 65 % and 120 % and the total suspension growth between 8-32 for the 4-hour exposure and 20-180 for the 24-hour exposure.

Positive Controls
The mutant frequency for at least one dose of the positive controls must meet the criteria for a positive response and induce an increase in small colony mutants according to the following criteria: Induced Mutant Frequency (IMF) positive control = 300 x 10E-6 mutants with 40 % small colonies or small colony IMF for positive control = 150 x 10E-6.

Test Article-Treated Cultures:
Cultures treated with a minimum of four concentrations of test article must be attained and their mutant frequencies reported. The highest test article concentration must produce 80 % to 90 % toxicity unless limited by solubility or the maximum required concentration as described in section 7.2 of the protocol. In the case of a test article with a steep toxicity curve (no concentrations with 10-20 % survival), the results may be considered acceptable if a concentration spacing of = 2-fold is used and the highest concentration tested showed <20 % survival or total kill. For example, the test is considered acceptable if the highest concentration cloned for mutant selection exhibits > 20 % survival and the next highest concentration, which is = 2 times the cloned concentration, is too toxic to clone.

EVALUATION OF RESULTS
The cytotoxic effects of each treatment condition were expressed relative to the solvent-treated control for suspension growth over two days post-treatment and for total growth (suspension growth corrected for plating efficiency at the time of selection). The mutant frequency (number of mutants per 10E6 surviving cells) for each treatment condition was determined by dividing the average number of colonies in the three TFT plates by the average number of colonies in the three corresponding VC plates and multiplying by the dilution factor (2x10E-4) then multiplying by 10E6. For simplicity, this is described as: (Average # TFT colonies / average # VC colonies) x 200 in the tables. The induced mutant frequency (IMF) is defined as the mutant frequency of the treated culture minus the mutant frequency of the solvent control cultures.

In evaluation of the data, increases in induced mutant frequency that occurred only at highly toxic concentrations (less than 10 % total growth) were not considered biologically relevant. All conclusions were based on scientific judgment; however, the following criteria are presented as a guide to interpretation of the data:

A result was considered positive if a concentration-related increase in induced mutant frequency was observed in the treated cultures and one or more treatment conditions with 10 % or greater total growth exhibited induced mutant frequencies of = 90 mutants per 10E6 clonable cells (based on the average mutant frequency of duplicate cultures). If the average solvent control mutant frequency was > 90 mutants per 10E6 clonable cells, a doubling of mutant frequency over the background will also be required.

A result was considered negative if the treated cultures exhibited induced mutant frequencies of less than 90 mutants per 10E6 clonable cells (based on the average mutant frequency of duplicate cultures) and there was no concentration-related increase in mutant frequency.

There are some situations in which a chemical would be considered negative when there was no culture showing between 10-20 % survival: 1) There was no evidence of mutagenicity (for example, no dose response or increase in induced mutant frequencies between 45 and 89 mutants per 10E6) in a series of data points within 100 % to 20 % survival and there was at least one negative data point between 20 % and 25 % survival. 2) There was no evidence of mutagenicity (for example, no dose response or increase in induced mutant frequencies between 45 and 89 mutants per 10E6) in a series of data points between 100 % to 25 % survival and there was also a negative data point between 10 % and 1 % survival. In this case it would be acceptable to count the TFT colonies of cultures exhibiting <10 % total growth.

ELECTRONIC DATA COLLECTION SYSTEMS
The primary computer or electronic systems used for the collection or analysis of data included but were not limited to LIMS Labware version 5, Excel 2003 (Microsoft Corporation) and Kaye Lab Watch Monitoring System (Kaye GE).

Evaluation criteria:

See above

Statistics:

No data

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Based on the results of the toxicity test, the concentrations in the mutagenesis assay ranged from 2.5 to 250 ug/mL for non-activated cultures (4 h), 1.0 to 250 ug/mL for S9 activated cultures (4 h) and 0.5 to 100 ug/mL for non-activated cultures (24 h)

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

JUSTIFICATION FOR USE OF READ-ACROSS DATA

Comparison of overall physico-chemical and toxicity profiles for target and source chemicals indicates it is appropriate to apply read-across data from the structural analogue when considering gene mutation in mouse lymphoma cells

SOLUBILITY TEST
Sterile distilled water was selected as the solvent of choice based on workability of the test article and compatibility with the target cells. The test article was workable in sterile distilled water at a maximum concentration of approximately 8.33 mg/mL with extensive Polytron mixing.

PRELIMINARY TOXICITY TEST
The results of the preliminary toxicity assay are presented in Table 1 (attached). The maximum concentration tested in the preliminary toxicity assay was 700 µg/mL. No visible precipitate was present at any concentration in treatment medium. The osmolality of the solvent control was 276 mmoL/kg and the osmolality of the highest soluble concentration, 700 µg/mL, was 283 mmoL/kg. Suspension growth relative to the solvent controls was 0 % at = 150 µg/mL with and without S9 activation with a 4 hour exposure and = 50 µg/mL without activation with a 24-hour exposure. Based on the results of the toxicity test, the concentrations treated in the mutagenesis assay ranged from 2.5 to 250 µg/mL for the non-activated cultures with a 4-hour exposure, 1.0 to 250 µg/mL for the S9-activated cultures with a 4-hour exposure, and 0.5 to 100 µg/mL for the non-activated cultures with a 24-hour exposure.

MUTAGENESIS ASSAYS
The results of the initial mutagenesis assay are presented in Tables 2 and 3 (attached). Colony size distributions for the positive and solvent control cultures are presented in Figures 1 and 2 (attached). No visible precipitate was present at any concentration in treatment medium. In the non-activated system, cultures treated with concentrations of 15, 25, 50, 60, and 80 µg/mL were cloned and produced a range in suspension growth from 24 % to 104 %. In the S9-activated system, cultures treated with concentrations of 10, 15, 25, 50, and 60 µg/mL were cloned and produced a range in suspension growth from 14 % to 96 %.

No cloned cultures exhibited mutant frequencies that were = 90 mutants per 10E6 clonable cells over that of the solvent control. No concentration-related increase in mutant frequency was observed in the non-activated or S9-activated systems. The total growth ranged from 23 % to 87 % for the nonactivated cultures at concentrations from 15 to 80 µg/mL and 14 % to 90 % for the S9-activated cultures at concentrations from 10 to 60 µg/mL.

The results of the initial assay were negative in the absence and presence of S9 activation. Because no unique metabolic requirements were known about the test article, an extended treatment assay was performed only in the absence of S9 for a 24-hour exposure period.

The results of the extended treatment assay are presented in Table 4 (attached). Colony size distributions for the positive and solvent control cultures are presented in Figure 3 (attached). No visible precipitate was present at any concentration in treatment medium. Cultures treated with concentrations of 2.5, 5.0, 10, 15, and 20 µg/mL were cloned and produced a range in suspension growth from 12 % to 106 %.

No cloned cultures exhibited mutant frequencies that were = 90 mutants per 10E6 clonable cells over that of the solvent control. No concentration-related increase in mutant frequency was observed. The total growth ranged from 8 % to 109 % for non activated cultures with a 24-hour exposure at concentrations from 2.5 to 20 µg/mL. No mutant frequency data were generated from the cultures with <10% total growth.

The TFT-resistant colonies for the positive and solvent control cultures from both assays were sized according to diameter over a range from approximately 0.2 to 1.1 mm. The colony sizing for the MMS positive controls yielded the expected increase in small colonies (verifying the adequacy of the methods used to detect small colony mutants) and large colonies.

DOSING FORMULATION ANALYSIS
At the request of the Sponsor, dosing formulation analysis for concentration and stability was not conducted. Due to the lack of dose formulation analysis, the interpretation of the study data is based on the targeted dose levels and not on the actual formulated test article concentrations. The study director has concluded that the test article was tested to the maximum feasible concentration of test article because toxicity or precipitation was observed. The test article can be concluded to be negative.

No stability data were provided for the test article. The study director believes that the test article, as provided, was tested to the maximum feasible concentration based on the laboratory records. In the absence of stability data, the study director must conclude that the test article was negative with the caveat that the maximum required concentration may not have been achieved due to instability.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

See comparison of overall physico-chemical and toxicity profiles for target and source chemicals in the data matrix (attached).

Applicant's summary and conclusion

Conclusions:

The test material was determined to be negative in the L5178Y/TK+/- mouse lymphoma mutagenesis assay.