Fatty acids, coco, esters with oxybis(propanediol)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Five S. typhimurium strains but neither the tester strain TA102 nor an additional E. Coli strain was included in the testing.

Data source

Reference

Reference Type:

publication

Title:

Genetic Toxicology Studies of SALATRIM Structured Triacylglycerols. 1. Lack of Mutagenicity in the Salmonelk/Microsome Reverse Mutation Assay

Author:

Johnnie R. Hayes and Edward S. Riccio

Year:

1994

Bibliographic source:

J. Agric. Food Chem. 1004, 42, 515-520

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

short- and long-chain triacylglycerols (SALATRIM 234CA and SALATRIM 234CS)

IUPAC Name:

short- and long-chain triacylglycerols (SALATRIM 234CA and SALATRIM 234CS)

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Members of the SALATRIM family were produced and provided by the Nabisco Foods Group, East Hanover, NJ. The members of the SALATRIM family of structured triacylglycerols investigated in this study were SALATRIM 4CA lot A006 , SALATRIM 23CA lot A014, SALATRIM 32CA lot A015, SALATRIM 234CA lot A019, SALATRIM 234CS lot A018, and SALATRIM 23SO lot A020.
- Fatty acid composition of the SALATRIM fats was determined at EPL Bio-Analytical Services, Inc., Decatur, IL. Fatty acid compositon was obtained by saponification of the triacylglycerol mixture with methanolic sodium hydroxide followed by esterification with methanolic boron trifluoride. Methyl esters of the LCFA were quantitated by gas chromatography. SCFA were quantitated by direct gas chromatography of the saponified fat. Standard curves were constructed by bracketing the concentration level of the analyte with quantitation based upon peak height. Complete chemical characterization of these fats is presented elsewhere (Softly et al., 1994).

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
The test fats were shipped frozen on solid carbon dioxide to SRI International, Menlo Park, CA, and maintained at -20 °C at the testing laboratory until use.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test fats were thawed at room temperature overnight and then heated to 55°C until an apparent homogeneous mixture was obtained. Aliquots were weighed and frozen at -20°C. For each assay, a weighed aliquot was thawed at room temperature and then heated to 50-55°C. Acetone was added to produce concentrations that would deliver the fat at doses of 62.5,125, 250, 500, 750, and 1000 µg/plate. The dosing solution was cooled to room temperature before addition to the preincubation mixture.
SALATRIM 4CA lot A006 formed a fine suspension in all dosing solutions and was vortexed to assure homogeneity. No precipitate was noted on the plates after addition of the dosing solution.
SALATRIM 23CA lot A014 was soluble at all of the dosing concentrations. However, a slight turbidity was observed at all doses after the dosing solution was added to the preincubation mixtures in the absence of metabolic activation. With metabolic activation, a slight increase in turbidity was noted at the 750 and 1000 µg/plate dose levels.
SALATRIM 32CA lot A015, SALATRIM 234CA lot A019, and SALATRIM 234CS lot A018 behaved like SALATRIM 23CA lot A014 except an increase in turbidity could not be detected when metabolic activation was used because of the turbidity of the S-9 preparation.
SALATRIM 23SO lot A020 behaved similarly to SALATRIM 23CA lot A014 except the slight increase in turbidity at 750 and 1000 µg/plate with metabolic activation could be detected in the 4% S-9 assay but not in the 10% S-9 assay. Also, after 35-45 min, a fine precipitate formed in the 1000 µglplate dosing solution. This suspension was vortexed to ensure homogeneity before it was added to the assay with metabolic activation. These differences in behavior in the dosing and assay mixtures probably result from the different physical characteristics of individual members of the SALATRIM family. Corn oil produced a slight turbidity at all doses when added to the preincubation mixtures without metabolic activation. In the presence of S-9, no increase in turbidity could be detected above the background turbidity. Precipitation in the assay mixtures indicates that the doses were beyond the limits of solubility.

Method

Target gene:

His operon

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

The S. typhimurium LT2 tester strains were TA98, TA100, TA1535, TA1537, and TA1538 obtained from Dr. Bruce Ames, University of California at Berkeley.

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

The S. typhimurium LT2 tester strains were TA98, TA100, TA1535, TA1537, and TA1538 obtained from Dr. Bruce Ames, University of California at Berkeley.

Metabolic activation:

with and without

Metabolic activation system:

The Aroclor 1254- induced rat liver S-9 preparation (from Sprague-Dawley rata) was obtained from Molecular Toxicology, Inc., Annapolis, MD.

Test concentrations with justification for top dose:

0, 62.6, 125, 250, 500, 750 and 1000 µg/plate.

Vehicle / solvent:

acetone

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-anthramine: strain strain TA98, TA100, TA1535, TA1537 and TA1538 with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Basically, 0.50 mL of either the metabolic activation mixture (+S-9) or buffer (-S-9) was added to a test tube in a 37°C heating block. This was followed by 0.05 mL of the specific tester strain (- 10E8 bacteria) followed by the appropriate amount of test fat in acetone. The mixture was allowed to incubate with agitation for 20 min. Then, 2 mL of 0.6 % agar containing 0.6% NaC1,0.05 mM biotin and 0.05 mM histidine was added, stirred, and poured onto plates containing 25 mL of minimal glucose agar.
During the first assay, a metabolic activation system using 4 % S-9 was used. If the test fat was negative in the assay, the second assay used a metabolic activation system with 10 % S-9.
- Cell density at seeding: 10E8 bacteria

DURATION
- Preincubation period: 20 min
- Selection time: The plates were incubated for 48 h at 37 °C

SELECTION AGENT (mutation assays): Histidine

NUMBER OF REPLICATIONS: After the range-finding study, the fats were assayed twice using the five tester strains, three plates per dose level, with and without metabolic activation.

NUMBER OF CELLS EVALUATED: Revertant colonies per plate were determined by automated counting.


DETERMINATION OF CYTOTOXICITY
A preliminary assay was conducted with strain TA100 with and without metabolic activation to determine an appropriate dose range and to determine potential cytotoxicity. The SALATRIM triacylglycerols showed no evidence of cytotoxicity in these studies.

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):

- OTHER:

Rationale for test conditions:

A preliminary assay was conducted with strain TAl100 with and without metabolic activation to determine an appropriate dose range and to determine potential cytotoxicity.
Although the SALATRIM triacylglycerols showed no evidence of cytotoxicity in these studies, they generally showed evidence of insolubility at least at the high-dose concentration (1000 µg/plate). Therefore, 1000 µg/plate is the limit dose for these fats in this assay.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Remarks:

Corn oil

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Remarks:

corn oil

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Remarks:

corn oil

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Remarks:

corn oil

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Remarks:

corn oil

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Several members of the SALATRIM family of structured triacylglycerols were tested in the Salmonella/mammalian microsome reverse mutation assay (Ames assay). The data from the assays indicate that SALATRIM triacylglycerols have no potential for mutagenic activity under the conditions of the assay when tested both with and without metabolic activation. Also, no indication of bacterial cytotoxicity was evident under any condition of the assays.