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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented publication meeting basic scientific principles. Test compound was a mixture of short- and long-chain acyl triglyceride.

Data source

Reference
Reference Type:
publication
Title:
Genetic Toxicology Studies of SALATRIM Structured Triacylglycerols. Lack of Genetic Damage in in Vitro Mammalian Cell Assays and the in Vivo Micronucleus Assay
Author:
Hayes, J.R. et al.
Year:
1994
Bibliographic source:
J. Agrlc. Food Chem. 42, 521-527

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:
exposure duration not reported;
Principles of method if other than guideline:
The SALATRIM family of triacylglycerols differs from other fats in the ratio of short-chain fatty acids (SCFA) to long-chain fatty acids (LCFA) and in that stearic acid is the major LCFA. These fats have caloric availability values (4.5-6 kcal/g) lower than that of corn oil (9 kcal/g). SALATRIM 23CA Lot
A014, a typical SALATRIM fat, was tested in in vitro mammalian cell genotoxicity assays including the chromosomal aberration, unscheduled DNA synthesis, and HPRT mammalian cell mutagenesis assays. Corn oil also was tested as a reference fat. Both the SALATRIM fat and corn oil were negative
in the three assays.
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Reference substance name:
short- and long-chain fatty acid triacylglycerols
IUPAC Name:
short- and long-chain fatty acid triacylglycerols
Details on test material:
- Name of test material (as cited in study report): SALATRIM 23CA lot A014 (Short- and long-chain acyl triglyceride molecules)

The studies reported here used SALATRIM 23CA lot A014 in the in vitro mammalian cell studies as a representative of SALATRIM fats. SALATRIM 23CA lot A014 was produced from triacetin, tripropionin, and hydrogenated canola oil, with triacetin being the predominant source of the SCFA. As a result, acetic acid is the predominant short-chain fatty acid and occurs in the vast majority of triacylglycerols that comprise this fat.

Members of the SALATRIM family of structured triacylglycerols are typical triacylglycerols composed of fatty acids esterified to a glycerol backbone. The unique characteristic of this family of fats is the triacylglycerols contain a preponderance of stearic, acetic, propionic, and/
or butyric acids. Triacylglycerols that contain one stearic acid and two short-chain fatty acids (SCFA) predominate among the mixed triacylglycerols that comprise these fats. Because of the limited absorption of stearic acid compared to certain other fatty acids and the fewer carbons available for energy production from the SCFA, these fats have lower caloric availabilities (4.5-6 kcal/g) than fats such corn oil (9 kcal/g). SALATRIM fats are produced by interesterification among high-stearate fats, such as hydrogenated canola oil and hydrogenated soybean oil, and triacetin, tripropionin, and tributyrin. Because the natural precursor fats contain mixed triacylglycerols, SALATRIM fats contain small quantities of other fatty acids such as oleic and palmitic, among others. SALATRIM fats can have different physical and functional characteristics because the ratios of the SCFA can be varied.


Method

Target gene:
HPRT
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Chinese hamster ovary (CHO) CHO-K1 cells obtained from the American Type Culture Collection
- Type and identity of media: in Ham’s F12 medium with 31 µg/mL penicillin, 50 µg/mL streptomycin sulfate, and 5 % heatinactivated
fetal bovine serum (F12/5).
Metabolic activation:
with and without
Metabolic activation system:
final S-9 concentration of 1 %
Test concentrations with justification for top dose:
0, 31.25, 62.5, 125, 250, 500 and 1000 μg/mL
The high dose was limited by the low solubility of the fats in the assay medium.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Stock solutions of SALATRIM 23CA lot A014 and corn oil were made in acetone such that the acetone never exceeded 1 % of the culture.


Controls
Untreated negative controls:
yes
Remarks:
Corn oil doses were 327.7-1000 μg/mL.
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Positive controls were EMS (200 μg/mL of culture) without metabolic activation and 3-MC (5 μg/mL of culture) with metabolic activation.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium;

DURATION
- Exposure duration: not reported
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 14-17 days

Phenotypic expression of induced mutants was conducted by incubating the cells for 7 days. Cells were subcultured every 2-3 days by trypsinizing the flask, counting the cells, and replating 2 x 10(6) cells. After the expression period, approximately 3 x 10(6) cells from each culture were seeded in 100 mL of cloning medium supplemented with TG for selection of resistant cells. Approximately 600 cells were seeded in 100 mL of TG-free medium to determine the percentage of viable cells. The cells were incubated for 14-17 days and cell colonies counted with an ARTEK colony counter. Mutant frequency (ratio of mutant cells to nonmutant cells) was calculated by dividing the number of resistant colonies by the number of unselected viable colonies.

SELECTION AGENT (mutation assays): The selective cloning medium contained 30 µM 6-thioguanine (TG).

NUMBER OF REPLICATIONS: The definitive study was done in duplicate using duplicate cultures for each replicate.

DETERMINATION OF CYTOTOXICITY
Cytotoxicity was determined by detaching the cells from the culture flask with 0.05% trypsin-0.02% EDTA. A Model 2F Coulter counter was used to determine cell numbers, and an aliquot containing at least 1-2 x 10(6) cells was added to 20 mL of F12/5 to determine phenotypic expression. The remaining cell suspension was diluted in approximately 35 mL of medium so that 500 cells were plated into two Petri dishes. After incubation for 14-17 days, cell colonies were determined. Survival was expressed as the cloning efficiency (CE) relative to the solvent control.
Evaluation criteria:
The test results were considered positive if a dose-related increase in the number of mutant colonies occurred and the mutant frequencies of duplicate cultures treated with one or more concentrations of the test article were at least 3 times the average of those from the solvent control cultures.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Although the test material was soluble in the acetone, turbidity was noted in the cultures, indicating solubility had been exceeded.

RANGE-FINDING/SCREENING STUDIES:
Preliminary experiments were done to determine cytotoxicity and aid in selection of the dose range.

Any other information on results incl. tables

Table 3: Results of the HPRT Mammalian Cell Gene Mutation Assay of SALATRIM 23CA lot A014

with and without Metabolic Activation*

SALATRIM

Dose

(μg/mL)

without metabolic activation

with metabolic activation

rel. cloning efficiency

(% of control)

mutant frequency

(per 106cells)

rel. cloning efficiency

(% of control)

mutant frequency

(per 106cells)

0

100

15

100

10

31.3

96

7

94

7

62.5

100

18

84

6

125

89

11

93

10

250

90

17

97

15

500

78

12

69

9

1000

86

17

75

10

corn oil 1000**

91

6

79

15

EMS200

56

170

-

-

3MC 5

-

 

79

170

 

 

*Data represent the mean of two cultures with the exception of the 0 dose (solvent control), which represents the mean of triplicate cultures.

**Data from a independent experiment with corn oil as test substance instead of SALATRIM

EMS= Ethyl methanesulfonate; positive control for the assay without metabolic activation.

3MC = 3-Methylcholanthrene; positive control for the assay with metabolic activation.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

SALATRIM 23CA lot A014 presented no evidence of mutagenic potential under the conditions of the assay.
Positive controls gave the expected results, validating the assay.
Corn oil did not alter either the relative cloning efficiency or the mutation frequency of the cells.