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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
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:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
, only 100 cells per dose scored.
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:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
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:
not applicable
Species / strain
Species / strain:
Chinese hamster Ovary (CHO)
Details on mammalian cell lines (if applicable):
Chinese hamster ovary cells (CHO ETCC CCL 61 CHO-KL, proline-requiring)
- Type and identity of media: Cells were grown in a 5% C02 atmosphere at 37 "C in McCoy's 5a medium.
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
0, 250, 500, 1000 µg/mL.
The high dose was limited by the low solubility of the fats in the assay medium.
Vehicle:
- Vehicle(s)/solvent(s) used: acetone
Acetone was used as the solvent for both SALATRIM 23CA lot A014 and corn oil.



Controls
Negative controls:
yes
Remarks:
Corn oil doses were 500, 750 and 1000 µg/mL
Solvent controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: methylmethanesulfonate (-S9) and cyclophosphamide (+S9)
Details on test system and conditions:
METHOD OF APPLICATION: in medium;

DURATION
- Exposure duration: 8h (-S9); 2h (+S9);

In the absence of metabolic activation, the fats and 0.01 mM deoxybromouridine (BrdU) were added to the cell culture and incubated at 37ºC for 8h. The cells were washed and then incubated in fresh medium containing 0.01 mM BrdU for 13.5 h.
When metabolic activation was used, the cultures were exposed to the fats for 2 h at 37ºC, washed, and then incubated in fresh medium containing
0.01 mM BrdU for 6-8 h.
After incubation in BrdU, colchicine was added to the cultures at 0.4 μg/mL followed by incubation for 2.5 h at 37 ºC.

SPINDLE INHIBITOR (cytogenetic assays): colchicine 0.4 µg/mL
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: 50 cells per flask were evaluated for chromosomal aberrations, resulting in a total of 100 cells evaluated per dose.

DETERMINATION OF CYTOTOXICITY
- Method: The mitotic index was evaluated on the basis of at least 1000 cells per flask.
Evaluation criteria:
A test material would be considered positive if there was a statistically significant (p < 0.05) increase in the frequency of cells with chromosomal damage and if this increase was dose-dependent.
Statistics:
The number of cells with chromosomal damage in the test fat groups and positive control group were compared to the concurrent solvent control by Fischer's exact test (Gad and Weil, 1991).

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The SALATRIM fat appeared to be soluble in the culture medium for the chromosomal aberration assay at a concentration of 40 μg/mL but produced a suspension at higher concentrations.
The pH of the high dose in the medium was approximately 7.0. The preliminary dose range/cytotoxicity study used a dose range of 0-1000 μg/mL and was limited by the lack of solubility of the fat. Corn oil behaved in a manner similar to that of SALATRIM 23CA lot A014 and was tested using the same criteria. No significant cell cycle delay or reduction in the mitotic index was noted with either corn oil or the SALATRIM fat. There was a slight increase in the number of cells in metaphase 1 at a SALATRIM dose of 1000 μg/mL and at corn oil doses of 40 and 200 μg/mL, but these were not significant on the basis of assay criteria. Therefore, for the definitive assay, a dose range of 0 (solvent control), 250, 500, and 1000 μg/mL was chosen for the SALATRIM and 500,750, and 1000 μg/mL for corn oil.

RANGE-FINDING/SCREENING STUDIES:
A preliminary dose range study to determine cytotoxicity and optimal cell fixation time was done before the definitive study was performed. Doses of 8.0, 40.0, 200.0, 500.0, and 1000 μg/mL were tested.
On the basis of the results of the range-finding study, doses of 250,500, and 1000 μg/mL for the SALATRIM fat and 500,750, and 1000 μg/mL for corn oil were used for the definitive study and a harvest time of 8-10 h was selected.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of metabolic activation, no major changes were noted in the mitotic index with either SALATRIM 23CA lot A014 or corn oil, indicating a lack of cytotoxicity.

Any other information on results incl. tables

 

Table 1:Summary of results of the chromosome aberration study in CHO cells treated with SALATRIM 23CA Lot A014

 

 

S9

Conc.

in μg/mL

Cells scored

mitotic

index (%)

cells with abnormal chromosomes

(%)

cells with chromatid deletions

/exchanges

(%)

structural aberrations

per cell

corn oil**

-

1000

100

9.6

2

2/0

0.02

MMS

-

20

100

4.7

18*

13/5

0.22

Acetone

-

0

100

5.9

4

3/0

0.04

Test item

-

250

100

4.8

2

1/0

0.01

Test item

-

500

100

5.0

4

3/0

0.06

Test item

-

1000

100

4.4

4

3/0

0.05

corn oil**

+

1000

100

4.7

5

4/0

0.05

CP

+

50

100

0.4

26*

16.2/6.1

0.42

Acetone

+

0

100

3.6

4

3/0

0.06

Test item

+

250

100

3.9

0

0/0

0.00

Test item

+

500

100

3.9

2

2/0

0.03

Test item

+

1000

100

5.1

3

3/0

0.03

*p<0.05

**corn oil was tested in an independent experiment

 

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative