Oils, menhaden

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Data source

Reference

Reference Type:

publication

Title:

Safety assessment of EPA-rich triglyceride oil produced from yeast: Genotoxicity and 28-day oral toxicity in rats

Author:

Leigh A. Belcher, Susan A. MacKenzie, Maria Donner, Greg P. Sykes, Steven R. Frame, Peter J. Gillies

Year:

2011

Bibliographic source:

Regulatory Toxicology and Pharmacology, 59, 2011,53–63

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: FDA Redbook 2000, IV.C.1.a Bacterial Reverse Mutation Test

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Test material form:

liquid

Specific details on test material used for the study:

The source of the EPA oil used in these studies was a biotechnology-derived Y. lipolytica yeast. The EPA oil was extracted from the yeast and processed under Good Manufacturing Practice (GMP; food, 21 CFR110) standards at Pilot Plant Corporation (POS), Saskatoon, Saskatchewan, Canada. The EPA oil contained residual antioxidants (e.g., approximately 300 ppm tocopherols).

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 5

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

10% S9

Test concentrations with justification for top dose:

1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg per plate. Top dose choice was based on an initialtoxicity test. This dose was achieved using a concentration of 100 mg/mL EPA oil and a 50 lL plating aliquot.

Vehicle / solvent:

DMSO

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

other: 2-Aminoanthracene, Acridine mutagen ICR-191

Details on test system and experimental conditions:

EPA oil was evaluated for mutagenicity in the Ames assay using the plate incorporation method. The study was conducted as two trials. DMSO was chosen as the dosing vehicle based on the solubility of the test substance and compatibility with the target cells. Appropriate positive controls were included in the study. Revertant colonies were counted with an automated counter (Sorcerer, Perceptive Instruments Ltd., Suffold, United Kingdom).

Evaluation criteria:

Data were judged positive if the increase in mean revertants at the highest numerical dose response was >=2.0-fold, the mean concurrent negative control value (vehicle control) for strains TA98, TA100, and WP2uvrA, and >=3.0-fold for strains TA1535 and TA1537.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

2-Nitrofluorene at 1 µg/plate.

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

2-Aminoanthracene at 2.5 µg/plate

Test results 3

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

Sodium azide at 2 µg/plate.

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

2-Aminoanthracene at 2 µg/plate

Test results 5

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

Sodium azide at 2 µg/plate

Test results 6

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

2-Aminoanthracene at 2.5 µg/plate

Test results 7

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

Acridine mutagen ICR-191 at 2 µg/plate

Test results 8

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

2-Aminoanthracene at 2.5 µg/plate

Test results 9

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

4-Nitroquinole-N-oxide at 1 µg/plate

Test results 10

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks:

2-Aminoanthracene at 25 µg/plate

Additional information on results:

EPA oil did not increase the number of revertants in any of the tester strains used in the Ames assay when compared to the concurrent negative controls either in the absence or presence of S9, and tested to dose levels up to 5000 µg/plate. No appreciable toxicity was observed, but test substance precipitation was seen at 1500 or 5000 µg per plate.

Any other information on results incl. tables

Dose (µg/plate)	TA98	TA100	TA1535	TA1537	WP2uvrA
	Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD
DMSO-S9	18 ± 3	142 ± 19	11 ± 3	14 ± 4	22 ± 1
+S9	19 ± 9	109 ± 16	13 ± 3	10 ± 3	29 ± 0
Positive-S9	261 ± 65	593 ± 59	455 ± 28	1176 ± 71	207 ± 15
Control + S9	700 ± 533	438 ± 13	98 ± 25	54 ± 20	242 ± 38
50-S9	16 ± 3	144 ± 12	10 ± 5	10 ± 2	22 ± 3
+S9	31 ± 2	118 ± 9	14 ± 4	9 ± 6	23 ± 4
150-S9	16 ± 3	134 ± 7	13 ± 2	13 ± 5	23 ± 4
+S9	22 ± 5	120 ± 6	12 ± 1	10 ± 3	31 ± 2
500-S9	18 ± 1	149 ± 7	15 ± 3	10 ± 2	22 ± 1
+S9	23 ± 7	111 ± 2	11 ± 2	9 ± 3	26 ± 6
1500-S9	23 ± 3	137 ± 10	15 ± 1	10 ± 3	25 ± 3
+S9	26 ± 7	107 ± 25	12 ± 1	8 ± 6	23 ± 6
5000-S9	17 ± 4	138 ± 28	16 ± 5	11 ± 4	21 ± 3
+S9	26 ± 3	120 ± 20	11 ± 3	12 ± 3	25 ± 1

Applicant's summary and conclusion

Conclusions:

The oil was not mutagenic in the in vitro Ames assay.

Executive summary:

In this publication by Belcher et al. 2011 the safety of an oil produced from yeast is evaluated which contains a high amount of polyunsaturated fatty acids. An Ames test is used to investigate the genotoxicity of this oil. Five test strains were used in the experiments and each assay was conducted with and without metabolic activation. The results of all tests are negative. The validity of the experiments was demonstrated by positive reactions induced by reference mutagens.