Oils, menhaden

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Tests of the genotoxicity of several oils produced from algae or yeast which consist of fatty acids also present in menhaden oil are negative in all cases. Therefore, menhaden oil is expected to be not genotoxic, as well.

Link to relevant study records

Reference

Reference 1

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

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

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).

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

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

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.

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.

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

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.

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

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

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

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

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

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

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.

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

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.

Reference 2

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:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

DHA-algal oil is a refined food grade oil that is derived from Ulkenia sp. SAM2179, a thraustochytrid microalgae. It is manufactured through a multi-step fermentation process and a standard edible oil refining method. DHA-algal oil contains approximately 45% (w/w) of docosahexaenoic acid (DHA), as well as smaller amounts of palmitic acid (~35%) and docosapentaenoic acid (DPA) (~11%). The polyunsaturated fatty acids identified in DHA-algal oil are similar to those in fish oils.

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from the livers of Aroclor-induced rats

Test concentrations with justification for top dose:

0.062, 0.185, 0.556,1.667, and 5 mg DHA-algal oil/plate

Vehicle / solvent:

DMSO/Water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

ethylnitrosurea

other: 2-aminoanthracene

Details on test system and experimental conditions:

Just before use, the test substance was suspended in dimethylsulfoxide (DMSO) vehicle at 50 mg/ml, warmed to 37 °C and mixed by shaking for 1 h. This suspension was used to prepare serial dilutions in water. The test substance suspension (0.1 ml), 0.1 ml of fully grown bacterial culture and 0.5 ml S9-mix or 0.5 ml of 100 mM sodium phosphate (pH 7.5) were added to 2 ml molten top agar (containing 0.6% agar, 0.5% sodium chloride and 0.05 mM L-histidine*HCl/0.05 mM biotin for the S. typhimurium strains and supplemented with 0.05 mM tryptophan for the E. coli strain) maintained at 46 °C. The ingredients were thoroughly mixed and immediately poured onto minimal glucose agar plates (1.5% agar in Vogel and Bonner medium E with 2% glucose). Following incubation at 37 °C for 3 days, the number of his+ and trp+ revertants were counted. The test sample was assayed in triplicate at five concentrations. Negative (solvent) and positive control experiments were also conducted.

Evaluation criteria:

If a twofold or greater increase in the mean number of revertants was observed compared to the number of revertants on negative control plates, the test substance was considered to be mutagenic.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the second experiment, DHA-algal oil did not cause a twofold or greater increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed with the negative control. There was no evidence of a dose–response relationship. The mean number of revertant colonies of the negative controls was within the acceptable range, and the positive control mutagens gave the expected increase in the mean number of revertant colonies. The test substance was not toxic to any strain tested, as evidenced by the absence of a decrease in the mean number of revertant colonies.

Dose (µg/pl)	Number of revertants (number of colonies/plate)
	T A1535		TA1537		TA98		TA100		E. coli
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
0	24 ± 2	18 ± 1	18 ± 5	19 ± 9	23 ± 4	45 ± 14	154 ± 12	145 ± 4	26 ± 3	31 ± 4
62	21 ± 5	27 ± 7	18 ± 1	18 ± 1	27 ± 3	34 ± 8	145 ± 12	157 ± 11	25 ± 5	28 ± 6
185	20 ± 5	15 ± 2	12 ± 7	14 ± 2	28 ± 10	37 ± 11	150 ± 14	148 ± 6	23 ± 5	31 ± 8
556	18 ± 6	26 ± 6	13 ± 5	16 ± 6	19 ± 9	50 ± 3	124 ± 37	136 ± 18	23 ± 12	29 ± 8
1667	22 ± 6	24 ± 6	15 ± 2	16 ± 3	25 ± 4	46 ± 9	132 ± 14	147 ± 9	23 ± 7	25 ±6
5000	29 ± 6	30 ± 2	13 ± 7	13 ± 2	21 ± 9	38 ± 7	120 ± 11	160 ± 10	30 ± 6	28 ± 2
Positive control	473 ± 82	370 ± 42	1114 ± 167	268 ± 13	1008 ± 106	652 ± 123	533 ± 21	1509 ± 59	196 ± 10	464 ± 12

Conclusions:

The test substance was not toxic to any strain tested.

Executive summary:

In this publication by Blum et al. 2007 the genotoxicity and subchronic toxicity of an algal oil which contains a high amount of polyunsaturated fatty acids is evaluated. 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.

Reference 3

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:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Algal Oil from Schizochytrium sp. Algal Oil (Lot No. 98–5814E)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

up to 5000 µg/plate

Positive controls:

yes

Positive control substance:

not specified

Details on test system and experimental conditions:

In the Ames test, Algal Oil was investigated for its potential to induce gene mutations according to the plate incorporation test using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and tester strain Escherichia coli WP2 uvrA. In two independent experiments several concentrations up to 5000 µg/plate of the test item were used. Each assay was conducted with and without metabolic activation (S9 mix).

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Positive controls validity:

valid

Conclusions:

In the Ames test, no toxic effects of the test item were noted in any of the five tester strains. The reference mutagens induced a distinct increase of revertant colonies as indication of the validity of the experiments.

Executive summary:

In this publication by Fedorova-Dahms et al. 2011 the safety of an algal oil containing a high amount of polyunsaturated fatty acids is evaluated. The genotoxicity of this oil is investigated using an Ames test with five different test strains. Each assay was conducted with and without metabolic activation. Concentrations up to 5000 µg/plate were used. The results of all tests are negative.

Reference 4

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:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

The material tested was DHA-rich Algal Oil from Schizochytrium sp. DHA-rich Algal Oil (lot # 9800005999) contained 43% Docosahexaenoic acid (DHA) and 8% Eicosapentaenoic acid (EPA) of total fatty acids in triglyceride form.

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Several concentrations up to 5000 µl/plate of the test item

Positive controls:

yes

Positive control substance:

not specified

Details on test system and experimental conditions:

In the Ames test, DHA-rich Algal Oil was investigated for its potential to induce gene mutations according to the plate incorporation test using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and tester strain Escherichia coli WP2 uvrA. Several concentrations up to 5000 µg/plate of the test item were used in two independent experiments. Each assay was conducted with and without metabolic activation (S9 mix).

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Positive controls validity:

valid

Conclusions:

In the Ames test, no toxic effects of DHA-rich Algal Oil were noted in any of the five tester strains. No biologically relevant increases in revertant colony numbers were observed following treatment with DHA-rich Algal Oil at any concentration level, neither in the presence nor absence of metabolic activation. The reference mutagens induced a distinct increase of revertant colonies and demonstrated the validity of the experiments.

Executive summary:

In this publication by Fedorova-Dahms et al. 2011 the safety of an algal oil rich in polyunsaturated fatty acids is evaluated. 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 genotoxicity tests are negative. The validity of the experiments was demonstrated by positive reactions induced by reference mutagens.

Reference 5

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:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

ONC T-18 DHA-rich algal oil (lots no. 22630 and 22740; approximately 39–42% docosahexaenoic acid (DHA); total omega-3 fatty acids, approximately 41–44%; storage condition, frozen, -10 to -30 °C) was produced and received from Ocean Nutrition Canada Limited, Dartmouth, Nova Scotia, Canada. Ascorbyl palmitate and mixed natural tocopherols were added to the algal oil to prevent oxidation.

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

313, 625, 1250, 2500, 5000 µg/plate. Choice of concentrations based on an initial dose range-finding assay.

Vehicle / solvent:

DMSO

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: ICR-191, 2-aminoanthracene

Details on test system and experimental conditions:

DHA-rich algal oil was tested in a plate incorporation assay and an independent confirmatory assay performed under identical conditions. In the plate incorporation method, molten agar was utilized as the medium for transference of the test and control articles, metabolic activation system, and the bacterial culture to the agar plate, allowing colony growth. Three test plates per strain per treatment condition were used. Appropriate vehicle and positive controls were tested with each strain. Treatments were performed by adding 100 µL tester strain and 50 µL of test or control article to 2.5 mL of molten selective top agar (maintained at 45 ± 2 °C). After the required components had been added, the mixture was vortexed and overlaid onto the surface of 25 mL minimal bottom agar in a 15 x 100 mm petri dish. After the overlay solidified, the plates were inverted and incubated for 52 ± 4 h at 37 ± 2 °C. Cultures were treated in the presence of S9 in an identical manner, except using 2.0 mL undiluted molten selective top agar and adding 500 µL S9 mix.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Positive controls validity:

valid

Remarks:

2-nitrofluorene

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Positive controls validity:

valid

Remarks:

benzo(a)pyrene

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Positive controls validity:

valid

Remarks:

sodium azide

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Positive controls validity:

valid

Remarks:

2-aminoanthracene

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Positive controls validity:

valid

Remarks:

sodium azide

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Positive controls validity:

valid

Remarks:

2-aminoanthracene

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Positive controls validity:

valid

Remarks:

ICR-191

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Positive controls validity:

valid

Remarks:

2-aminoanthracene

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Positive controls validity:

valid

Remarks:

4-nitroquinoline-N-oxide

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

negative

Positive controls validity:

valid

Remarks:

2-aminoanthracene

Additional information on results:

The results of the bacterial reverse mutation test with a confirmatory assay (Ames assay) indicate that under the conditions of the study, DHA-rich algal oil did not cause an increase in the number of histidine revertants (Salmonella strains) or tryptophan revertants (E. coli) per plate in the presence or absence of S9 microsomal enzymes. The positive control chemicals produced the expected increase in mutation frequency, and all criteria for a valid study were met.

Conclusions:

DHA-rich algal oil does not possess genotoxic potential under the conditions of the study.

Executive summary:

In this publication by Schmitt et al. 2012 some toxicological properties of an algal oil which contains a high amount of docosahexaenoic acid (DHA) are evaluated. 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Tests of the genotoxicity of several oils produced from algae or yeast which consist of fatty acids also present in menhaden oil are negative in all cases. Therefore, menhaden oil is expected to be not genotoxic, as well.