Oils, fish, oxidized

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

end on January 22, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed according to OECD guideline and GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Oils, fish, oxidized

EC Number:

269-122-1

EC Name:

Oils, fish, oxidized

Cas Number:

68187-75-7

Molecular formula:

Not necessary

IUPAC Name:

Triglycerides C12-24, even, saturated and unsaturated, oxidized

Details on test material:

- Name of test material (as cited in study report): Triglycerides, C12-24, saturated and unsaturated, oxidized- Substance type: UVCB- Physical state: liquid- Stability under test conditions: data not available- Storage condition of test material: at room temperature

Method

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100, TA 102

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-Naphthoflavone induced rat liver S9 is used as the metabolic activation system.

Test concentrations with justification for top dose:

- Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate- Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Controls

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: WITH S9-MIX: sodium azide (TA 1535, TA 100, 10 µg/pl), 4-nitro-o-phenylene-diamine (10 µg/pl in TA 98, 50 µg/pl in TA 1537), methyl methane sulfonate (TA 102, 3.0 µL/pl) - WITHOUT S9-MIX: 2-aminoanthracene (2.5 µg/pl (10.0 µg/pl in TA 102))

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation test (experiment I) and pre-incubation test (experiment II)DURATION- Preincubation period: 60 minutes in the pre-incubation test- Exposure duration: at least 48 hours (at 37 °C in the dark)NUMBER OF REPLICATES: three at each concentrationOTHER: the colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB9 7BN, UK) with the software program Ames Study Manager.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.A dose dependent increase in the number of revertant colonies below the threshold isregarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100, TA 102

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- Precipitation: the test item precipitated in the overlay agar in the test tubes from 1000 µg/plate up to 5000 µg/plate. Precipitation of the test item on the incubated agar plates was observed from 2500 µg/plate up to 5000 µg/plate. The undissolved particles of the test item had no influence on the data recording.- Effects of pH, effects of osmolality, evaporation from medium,water solubility, other confounding effects: data not availableRANGE-FINDING/SCREENING STUDIES: in the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. The pre-experiment is reported as experiment I. Since no toxic effects were observed 5000 µg/plate were chosen as maximal concentration.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 : Summary of Results Pre-Experiment and Experiment I

Metabolic acitivation	Test Group	Dose level (per plate)	Revertant colony counts (Mean)
			TA 1535	TA 1537	TA 98	TA 100	TA 102
Without activation	DMSO		16	12	26	108	349
	Untreated		11	10	31	110	370
	Oxidized fish oil	3 µg	14	12	27	114	339
		10 µg	15	11	25	113	337
		33 µg	14	13	24	117	366
		100 µg	15	11	28	115	350
		333 µg	14	8	23	129	327
		1000 µg	12	6	31	125	336
		2500 µg	15 P	7 P	29 P	137 P	335 P
		5000 µg	14 P	9 P	23 P	167 P	325 P
	NaN3	10 µg	1566			1851
	4-NOPD	10 µg			276
	4-NOPD	50 µg		103
	MMS	3.0 µL					1916
With activation	DMSO		17	12	33	133	446
	Untreated		14	13	40	148	567
	Oxidized fish oil	3 µg	19	11	33	147	521
		10 µg	17	15	31	149	471
		33 µg	16	15	30	131	473
		100 µg	16	14	31	140	458
		333 µg	14	12	37	141	458
		1000 µg	14	11	31	142	440
		2500 µg	22 P	8 P	27 P	144 P	476 P
		5000 µg	18 P	8 P	35 P	143 P	472 P
	2 –AA	2.5 µg	295	353	1702	2683
	2 –AA	10 µg					1702

P = precipitates

 

Table 2 : Summary of Results Experiment II

Metabolic acitivation	Test Group	Dose level (per plate)	Revertant colony counts (Mean)
			TA 1535	TA 1537	TA 98	TA 100	TA 102
Without activation	DMSO		13	9	29	121	405
	Untreated		12	11	27	133	395
	Oxidized fish oil	33 µg	12	11	28	131	382
		100 µg	12	8	27	115	428
		333 µg	13	8	25	118	430
		1000 µg	10	8	29	128	379
		2500 µg	17 P	8 P	27 P	147 P	413 P
		5000 µg	14 P	7 P	32 P	168 P	371 P
	NaN3	10 µg	1897			2022
	4-NOPD	10 µg			318
	4-NOPD	50 µg		109
	MMS	3.0 µL					1661
With activation	DMSO		15	8	34	135	534
	Untreated		16	11	33	153	591
	Oxidized fish oil	33 µg	21	11	36	142	502
		100 µg	16	12	36	152	537
		333 µg	18	7	32	148	553
		1000 µg	13	13	34	151	538
		2500 µg	17	7	42	161	547
		5000 µg	28	12	47	155	513
	2 –AA	2.5 µg	345	221	1849	2167
	2 –AA	10 µg					3200

P = precipitates

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):negative with and without metabolic activationTriglycerides, C12-24, even, saturated and unsaturated, oxidized is non-mutagenic by Ames bacterial reverse mutation assay under the condition of the test employed.

Executive summary:

In a reverse gene mutation assay in bacteria (Sokolowski A, 2010), strains TA 1535, TA 1537, TA 98, TA 100, TA 102 of S. typhimurium were exposed to Triglycerides, C12-24, even, saturated and unsaturated, oxidized in DMSO at concentrations of 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate (Pre-Experiment/Experiment I, plate incorporation) and 33; 100; 333; 1000; 2500; and 5000 µg/plate (Experiment II, pre-incubation method) in the presence and absence of mammalian metabolic activation. 

 

Triglycerides, C12-24, even, saturated and unsaturated, oxidized oil was tested up to limit concentration (5000 µg/plate).

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Oxidized fish oil at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase

of induced revertant colonies.

 

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.