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Toxicological information

Genetic toxicity: in vitro

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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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
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 concentration

OTHER: 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 available


RANGE-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 activation

Triglycerides, 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.