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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:
from 31 May 2011 to 14 October 2011
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of 1-phenyloctadecane-1,3-dione and phenylicosane-1,3-dione
EC Number:
915-316-2
Molecular formula:
C50H80O4
IUPAC Name:
Reaction mass of 1-phenyloctadecane-1,3-dione and phenylicosane-1,3-dione
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 from the liver of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
Concentration tested in Experiments 1 and 2 (plate incorporation, with and without metabolic activation): 12.3, 37, 111.1, 333.3 and 1000 µg/plate (A strong precipitate was observed in the Petri plates at dose levels > or = at 1000 µg/plate).
Due to a severe toxicity observed with S9 mix using the pre-incubation method, a third experiment was undertaken using a lower range of dose levels. The selected dose-levels for this third experiment were:
- 0.137, 0.412, 1.23, 3.7, 11.1 and 33.3 µg/plate for the TA 102 strain,
- 0.412, 1.23, 3.7, 11.1, 33.3 and 100 µg/plate for the TA 1537 strain,
- 0.8, 2.5, 7.4, 22.2, 66.7 and 200 µg/plate for the TA 98 strain,
- 1.23, 3.7, 11.1, 33.3, 100 and 300 µg/plate for the TA 100 strain.
Vehicle / solvent:
- Solvent used: Tetrahydrofuran (THF)
- Justification for choice of solvent: THF was chosen because of its solubility properties.
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: see details below
Details on test system and experimental conditions:
Positive controls:
Without S9 mix:
- sodium azide for strains TA 1535 and TA 100 (1 µg/plate),
- 9-Aminoacridine for strain TA 1537 (50 µg/plate),
- 2-Nitrofluorene for strain TA 98 (0.5 µg/plate),
- Mitomycin C for strain TA 102 (0.5 µg/plate).

With S9 mix:
- 2-Anthramine for strains TA 1535, TA 1537, TA 98 (2 µg/plate) and TA102 (10 µg/plate),
- Benzo(a)pyrene for strain TA 100 (5 µg/plate).
Evaluation criteria:
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result.
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 TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of Results Experiment I

Direct plate incorporation method

 

Metabolic activation

Test group

Dose level per plate

Mean revertant colony counts

TA 1535

TA 1537

TA 98

TA 100

TA 102

Without activation

THF

15

11

18

108

272

Rhodiastab 55P

12.3 µg

17

14

23

113

312

37 µg

23

7

22

112

304

111.1 µg

31

12

22

115

311

333.3 µg

39

16

32

111

307

1000 µg

29

14

27

101

366

NAN3

1 µg

669

 

 

452

 

9AA

50 µg

 

337

 

 

 

2NF

0.5 µg

 

 

163

 

 

MMC

0.5 µg

 

 

 

 

1872

With activation

THF

10

5

23

147

438

Rhodiastab 55P

12.3 µg

14

8

28

123

411

37 µg

10

15

28

114

483

111.1 µg

14

8

29

116

485

333.3 µg

16

9

27

164

429

1000 µg

29

7

23

150

500

2AM

2 µg

241

57

895

 

 

10 µg

 

 

 

 

3065

BAP

5 µg

 

 

 

565

 

 

Summary of Results Experiment II

Direct plate incorporation method (without S9 mix) and Pre-incubation method (with S9 mix)

 

Metabolic activation

Test group

Dose level per plate

Mean revertant colony counts

TA 1535

TA 1537

TA 98

TA 100

TA 102

Without activation

THF

17

7

24

110

410

Rhodiastab 55P

12.3 µg

19

10

26

93

370

37 µg

17

12

26

93

345

111.1 µg

19

6

26

88

405

333.3 µg

30

12

19

102

318

1000 µg

33

2

21

86

347

NAN3

1 µg

443

 

 

516

 

9AA

50 µg

 

132

 

 

 

2NF

0.5 µg

 

 

128

 

 

MMC

0.5 µg

 

 

 

 

2089

With activation

THF

14

10

32

139

387

Rhodiastab 55P

12.3 µg

10

6

24

137

215

37 µg

23

13

31

137

186

111.1 µg

18

2

26

126

110

333.3 µg

19

14

22

97

55

1000 µg

27

13

51

41

37

2AM

2 µg

170

203

1659

 

 

10 µg

 

 

 

 

2085

BAP

5 µg

 

 

 

541

 

 

Summary of Results Experiment III

With metabolic activation, Pre-incubation method

 

Metabolic activation

Test group

Dose level per plate

Mean revertant colony counts

TA 102

THF

483

Rhodiastab 55P

0.137 µg

359

0.412 µg

349

1.23µg

378

3.7 µg

319

11.1 µg

357

33.3 µg

206

2AM

10 µg

1570

TA 1537

THF

7

Rhodiastab 55P

0.412 µg

9

1.23µg

4

3.7 µg

2

11.1 µg

5

33.3 µg

4

100 µg

3

2AM

2 µg

99

TA 98

THF

31

Rhodiastab 55P

0.8 µg

30

2.5 µg

29

7.4 µg

22

22.2 µg

17

66.7 µg

21

200 µg

16

2AM

2 µg

1572

TA 100

THF

109

Rhodiastab 55P

1.23µg

141

3.7 µg

112

11.1 µg

100

33.3 µg

120

100 µg

142

300 µg

117

BAP

5 µg

590

 

 Vehicle:

THF: Tetrahydrofuran

 

Positive Controls:

NAN3: sodium azide

9-AA: 9-aminoacridine

2NF: 2-Nitrofluorene

MMC: Mitomycin C

2AM: 2-Anthramine

BAP: Benzo(a)pyrene

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Reaction mass of Stearoylbenzoylmethane and Palmitoylbenzoylmethane is considered to be non-mutagenic in Ames test, with and without metabolic activation.
Executive summary:

This study was performed to investigate the potential of the test item, RHODIASTAB 55 P, to induce reverse mutation in Salmonella typhimurium. The study was performed according to OECD guideline no. 471 and EC guideline n° B13/14 and in compliance with the Principles of Good Laboratory Practice. 

 

A preliminary toxicity test was performed to define the dose-levels of RHODIASTAB 55 P to be used for the mutagenicity study. The test item was then tested in three independent experiments, with and/or without a metabolic activation system, the S9 mix, prepared from a liver post‑mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254. The negative control was the vehicle (Tetrahydrofuran).

The preliminary test, both experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method. The second and third experiments with S9 mix were performed according to the preincubation method (60 minutes,37°C).

Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to at least five dose-levels of the test item (three plates/dose‑level). After 48 to 72 hours of incubation at37°C, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

 

Since the test item was found poorly soluble in the preliminary test, the choice of the highest dose-level was based on the level of precipitate, according to the criteria specified in the international guidelines.

The selected treatment-levels were 12.3, 37, 111.1, 333.3 and 1000 µg/plate for the first and second experiments with and without S9 mix. Due to a severe toxicity observed with S9 mix using the preincubation method, a third experiment was undertaken using a lower range of dose-levels. The selected dose-levels for this third experiment were:

.           0.137, 0.412,1.23,3.7, 11.1 and 33.3 µg/plate for the TA 102 strain,

.           0.412,1.23,3.7, 11.1, 33.3 and 100 µg/plate for the TA 1537 strain,

.           0.8, 2.5, 7.4, 22.2, 66.7 and 200 µg/plate for the TA 98 strain,

.           1.23,3.7, 11.1, 33.3, 100 and 300 µg/plate for the TA 100 strain.

 

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered to be valid. 

The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains and in either experiment.

In conclusion, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium, in the absence or in the presence of a rat metabolising system therefore RHODIASTAB 55 P is not classified according to Annex VI of the Directive 67/548/CEE and according to EU Regulation 1272/2008 (CLP).