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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:
2012-10-25 to 2013-02-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
(2008)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
(2005)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Brassylic acid/Tridecanedioic acid
- Physical state: White powder
- Analytical purity: 99.42 %
- Lot/batch No.: 3311050098 of 22 August 2011
- Expiration date of the lot/batch: 27 Jun 2013
- Storage condition of test material: At room temperature

Method

Target gene:
mutated gene loci resposible for histidine auxotropy
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
other: histidine auxotroph
Metabolic activation:
with and without
Metabolic activation system:
Arochlor 1254 induced rat liver S9; male rats
Test concentrations with justification for top dose:
Test concentration with and without metabolic activation
Plate incorporation test: 3.16, 10.0, 31.6, 100, 316, 1000 µg per plate;
Preincubation test: 3.16, 10.0, 31.6, 100, 316, 1000 µg per plate;
Vehicle / solvent:
DMSO
The test item was completely dissolved in dimethylsulfoxide (DMSO). The vehicle served as the negative control. A correction factor of 1.01 was used in order to correct for the purity of the test item of 99.0%. Fresh preparations of the test item were used for the treatment in all experimental parts.
Controls
Untreated negative controls:
no
Remarks:
solvent test will be used as negative reference item
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
for details see below
Positive control substance:
other: without metabolic activation: sodium azide in aqua ad iniectabilia for TA 1535 and TA 100, 2-nitroflurene in DMSO for TA 98, 9-amino-acridine in ethanol abs. for TA 1537, Mitomycin C in DMSO for TA 102
Remarks:
with metabolic activation: Benz(a)pyrene for TA 98, TA 102, TA 1537, 2-aminoanthracene for TA 100, TA 1535
Details on test system and experimental conditions:
Bacterial Reverse Mutation Test
SYSTEM OF TESTING
- Pre-Experiment:
Tridecanedioic acid was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain
TA 100. Ten concentrations ranging from 0.316 to 5000 μg Tridecanedioic acid/plate were tested. Cytotoxicity (reduction of the number of
revertants by more than 50%) was noted at the top concentration of 5000 μg/plate. In addition, test item precipitation was noted starting
at 1000 μg/plate.
Hence, 1000 μg Tridecanedioic acid/plate were chosen as top concentration for the main study in the plate incorporation test and in the
preincubation test.
- Main test: 1st - Standard plate incorporation method, 2nd - Preincubation method
- Metabolic activation assay: Arochlor 1254 induced rat liver S9 fraction, the protein content of the S9 fraction was 33.1 mg/mL S9, cytochrome
P-450: 0.40 nmol/mg protein
ADMINISTRATION
- Dosing:
* Plate incorporation test: 3.16, 10.0, 31.6, 100, 316, 1000 µg per plate;
* Preincubation test: 3.16, 10.0, 31.6, 100, 316, 1000 µg per plate;
- Data : 2 independent experiments with and without metabolic activation
- Number of replicates: 3 per concentration and experiment
- Positive and negative control groups and treatment:
- without metabolic activation:
* sodium azide in aqua ad iniectabilia for TA 1535 and TA 100, 10 µg/plate
* 2-nitroflurene in DMSO for TA 98, 10 µg/plate
* 9-amino-acridine in ethanol abs. for TA 1537, 100 µg/plate
* Mitomycin C in DMSO for TA 102, 10 µg/plate
- with metabolic acivation
* 2-aminoanthracene in DMSO for TA 100, TA 1535, 2 µg/plate
* Benzo(a)pyrene in DMSO for TA 98, TA 102, TA 1537, 10 µg/plate
- negative control: the vehicle DMSO was used as negative reference item (all test strains).
- Incubation time: 48 h to 72 h at 37 °C in the dark
- Pre-incubation time: 20 min at 37 °C;

NUMBER OF REPLICATIONS: 3 per concentration and experiment

NUMBER OF CELLS EVALUATED: approximately 10E8 viable cells in the late exponential or early stationary phase

DETERMINATION OF CYTOTOXICITY
- Method: In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation test item precipitation was noted at the top concentration of 1000 μg/plate in all test strains. No signs of cytotoxicity were noted up to the top concentration
of 1000 μg/plate.
Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the solvent control and/or a scarce background
lawn.
Evaluation criteria:
The test item is considered to show a positive response if:
- the number of revertants is significantly increased (p ≤ 0 .05, U-test according to MANN and WHITNEY) compared to the solvent control to at least
2-fold of the solvent control for TA 98, TA 100 and TA 102 and 3-fold of the solvent control for TA 1535 and TA 1537 in both independent
experiments.
- or, a concentration-related increase of the revertants is observed. The Spearman's rank correlation coefficient may be applied.

Positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on
histidine-free agar plates.
Statistics:
Statistical analysis using tests described in 'Evaluation criteria' was applied as appropriate.

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
Remarks:
precipitating at 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
GENTOXIC EFFECTS:
- With metabolic activation: negative
- Without metabolic activation: negative

CYTOTOXICITY EFFECTS:
in plate incorporation and preincubation test
- With metabolic activation: negative up to 1000 µg/plate
- Without metabolic activation: negative up to 1000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA: A History Profile of Negative and Positive Control Values (n = 37 studies) of the year 2012 from the laboratory is presented below.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

History Profile of Negative and Positive Control Values (n = 37 studies) of the year 2012

Data obtained from plate incorporation and preincubation tests

Negative Reference Item

Strain

S9-Mix

TA98

TA100

TA102

TA1535

TA1537

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Mean

29.9

31.5

137.7

134.1

276.0

279.2

18.3

17.8

6.6

6.9

SD

6.3

6.8

21.8

18.3

16.0

17.2

4.8

4.8

2.3

2.5

Min

20

20

100

101

224

245

10

10

2

0

Max

49

50

191

188

317

315

31

33

11

18

Positive Reference Item

Strain

S9-Mix

 

TA98

 

TA100

 

TA102

 

TA1535

 

TA1537

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

2-nitro-fluorene

Benzo(a)

pyrene

Sodiumazide

2-amino-anthracene

Mito-mycinC

Benzo(a)

pyrene

Sodiumazide

2-amino-anthracene

9-amino- acridine

Benzo(a)

pyrene

Mean

214.4

211.6

947.4

952.9

931.7

924.6

152.7

157.0

101.2

99.3

SD

82.7

83.7

65.9

69.1

56.1

54.0

58.8

59.0

47.1

45.7

Min

94

83

795

802

797

829

61

69

30

35

Max

434

433

1135

1144

1102

1075

382

371

272

257

Study report attachment:

LPT 28722 (Tables)

Applicant's summary and conclusion

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

In conclusion, under the present test conditions tridecanedioic acid tested up to a concentration of 1000 µg/plate, that led to test item precipitation, caused no mutagenic effect in the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation
test
nor in the preincubation test each carried out without and with metabolic activation.
 
Executive summary:

The purpose of this study was to evaluate the test item for mutagenic activity (gene mutation) in bacteria without and with the addition of a mammalian metabolic activation system as originally described by AMES et al. (1973, 1975) and revised by MARON and AMES (1983). Tridecanedioic acid was examined in the 5 Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254 -induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test. Tridecanedioic Acid was completely dissolved indimethylsulfoxide (DMSO). A correction factor of 1.01 was used in order to correct for the purity of the test item of 99.0%. The vehicle served as the negative control.

Tridecanedioic acid was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA 100. Ten concentrations ranging from 0.316 to 5000 µg tridecanedioic acid/plate were tested.Cytotoxicity (reduction of the number of revertants by more than 50%) was notedat the top concentration of 5000 µg/plate. In addition, test item precipitation was noted starting at 1000 µg/plate. Hence, 1000 µg tridecanedioic acid/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

Six concentrations ranging from 3.16 to 1000 µg tridecanedioic acid/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.

In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activationtest item precipitationwas noted at the top concentration of 1000 µg/plate in all test strains.No signs of cytotoxicity were noted up to the top concentration of 1000 µg/plate.

No increase in revertant colony numbers as compared with control counts was observed for tridecanedioic acid, tested up to a concentration of 1000 µg/plate, that led to test item precipitation, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test). The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.  

In conclusion, under the present test conditions tridecanedioic acid tested up to a concentration of 1000 µg/plate, that led to test item precipitation, caused no mutagenic effect in the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.