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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is conducted as a guideline study under GLP-conditions. The study is reliable without restrictions and fully sufficient for endpoint evaluation.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
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): 3,7,11-trimethyldodecyn-3-ol, CAS: 1604-35-9
- Physical state: Colorless, liquid
- Analytical purity: 99.0 area-%
- Lot/batch No.: 05-0001
- Stability under test conditions: lt is specified in the report that the results of the reanalysis will be reported in an amendment. According to the analytical report 06L00128, dated 24 Jul 2006 of the Analytical Department, BASF AG, the test substance was stable over the study period.
- Storage condition of test material: Room temperature

Method

Target gene:
- Test on mutagenic potential based on the ability to induce point mutations in selected Ioci of several bacterial strains, e.g. amino acid-requiring Salmonella typhimurium and Escherichia coli strains, in a reverse mutation assay (his+ or trp+ revertants). The principle is that mutations that lead to a restoration of the functional capability of the bacteria to synthesize the essential amino acid and thus to the ability to grow in the absence of the amino acid required by the parent strains.
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
cofactor-supplemented postmitochondrial fraction (S-9) obtained from Iivers of rats treated with an enzyme-inducing agent, this case: Aroclor 1254.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
cofactor-supplemented postmitochondrial fraction (S-9) obtained from Iivers of rats treated with an enzyme-inducing agent, this case: Aroclor 1254.
Test concentrations with justification for top dose:
20 µg - 5000 µg/plate (SPT) and 4 µg - 2500 µg/plate (PIT)
Vehicle:
- Vehicle/solvent) used: DMSO
- Justification for choice of solvent/vehicle: Due to the Iimited solubility of the test substance in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical contral data are available.
Controls
Negative controls:
yes
Solvent controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: see any other information on results for details
Details on test system and conditions:
METHOD OF APPLICATION:
1. in agar (plate incorporation) - standar plate incubation test (SPT)
2. preincubation test (PIT)

DURATION - Preincubation test based on the method described by Yahagi et al. () and Matsushima et al. ().
- Preincubation period: 0.1 ml test solution or vehicle, 0.1 ml bacterial suspension and 0.5 ml S-9 mix or phosphate buffer are incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 ml of soft agar is added and, after mixing, the samples are poured onto the agar plates within approx. 30 seconds.
- Exposure duration: incubation at 37°C for 48 - 72 hours in the dark

SELECTION AGENT (mutation assays): Histidine and tryptophan auxotrophy

NUMBER OF REPLICATIONS: 3 test plates per dose or per control

NUMBER OF CELLS EVALUATED: The titer of viable bacteria was > 1081 ml.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:

Evaluation criteria:
- Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments.
- In general, five doses of the test substance are tested with a maximum of 5 mg/plate, and triplicate plating is used for all test groups at least in the
1st experiment.
- Dose selection and evaluation as weil as the number of plates used in repeat studies or further experiments are based on the findings of the ist experiment.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
A slight decrease in the number of his+ revertants was observed in the standard plate test depending an the strain and test canditions at doses > 2500 µg/plate
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Remarks:
anly a slight decrease in the number af trp~ revertants and a reduction in the titer in the experimental part with S-9 mix from about 500 µg/plate onward.
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: see below
- Precipitation: Test substance precipitation was found from about 2 500 µg/plate onward.

RANGE-FINDING/SCREENING STUDIES: yes


COMPARISON WITH HISTORICAL CONTROL DATA: yes


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Toxicity detected by a
- decrease in the number of revertants
- clearing or diminution of the background lawn (= reduced his or trp background growth)
- reduction in the titer is recorded for all test groups both with and withaut S-9 mix in all experiments and indicated in the tables.

Any other information on results incl. tables

Table 1: Results of the Salmonella typhimurium /  Escherichia coli reverse mutation assay

Dose level in µg/plate

- S9 mix

+ S9 mix

TA1535

TA100

TA1537

TA98

EC WP2 urvA

TA1535

TA100

TA1537

TA98

EC WP2 urvA

DMSO

19±2

112±9

10±3

28±3

34±3

19±4

107±6

11±1

33±5

38±4

20

20±1

106±8

10±1

28±2

35±9

17±1

114±16

12±5

37±10

34±7

100

16±3

111±6

10±2

27±4

28±5

16±3

101±9

9±2

32±2

35±5

500

19±6

103±4

7±1

26±4

30±5

11±1

103±12

10±1

28±3

36±6

2500

14±2

106±15

5±1

17±1

28±6

10±2

84±12

4±2

24±3

34±3

5000

10±3

75±9

3±2

15±2

29±2

6±2

64±11

4±1

15±4

32±2

Positive control

-S9 mix

640±45

848±32

477±

71

548±

78

549±

16

/

/

/

/

/

Positive control

+S9 mix

/

/

/

/

/

135±26

780±50

118±8

627±23

221±27

Negative controls

Parallel with each experiment, negative controls are carried out in order to check for possible contaminants (sterility control) and to determine the spontaneous mutation rate (vehicle control).

Sterility control

Additional plates are treated with soft agar, S-9 mix, buffer, vehicle or the test substance

but withaut the addition of tester strains (See Appendix 3).

Vehicle control

The vehicle control with and without S-9 mix only contains the vehicle used for the test

substance at the same concentration and volume for all tester strains.

Positive controls

The following positive controls are used to check the mutability of the bacteria and the activity of the 5-9 mix:

With S-9 mix

- 2-aminoanthracene (2-AA)(SIGMA, A-1 381): 2.5 µg/plate, dissolved in DMSO,-strains: TA 1535, TA 100, TA 1537, TA 98; 60 pg/plate, dissolved in DMSO, strain: Escherichia coli WP2 uvrA

Without S-9 mix

- N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (FLUKA, 68051): 5 µg/plate, dissolved in DM50,  strains: TA 1535, TA 100

- 4-nitro-o-phenylendiamine (NOPD) (SIGMA, N-9504): 10 µg/plate, dissolved in DM50,  strain: TA 98

- 9-aminoacridine (AAC) (SIGMA, A-1135): 100 µglplate, dissolved in DMSO, strain: TA 1537

- 4-nitroquinoline-N-oxide (4-NQO) (SIGMA, N-8141): 5 µg/plate, dissolved in DMSO, strain: E.coliWP2uvrA

The stability of the selected positive controls is weII-defined under the selected culture conditions, since they are weII-established reference mutagens.

Applicant's summary and conclusion

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

3,7,11-trimethyldodecyn-3-ol is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen here.
Executive summary:

The study is reliable without restrictions and was conducted according to OECD TG 471 und GLP conditions.

Genetic toxicity in vitro was tested in 4 strains of Salmonella typhimurium (TA1535, TA100, TA1537, TA98) and 1

E. coli WP2 uvrA strain. The test substance was tested for mutagenicity in the Salmonella typhimurium / 

Escherichia coli reverse mutation assay both in the standard plate test and in the preincubation test with and

without the addition of a metabolizing system (S-9 mix) obtained from rat liver using the Salmonella strains

TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA. According to the results of the present study,

the test substance did not Iead to an increase in the number af revertant colonies either without S-9 mix or after

adding a metabolizing system in two experiments carried out independently of each ather.

An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the
 preincubation test either without S-9 mix or after the addition of a metabolizing system.

The test substance was non mutagenic in the Ames test.