2,5,7,10-tetraoxaundecane

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:

From 2010-03-26 to 2010-04-26

Reliability:

1 (reliable without restriction)

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)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,5,7,10-tertaoxaudecane

IUPAC Name:

2,5,7,10-tertaoxaudecane

Details on test material:

- Name of test material (as cited in study report): 2,5,7,10-tertaoxaudecane (TOU)
- Substance type: colourless liquid
- Physical state:
- Analytical purity: 99.632% (w/w)
- Impurities (identity and concentrations): alcohols (0.064%); aldehydes (not found); water (0.142%)
- Lot/batch No.: 0804061200
- Expiration date of the lot/batch: 06/04/2011
- Storage condition of test material: stored in a glas vial at laboratory temperature during the study

Method

Target gene:

his (trp) locus

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

- Type and identity of media: Nutrient Broth (Merck); Agar-agar (Fluka)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not specified
- Periodically checked for karyotype stability: yes, genotypes of each strain were controlled (plasmid pKM 101 - ampicillin resistance, uvr mutation, rfa mutation, his/trp mutation - spontaneous reversions)
- Periodically "cleansed" against high spontaneous background: not specified

S. typhimurium strains TA 1537 and TA 98 serve to detect frame shift mutations, strains TA100 and TA 1535 serve to detect base-pair substitution mutations.

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Type and identity of media: Nutrient Broth (Merck); Agar-agar (Fluka)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not specified
- Periodically checked for karyotype stability: yes, genotypes of each strain were controlled (plasmid pKM 101 - ampicillin resistance, uvr mutation, rfa mutation, his/trp mutation - spontaneous reversions)
- Periodically "cleansed" against high spontaneous background: not specified

E. coli strain WP2uvrA serve to detect cross-linking mutagens

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of rat liver homogenate and mixture of cofactors

Test concentrations with justification for top dose:

Toxicity test: 10, 100, 500, 1000, 2500, 5000 µg/plate with TA 100 without metabolic activation
First and second mutation experiments: 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation
(please refer to details to test system and conditions)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: not specified

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

(whitout solvent)

Negative solvent / vehicle controls:

yes

Remarks:

(contains 0.1 ml ofwater)

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Migrated to IUCLID6: 1.5 µg/plate; TA 100 and TA 1535 without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenilenediamine (20 µg/plate;TA 98 without metabolic activation)

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminofluorene (10 µg/plate; TA 100 and TA 98 with metabolic activation)

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthacene (1.0 µg/plate; TA 1535 - 2.5 µg/plate; TA 1537 - 25 µg/plate; E. coli with metabolic activation)

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 9-aminoacridine hydrochloride monohydrae (100 µg/plate; TA 1537 without metabolic activation)

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Migrated to IUCLID6: (20 µg/plate; E. coli without metabolic activation)

Details on test system and experimental conditions:

TEST PROCEDURE

100 µl of TOU of required concentration, 0.1 ml 16-18h culture of tester strain, 0.5 ml relevant buffer and 30 or 100 µl ofS9 postmitochondrial fraction were added to the 2 ml top agar (with trace of hisdine or tryptophan) kept in a testtube at 45 +/- 3°C. After shaking the mixture was poured into a minimal glucose agar plate. After incubation of 48-78h at 37+/- 1°C, the number of revertant colonies on the plate was counted manually with exception of positive controls, which were counted by an AccuCount 1000.

For an adequate estimate of variation, triplicate plating was used ateach dose level except in the toxicity test with strain TA100, where the substance was tested in duplicate. Each experiment was repeated0 As there was no toxicity, no precipitation or dose-responsiveness, doses in the second experiments remained the same as in the first experiment. In case of toxicity or precipitation, toxic (precipitating) doses would be omitted0 In case of mutagenicity, such doses would be chosen which allow construction of a dose-response curve.

SELECTION OF DOSE / TOXICITY

The test substance was dissolved in water for injections in the maximum cocentration given in gguidelines (5000 µg/plate).
- The highest concentration was further diluted. The concentration series (10-5000 µg/plate) was tested for toxicity in strain TA100 without metabolic activation.
- The first mutagenicity tests were done with doses of 50, 150, 500, 1500 and 5000 µg/plate.
- The second mutagenicity tests was performed with the same doses than the ones used in first mutagenicity tests.

All concentrations of the test substance suspension were dosed in the volume of 0.1 ml/plate. Fresh suspensions of test substance were prepared before each experiment. The suspensions were shaken during dilution, before dosing to the top agar and before pouring onto the plates.

Evaluation criteria:

The main criterion for evaluation of results was modified 2-fold increase rule that is compatible with the application of statistical methods. Following application of this rule, the result is positive, if a reproductible dose-response effect occurs and/or a doubling of the ratio of revertants at tested dose to number of revertants at negative doseis reached.

Statistics:

Dunkel & Chu (1980), Claxton et al. (1987)

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

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

Test results 2

Species / strain:

E. coli WP2 uvr A

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:

Toxicity test: Precipitation and signs of toxicity were not observed at any dose.
First and second mutagenicity test: The results obtained did not show substantial (biologically significant) increases in the number of revertants versus solvent control and no experiment gave evidence of rising trend in the number of revertants with increasing dose.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the described experimental design, the test substance 2,5,7,10-tertaoxaudecane (TOU) was non mutagenic for all Salmonella typhimurium as well as Escherchia coli strains both in experiments with and without metabolic activation.

Executive summary:

Test substance2,5,7,10-tertaoxaudecane (TOU)was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The test was performed according to EU method B.13/14 Mutagenicity – Reverse mutation test using bacteria, which is analogous to the OECD Test Guideline No. 471.

Four indicator Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and one indicator Escherchia coli WP2 uvrA strain was used. The test substance was diluted in water and assayed in doses of 50-5000 µg which were applied to plates in volumes of 0.1 mL.

Two series of experiments were performed with each strain – without metabolic activation and with a supernatant of rat liver and a mixture of cofactors.

Under the described experimental design, the test substance 2,5,7,10-tertaoxaudecane (TOU) was non mutagenic for all Salmonella typhimurium as well as Escherchia coli strains both in experiments with and without metabolic activation.