Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
16-jun-2009 to 29-jun-2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
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): Oleyl dipropylenetriamine
- Substance type: Off white turbid liquid
- Physical state: Liquid
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark under Nitrogen

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
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:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Experiment 1:
Preliminary test (without and with S9, TA100 and WP2uvrA): 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Main study (TA1535, TA1537, TA98 and TA100):
Without S9-mix: 0.3, 1, 3, 10, 20 and 33 µg/plate
With S9-mix: 0.3, 1, 3, 10, 33 and 66 µg/plate
Experiment 2:
TA1535, TA1537, TA98 and TA100
(Without S9): 0.3, 1, 3, 10, 20 and 33 µg/plate
(With S9): 0.3, 1, 3, 10, 33 and 66 µg/plate
WP2uvrA:
(Without and with S9): 1, 3, 10, 33, 66 and 100 µg/plate


Vehicle:
- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: Accepted and approved by authorities and international guidelines
Controlsopen allclose all
Negative controls:
no
Solvent controls:
yes
Remarks:
ethanol
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9

Migrated to IUCLID6: 5 µg/plate in saline for TA1535
Positive control substance:
9-aminoacridine
Remarks:
without S9

Migrated to IUCLID6: 60 µg/plate in water for TA1537
Positive control substance:
2-nitrofluorene
Remarks:
without S9

Migrated to IUCLID6: 10 µg/plate in DMSO for TA98
Positive control substance:
methylmethanesulfonate
Remarks:
without S9

Migrated to IUCLID6: 650 µg/plate in DMSO for TA100
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9

Migrated to IUCLID6: 10 µg/plate in DMSO for WP2uvrA
Positive control substance:
other: 2-aminoanthracene in DMSO for all tester strains
Remarks:
with S9
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies

OTHER:
- Precipitation of test substance
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
b) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) The increase in the mean number of revertant colonies follows the concentration of test substance (dose-response relationship).

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
Negative controls valid:
yes
Positive controls valid:
yes
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Slight precipitate was observed at the top dose of 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
In tester strain TA100, toxicity was observed at dose levels of 33 μg/plate and above in the absence and presence of S9-mix. In tester strain WP2uvrA, toxicity was observed at dose levels of at 100 µg/plate in the absence and presence of S9-mix

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
TA1535: without S9: 20 µg/plate and above and with S9: 33 µg/plate and above
TA1537: without S9: 20 µg/plate and above and with S9: 10 µg/plate and above
TA98: without S9: 20 µg/plate and above and with S9: 33 µg/plate and above
TA100: without S9: 20 µg/plate and above and with S9: 33 µg/plate and above
WP2uvrA: without S9: 66 µg/plate and above and with S9: 100 µg/plate and above

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.
The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Based on the results of this study it is concluded that Oleyl dipropylene triamine (C18 dipropylene triamine) is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.