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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 20, 2004 - July 29, 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
(2000)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
453-140-3
EC Name:
-
Cas Number:
667465-46-5
Molecular formula:
C20H38N6O13 (not dissociated) C20H44N6O13 (dissociated in water)
IUPAC Name:
3,6,9-tris(2-{[(2-hydroxyethyl)amino]oxy}-2-oxoethyl)-3,6,9-triazaundecan-1,11-dioic acid
Constituent 2
Chemical structure
Reference substance name:
Water
EC Number:
231-791-2
EC Name:
Water
Cas Number:
7732-18-5
Molecular formula:
H2O
IUPAC Name:
Water
Test material form:
liquid
Details on test material:
- Name of test material: DHX2
- CAS no.: 667465-46-5
- Storage condition of test material: at room temperature in the dark

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by Aroclor 1254.
Test concentrations with justification for top dose:
Preliminary test (without and with S9) TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate

Experiment 1:
TA1535, TA1537, TA98 and TA100: Without and with S9-mix: 3, 10, 33, 100, 166 and 333 µg/plate
WP2uvrA: Without and with S9-mix: 3, 10, 33, 50, 75 and 100 µg/plate

Experiment 2:
TA1535 and TA1537: Without and with S9-mix: 0.3, 1, 3, 10, 33 and 100 µg/plate
TA98 (Without and with S9-mix) and TA100 (without S9-mix): 1, 3, 10, 33, 100 and 166 µg/plate
TA100 (with S9-mix): 3, 10, 33, 100, 166 and 333 µg/plate
WP2uvrA (Without S9-mix): 3, 10, 33, 50, 75 and 100 µg/plate
WP2uvrA (with S9-mix): 10, 33, 50, 75, 100 and 333 µg/plate

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Milli-Q water
- Justification for choice of solvent/vehicle: The test substance is an aqueous solution
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
650 μg/plate in DMSO for TA100
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
10 μg/plate in DMSO for TA98 and 15 μg/plate for TA1537
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
10 μg/plate in DMSO for WP2uvrA
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
5 μg/plate in saline for TA1535
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene in DMSO for all tester strains
Remarks:
with S9 all tester strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

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

NUMBER OF CELLS EVALUATED: 10E8 per plate

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 EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not biologically relevant.
b) The positive response should be reproducible in at least one independently repeated experiment.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
but tested up to the cytotoxic concentration of 333 μg/plate.
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
but tested up to the cytotoxic concentration of 333 μg/plate.
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
but tested up to the cytotoxic concentration of 333 μg/plate.
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
but tested up to the cytotoxic concentration of 333 μg/plate.
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
but tested up to the cytotoxic concentrations of 100 and 333 µg/plate.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose

RANGE-FINDING/SCREENING STUDIES:
- In tester strain TA100, toxicity was observed at dose levels of 333 μg/plate and above in the absence and presence of S9-mix. In tester strain WP2uvrA, toxicity was observed at dose levels of 100 μg/plate and above 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:
Experiment 1:
TA1535 and TA1537 : without S9 and with S9: 100 µg/plate and above
TA98 and TA100: without S9: 100 µg/plate and above and with S9: 166 µg/plate and above
WP2uvrA: without S9: 100 µg/plate
Experiment 2:
TA1535: without S9 and with S9: 100 µg/plate
TA1537: without S9: 100 µg/plate and with S9: 33 µg/plate and above
TA98: without S9: 33 µg/plate and above and with S9: 100 µg/plate and above
TA100: without S9 and with S9: 100 µg/plate and above
WP2uvrA: without S9 and with S9: 75 µg/plate and above

Applicant's summary and conclusion

Conclusions:
The substance DHX2 is not mutagenic in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay.
Executive summary:

DHX2 was tested in the Salmonella typhimurium reverse mutation assay with TA1535, TA1537, TA100 and TA98 and in the Escherichia coli reverse mutation assay with WP2uvrA, in accordance with OECD 471, EU Method B.14 and according to GLP principles. Toxicity was observed in all tester strains in the absence and presence of S9-mix, except in tester strain WP2uvrA in the presence of S9-mix in experiment 1. DHX2 did not induce a dose-related increase in the number of revertant (His+) colonies in each of the four tester strains and in the number of revertant (Trp+) colonies in the tester strain both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. Based on the results of this study it is concluded that DHX2 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.