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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:
1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
4 strains instead of 5 used
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
29th November, 1992
Deviations:
yes
Remarks:
4 strains instead of 5 used
Principles of method if other than guideline:
Four strains instead of five.
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
hexasodium 4-{2-[(9E)-12-hydroxyoctadec-9-enamido]ethoxy}-4-oxo-2-sulfobutanoate 4-{[(9E)-17-({2-[(3-carboxy-3-sulfopropanoyl)oxy]ethyl}carbamoyl)heptadec-9-en-7-yl]oxy}-4-oxo-2-sulfobutanoate
EC Number:
939-654-5
Molecular formula:
Molecular formula cannot be given as the substance is a mixture
IUPAC Name:
hexasodium 4-{2-[(9E)-12-hydroxyoctadec-9-enamido]ethoxy}-4-oxo-2-sulfobutanoate 4-{[(9E)-17-({2-[(3-carboxy-3-sulfopropanoyl)oxy]ethyl}carbamoyl)heptadec-9-en-7-yl]oxy}-4-oxo-2-sulfobutanoate
Test material form:
other: liquid

Method

Target gene:
histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: The strains TA98 and TA100 have an additional plasmid (pkM 101) which confers resistance against ampicillin. This plasmid also carries a gene (muc+), which is involved in recA+/lexA- genotypes in the SOS-DNA- repair system.
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 0.000; 0.780; 1.560; 3.125; 6.250; 12.500; 25.000; 50.000; 100.000; 200.00µL/plate (TA100)
1st Assay (Table II): 0.00; 0.04, 0.20, 1.00, 5.00 and 25.00 µL per plate (TA1535, TA1537, TA98, TA100 all +S9 and –S9)
2nd Assay (Table III): 0.00; 0.30; 0.93; 2.78; 8.33 and 25.00 µL/plate (TA1535, TA1537, TA98, TA100 all +S9 and –S9)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water (Aqua dest.)
- Justification for choice of solvent/vehicle: test substance dispersable in water
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
for all strains with as well as without S-9 mix
Negative solvent / vehicle controls:
yes
Remarks:
for all strains with as well as without S-9 mix
Positive controls:
yes
Positive control substance:
other: Na-azide 0.25 µL/plate
Remarks:
TA100 and TA1535 ; -S9
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA98 ; -S9
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA1537 ; -S9
Positive controls:
yes
Positive control substance:
other: 2-aminoantrhacene 0.5 µg/plate
Remarks:
TA100, TA98 ; +S9
Positive controls:
yes
Positive control substance:
other: 2-aminoantrhacene 2.0 µg/plate
Remarks:
TA1535 and TA1537; +S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 3 days (at 37°C in the dark)

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: 3


DETERMINATION OF CYTOTOXICITY
- Method: other: The criterion for a biologically significant bacteriotoxic effect is either a reduction of the survival of the cells to at least 50% or a reduction of the background growth of auxotrophic cells and the number of revertants.



Evaluation criteria:
In the Ames assay a test compound is considered as mutagenic if there is a dose dependent increase in the number of revertant colonies of one or more strains. For the highest non-toxic concentration an increase should be by a factor of about 2 and deemed to be biologically relevant. Any evidence of mutagenic activity must be reproducible in an independent experiment. In addition statistical evaluation may aid data interpretation.

Results and discussion

Test results
Key result
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
Remarks:
not in main study, however in the range finding study 25.00µL per plate (highest tested concentration in main study) reduced the survival to 5.3% of the control value.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: Preliminary Toxicity Test: 0.000; 0.780; 1.560; 3.125; 6.250; 12.500; 25.000; 50.000; 100.000; 200.00µL/plate (TA100)
The test compound [Trade name] reduced the survival at a concentration of 25 µl per plate to 5.3 % of the control value.
An increase in numbers of his+-revertants over the control value at a concentration of 200 µl per plate was due to histidine-auxotroph cells which were able to grow at reduced cell density.
Therefore the mutagenicity assays were performed in a concentration range from 0.04 to 25.00 µl per plate.

MAIN TEST
Concentrations between 0.04 and 25.00 µl/plate were applied to the bacterial strains TA 1535, TA 1537, TA 98 and TA 100.

No evidence of biologically significant mutagenic activity of [Trade name] was found. The results of the 1st assay and the 2nd assay indicate, that in the tested concentration range with and without metabolic activation no significant increase in the numbers of his+-revertants over the spontaneous values could be detected with the Salmonella tester strains TA 100, TA 1535, TA 98 and TA 1537.

No bacteriotoxic effects were induced at the tested concentrations.

STERILITY CONTROLS
The plates for the sterility control of top agar, S9-mix and test compound showed no growth.

NEGATIVE CONTROLS
In all experiments the control plates without mutagen showed a normal number of spontaneous revertants

POSITIVE CONTROLS
The positive controls demonstrated the sensitivity of the indicator strains and the activity of the metabolizing system.

COMPARISON WITH HISTORICAL CONTROL DATA: yes: Historical Spontaneous Mutation frequencies to Histidine-Protrophy of the Tester-Strains used in Labor L+S AG in 1993.


Remarks on result:
other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:
Evaluation of the results of the two independent experiments did not provide evidence of a mutagenic potency of the test item.
At up to 25.0 µL per plate (cytotoxic concentration), the test item, did not induce biologically relevant increases in revertants in any of the tester strains. This applied to both the activated and the non-activated assay system.
The test item is therefore not found to be genetically active under the aforementioned test conditions.
Executive summary:

The test item containing 39.8% active ingredient was assayed in a bacterial gene mutation assay (Ames test) using the strains TA100, TA1535, TA98 and TA1537. Induced his+-revertants were determined both in the absence and presence of metabolic activation by a rat liver post-mitochondrial fraction (S9) from Aroclor 1254 induced animals. In two independent mutation experiments, cells were exposed to concentrations of 0.04, 0.20, 1.00, 5.00 and 25.00 (cytotoxic concentration) µL per plate (2nd assay) in the absence and presence of S9. In order to demonstrate the sensitivity of the assay system, positive control agents were used and marked increases in his+-revertants were induced in all tester-strains. In the 1st and 2nd assay the test item induced neither cytotoxicity nor statistically significant increases in histidine-protrophy revertants in any tester-strains with and without metabolic activation in the tested concentration range.

It is thus concluded that the test item did not induce gene mutations in the bacterial mutagenicity assay with and without metabolic activation in vitro when tested under the experimental conditions reported.