Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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:
1999-04-13 to 1999-04-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Additional strain / cell type characteristics:
other: trptophan auxotroph
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: histidin auxotroph
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (phenobarbital/ß-naphtoflavone induced), Lot-No. 070898, micorsomal protein-content: 24.7 mg/ml; purchased by CCR GmbH & Co. KG, 64380 Roßdorf, Germany
Test concentrations with justification for top dose:
Preliminary assay with TA 100 : 0.38, 0.76, 1.52, 3.03, 6.06, 12.13, 24.25, 48.5, 97.00 mg/plate
1st assay performed on all strains: 24.25, 76.69, 242.50, 766.85, 2425.00 µg/plate
2nd assay performed on all strains: 75.78, 151.56, 303.13, 606.25, 1212.5 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Aqua dest.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, 0.5 µg/plate for TA 100, TA 98, 2.0 µg/plate for TA 1535, TA 1537, and 1.0 µg/plate for E. coli with metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Migrated to IUCLID6: 12.9 µg/plate for E. coli without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Migrated to IUCLID6: 50 µg/plate for TA 1537, without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene, 0.2 µg/plate for TA 98 without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Migrated to IUCLID6: 0.25 µg/plate for TA 100, TA 1535 without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Exposure duration: 48 h

SELECTION AGENT (mutation assays): histidine for s.typhimurium strains, tryptophan for E. coli

NUMBER OF PLATES EVALUATED: 3 parallel plates were performed for each experimental point. Two indipendent experiments.

DETERMINATION OF CYTOTOXICITY
- Method: The highest concentration used in the test must either reduce the survival of the cells to at least 50 % or reduce the background growth of auxotrophic cells and the number of revertants.

OTHER: Because of the reduced background lawn in the 1st assay the 2nd assay was performed at a concentration range from 75.78 to 1212.5 1 µg/plate.
Evaluation criteria:
Selection of the test compound:
The highest concentration used in the test must either reduce the survival of the cells to at least 50 % or reduce the background growth of auxotrophic cells and the number of revertants. If no toxicity is observed, the survey is carried out with the highest soluble concentration of the test article, however, not exceeding 5 mg/plate for solids and 10 mM of Iiquids or 200 µl/plate of liquids or extracts. The concentration intervals between the test doses have maximum a factor of sqrt. 10,

Analysis of results:
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 af mutagenic activity must be reproducible in an independent 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 / choice of top concentrations:
cytotoxicity
Remarks:
at 1212.5 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
1212.5 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
PRELIMINARY TOXICITY TEST:
The test substance reduced the survival at a concentration of 1.52 mg/plate to 46.3 % of the control value. The number of the his-cells of the strain TA 100 at the concentration of 1.52 mg/plate was reduced to 58.3 % of the control value. No precipitation could be observed up to 97 mg/plate.

MAIN TEST:
The results of the 1st assay and the 2nd assay indicate, that in the tested concentration range (1 st assay: 24.25, 76.69, 242.5, 766.85 and 2425.0 µg/plate, 2nd assay: 75.78, 151.56, 303.13, 606.25 and 1212.5 µg/plate) with and without metabolic activation no significant increase in the numbers of his+- or trp+-revertants over the spontaneous values could be detected with the Salmonella strains TA 100, TA 1535, TA 98, TA 1537
and E. coli WP2 uvrA pKM(101).

Because of the reduced background lawn in the 1st assay the 2nd assay was performed at a concentration range from 75.78 to 1212.5 1 µg/plate.

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

Applicant's summary and conclusion

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

Evaluation of the results of the two independent plate incorporation experiments did not provide evidence of a mutagenic potency of the substance.
Up to 2425.0 µg/plate, the substance did not induce biologically relevant increases in revertants in any of the tested strains. This applied to both the activated and the non-activated assay system. The substance is therefore found not to be genetically active under the aforementioned test conditions.
Executive summary:

In a reverse gene mutation assay in bacteria according OECD guidelines 471/472, and EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria), 1992, four strains of S. typhimurium (TA 1535, TA 1537, TA 100 and TA 98) and E. coli WP2 uvr A pKM 101 were exposed to the substance (48.5 % a.i. in Aqua dest.), at concentrations of 24.25, 76.69, 242.50, 766.85, 2425.00 µg/plate (1st assay) and 75.78, 151.56, 303.13, 606.25, 1212.5 µg/plate (2nd assay) in the presence and absence of mammalian metabolic activation (plate incorporation). Concentration selection for the first assay was based on a preliminary toxicity study with and without metabolic activation on strain TA 100 in which the concentration of 1.52 mg/plate was cytotoxic. Because of an reduced background lawn in the 1st assay the 2nd assay was performed at the concentration range from 75.78 to 1212.5 µg/plate.

No evidence of biologically significant mutagenic activity of the test item was found in the presence and absence of metabolic activation, up to and including the highest tested concentration of 2425 µg/plate. The test material was tested up to its toxic limit. The positive controls induced the appropriate responses in the corresponding strains and thus demonstrated the sensitivity of the indicator strains and the activity of metabolizing system..

There was no evidence of induced mutant colonies over background.

The adopted OECD TG 471 (1997) requires at least 5 test strains. These should include four strains of S. typhimurium (TA1535; TA137 or TA97a or TA97; TA98; and TA 100 and in addition the use of E. coliWP2 strains or Salmonella typhimurium TA 102 to detect certain oxidizing mutagens, cross-linking agents and hydrazines. This requirement is fulfilled by this test performed according to former versions of OECD guidelines 471/472 and EU Method B.13/14 (Version Commission Directive 92/69/EEC) on S. typhimurium (TA 1535, TA 1537, TA 100 and TA 98) and E. coliWP2 uvr A pKM 101.

Thus, this GLP test is considered as sufficient to evaluate the mutagenic activity of the substance in this bacterial test system.