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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:
From 12 August 1996 to 20 September 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline 471 and EU method B.14. GLP study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

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:
- Physical state: extremely pale straw coloured slightly viscous liquid
- Storage condition of test material: ambient temperature (< 25 ºC), shielded from light

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Experiment 1: 0, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2: 0, 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
(DMSO)
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without metabolic activation

Migrated to IUCLID6: (TA100 and TA1535)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without metabolic activation

Migrated to IUCLID6: (TA1537)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without metabolic activation

Migrated to IUCLID6: (TA98)
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine (TA1538)
Remarks:
Without metabolic activation
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (all strains)
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation). Known aliquots (0.1 ml) of one of the bacterial suspensions were dispensed into sets of sterile test tubes followed by 2.0 ml of molten, trace histidine supplemented, top agar at 45 ºC, 0.1 ml of the appropriately diluted test material or vehicle control and either 0.5 ml of the S9 liver rnicrosome mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates. All of the plates were incubated at 37 ºC for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

NUMBER OF REPLICATIONS: Triplicates

DETERMINATION OF CYTOTOXICITY
- Method: The plates were examined for a thinning of the background lawn.
Evaluation criteria:
For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in mutation rate in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at subtoxic dose levels. In the event of the two experiments giving conflicting or equivocal results, then a third experiment may be performed to confirm the correct response.

To be considered negative the number of induced revertants compared to spontaneous revertants should be less than twofold at each dose level employed, the intervals of which should be between two and five fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5000 µg/plate. In this case the limiting factor was the maximum recommended dose.
Statistics:
All data are statistically analysed using the methods recommended by the UKEMS and normally Dunnett's method of linear regression is used to evaluate the result.

Results and discussion

Test results
Species / strain:
other: all tested strains
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(at a dose level of 5000 µg/plate to the majority of strains of Salmonella tested)
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The dose range of the test material used in the preliminary toxicity study was 0, 50, 150, 500, 1500 and 5000 µg/plate. The test material exhibited toxicity at and above 1500 µg/plate to the strain of Salmonella used (TA100).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test material caused a visible reduction in the growth of the bacterial lawn at a dose level of 5000 µg/plate to the majority of strains of Salmonella tested.
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

No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test material, either with or without metabolic adivation. The test material was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were treated with the test material using the Ames plate incorporation method at up to six dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 15 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using a dose range of 50 to 5000 µg/plate, fresh cultures of the bacterial strains and fresh test material formulations. An extra dose was included in Experiment 1 to allow for the toxicity

of the test material. The method used conforms with the OECD Guidelines for the Testing of Chemicals, Protocol No. 471 and also with Method B14 in Commission Directive 92/69/EEC.

The vehicle (dimethyl sulphoxide) control plates produced counts of revertant colonies within the normal range.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without the metabolising system.

The test material caused a visible reduction in the growth of the bacterial lawn at a dose level of 5000 µg/plate to the majority of strains of Salmonella tested. The test material was tested up to the maximum recommended dose of 5000 µg/plate.

No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test material, either with or without metabolic adivation. The test material was considered to be non-mutagenic under the conditions of this test.