Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2001-10-24 to 2001-11-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

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)
Remarks:
Certificate from the Federal Republic of Germany
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Name of test material (as cited in study reports): T002102
- Physical state: solid
- Appearance: white (SDS)

Method

Target gene:
histidine locus and tryptophan locus
Species / strain
Species / strain:
other: S. typhimurium TA98, TA100, TA1535, TA1537, and E. coli WP2 uvrA
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and B-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Pre-experiment: concentration ranged from 3 to 5000 µg/plate
Experiments I & II: 33, 100, 333, 1000, 2500, and 5000 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
Controlsopen allclose all
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9; at 10 μg/plate with TA1535 and TA100
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-ophenylene-diamine
Remarks:
without S9; at 10 µg/plate with TA98, 50 µg/plate with TA1537
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9; at 5 µg/plate with WP2uvrA
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA)
Remarks:
with S9; at 2.5 μg/plate with TA 1535, TA 1537, TA 98 and TA 100, at 10 μg/plate with WP2 uvrA
Details on test system and conditions:
METHOD OF APPLICATION:
in agar (plate incorporation) (experiment I) and preincubation (experiment II)

Experimental performance:
The following materials were mixed in a test tube and poured onto the minmal agar plates:
- 100 µL of test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
- 500 µL of S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation)
- 100 µL of bacteria suspension (c.f. test system, pre-culture of the strains)
- 2000 µL overlay agar
In the pre-incubation assay 100 µL test solution, 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and shaken at 37°C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45°C) was added to each tube. The mixture was poured on minimal agar plates.
After solidification the plates were incubated upside down for at least 48 h at 37°C in the dark.

DURATION
- Preincubation period: 60 minutes (experiment II)
- Exposure duration: at least 48 hours
- Selection time: 48h (simultaneous with exposure)

SELECTION AGENT: tryptophan (E. coli) and histidine (S. typhimurium)

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
Method:reduction in the number of spontaneous revertants or a clearing of the bacteria background lawn.

Evaluation criteria:
A test substance is considered positive if a dose related increase in the number of revertants exceeding the threshold or twice the colony count of the corresponding solvent control is observed at more than one concentration. Furthermore, a biologically relevant and reproducible increase exceeding the threshold at one test concentration is considered as positive.
A test substance producing neither a dose-related increase in the number of revertants nor a biologically relevant positive response at any one of the test points is considered non-mutagenic in this system.

A biologically relevant response is described as follows:
An increase is considered relevant if in strains TA98, TA100, and WP2 uvrA the number of reversions is at least twice as high and in strains TA1535 and TA1537 at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent and reproducible increase in the number or revertants is regarded as an indication of possible existing mutagenic potential of the test substance regardless whether the highest dose will induce the above described enhancement factors or not.
Statistics:
No statistical evaluation was performed.

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA98, TA100, TA1535, TA1537, and E. coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: No precipitation of the test substance occurred up to the highest investigated dose.
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES:
To evaluate the toxicity of the test substance a pre-experiment was performed with all the strains. Eight concentrations (3-5000 µg/plate) were tested for toxicity and mutation induction with three plates each. This pre-experiment was reported as part of Experiment I based on the following criteria being met: evaluable plates (>0 colonies) at five concentrations or more in all strains used. This pre-experiment was used to select dose levels for Experiment II.

COMPARISON WITH HISTORICAL CONTROL DATA:
The vehicle and negative control values were within historical data control ranges in the absence of metabolic activation. No data was given for historical ranges in the presence of metabolic activation.
 The historical range of positive controls was exceeded without metabolic activation in strains TA1535, TA100 and WP2 uvrA. This effect indicates the sensitivity of the strains rather than compromising the assay. The positive controls showed a distinct increase of induced revertant colonies. Appropriate mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The plates incubated with the test substance showed normal background growth up to 5000 ug/plate with and without S9 in both experiments. Toxic effects, evident as a reduction in the number of revertants (below the factor of 0.5) were observed at the follow concentrations:
In experiment I, toxic effects were observed at 5000 µg/plate with and without S9 in TA1535 and TA1537, with S9 in TA98, and without S9 in WP2 uvrA.
In experiment II, toxic effects were observed at 333-5000 ug/plate without S9 in TA1535, 2500-5000 µg/plate with S9 in TA1535, 2500-5000 µg/plate without S9 in TA1537, 5000 µg/plate with and without S9 in TA98, 5000 µg/plate without S9 in TA100, 2500-5000 µg/plate without S9 in WP2 uvrA and 5000 µg/plate with S9 in WP2 uvrA.

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

During the described mutagenicity test and under the experimental conditions reported, the test substance did not induce gene mutation by base pair changes or frameshifts in the genome of the strains used in the Ames test in the presence or absence of metabolic activation. Based on these results, the test substance is not to be classified as mutagenic.