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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:
02 October 2003 to 23 October 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in accordance with recognised guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
other: OPPTS harmonised guidelines
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: colourless liquid
Details on test material:
- Physical state: colourless liquid
- Storage condition of test material: room temperature in the dark over silica gel under nitrogen

Method

Target gene:
Not required
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Non-mammalian study
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Non-mammalian study
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Range-finding test
0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate


Experiment 1: Main test
50, 150, 500, 1500, 5000 µg/plate

Experiment 2: Main test
15, 50, 150, 500, 1500, 5000 µg/plate

Experiment : Main test
Strain TA1535 without S9 only: 300, 500, 750, 1000, 1500 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test item was insoluble in water and DMSO.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Concurrent
True negative controls:
yes
Positive controls:
yes
Positive control substance:
ethylnitrosurea
Remarks:
2, 3 or 5 ug/plate for E.coli, TA100 and TA 1535 respectively
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Concurrent
True negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
80 ug/plate for TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Concurrent
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
0.2 ug/plate for TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Concurrent
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
1, 2 and 10 ug/plate for TA100, TA1535 and E.coli respectively
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Concurrent
True negative controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
5 ug/plate for TA98
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) at multiple 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 assay was performed by mixing 0.1 ml of bacterial culture (TAI00 or WP2uvrA-), 0.1 ml of the substance formulation, 0.5 ml of S9-mix or phosphate buffer and 2 ml of molten, trace histidine or tryptophan supplemented, top agar and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate). In total seven concentrations of the test material and a vehicle control (tetrahydrofuran) were tested. In addition, 0.1 ml of the maximum concentration of the test material and 2 ml of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.
DURATION
- Preincubation period: N/A
- Exposure duration: Approximately 48 hours
- Expression time (cells in growth medium): N/A
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): N/A

SELECTION AGENT (mutation assays): NDA
SPINDLE INHIBITOR (cytogenetic assays): N/A
STAIN (for cytogenetic assays): N/A

NUMBER OF REPLICATIONS: 3 replicates of each strain at each concentration both in the presence and absence of S9

NUMBER OF CELLS EVALUATED:
Cell viability at the end of pre-culture
RANGE FINDING TEST
All strains = 0.9 to 9.0 x 10^9/ml

MAIN TEST
All strains = 0.9 to 9.0 x 10^9/ml

DETERMINATION OF CYTOTOXICITY
- Method: growth of background lawn of bacteria and revertant colony count.

OTHER EXAMINATIONS:
N/A

OTHER:
Prior to the master strains being used, characterisation checks were carried out to confirm the amino-acid requirement, presence of rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate.

In order to select appropriate dose levels for use in the main test, a preliminary assay was carried out to determine the toxicity of the test material.
Evaluation criteria:
The test material may be considered positive in this test system ifthe following criteria are met:

The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression (5)) significant increase in the revertant count in at least one strain of bacteria.
Statistics:
Dunnett's linear regression.

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
Vehicle controls validity:
valid
Untreated negative 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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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

The substance was considered to be non-mutagenic under the conditions of this test.
Executive summary:
Introduction

The method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 “Bacterial Reverse Mutation Test”, Method B13/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines. 

Methods

Salmonella typhimurium strains TA1535, TAl537, TA98, TAlOO and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at a maximum of 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 50 to 5000 ug/plate in the first experiment. The experiment was repeated on a separate day using an amended dose range of 15 to 5000 ug/plate, fresh cultures of the bacterial strains and fresh test material formulations. An additional dose level was included in the second experiment to allow for test material induced toxicity, ensuring that a minimum of four non-toxic dose levels were achieved. A third, confirmatory experiment was performed using tester strain TA1535 (without S9 only) in an effort to confirm both reproducibility and a dose-response relationship. A dose range of 300, 500, 750, 1000 and 1500 ug/plate was employed using the plate incorporation method. 

Results

The vehicle (acetone) control plates gave 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 or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused a visible reduction in the growth of the bacterial background lawn of all the tester strains at 5000 ug/plate both with and without S9. A slight weakening in the integrity of the background lawn of TAl535 was noted at 1500 ug/plate (without S9 only). This response was slightly inconsistent however, as it was only observed in two out of three experiments. The test material was, therefore, tested up to the maximum recommended dose level of 5000 ug/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. Statistically significant, and moderately reproducible increases in revertant colony frequency were observed in tester strain TA1535, without S9 only, at the upper sub-toxic dose levels of the test material in Experiments 1 and 2. Therefore, a third, confirmatory experiment was performed, using the plate incorporation method. The third experiment was designed to enhance the weak dose-response relationship observed in the previous experiments. Small but statistically significant increases in revertant colony frequency were observed at only one dose level (1500 ug plate). However, in all three experiments, the increases never exceeded 1.75 times the concurrent solvent control and two of the statistically significant responses (1500 ug/plate in Experiments 1 and 2) were accompanied by weakened bacterial background lawns. Furthermore, there was no clear evidence of a dose-response relationship, even after the inclusion of a tightened dose range in Experiment 3. Therefore, the test material was considered not to be causing a mutagenic response. A statistically significant response was also noted in TA98, in the presence of S9 only, at 5000 pg/plate. This response was discounted however because it was non-reproducible and was accompanied by a weakened bacterial background lawn. 

Conclusion

The test material was considered to be non-mutagenic under the conditions of this test