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Genetic toxicity in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25.11.1999 to 06.12.1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Qualifier:
according to guideline
Guideline:
other: Japan Ministry of Agriculture, Forestry and Fisheries (1985) Notifcation of Director General, Agricultural Production Bureau. NohSan No. 4200
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
other: Joint Directives of J EPA, J MHW and J MITI (31 October 1997) Kanpoan No.287, Eisei No. 127 and Kikyoku No. 2 (31 October 1997)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine for Salmonella. Tryptophan for E.coli
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver, S9
Test concentrations with justification for top dose:
Test 1: 0, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Test 2: :0, 5, 15, 50, 150, and 500 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The solubility of the test item was assessed at 50mg/l in acetone, in which it was dissloved.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide
Remarks:
Without S9. : Sodium Azide 5μg/plate for TA1535 and TA100. 9-Aminoacridine 30μg/plate for TA1537. 2-Nitrofluorene 1μg/plate for TA98. 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide 0.05μg/plate for WP2uvrA/pKM101
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
with S9: 2-Aminoanthracene 2μg/plate for TA1535 and 10μg/plate for WP2uvrA/pKM101. Benzopyrene 5μg/plate for TA1537, TA98 and TA100
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) for Experiment 1 and pre-incubation for Experiment 2.

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 72 hrs


NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.
Evaluation criteria:
Evaluation Criteria:
The mutagenic activity was assessed by applying the following criteria:
1. If treatment with the test item produces an increase in revertant colony numbers of at least twice the concurrent control, with some evidence of a positive dose relationship, in two experiments, with any bacterial strain either in the presence or absence of S9 mix, the test item will be considered to show evidence of mutagenic activity in this test system..
2.If treatment with the test item does not produce reproducible increases of at least 1.5 times the concurrent control in either mutation test, the test item will be considered to show no evidence of mutagenic activity in this test system.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The absence of colonies on sterility check plates confirmed the absence of microbial contamination.

The total colony counts on nutrient agar plates confirmed the viability and high cell density of the cultures of the individual organisms.

The mean revertant colony counts for the solvent controls were within 99% confidence limits of the current historical control range for the laboratory. Appropriate positive controls induced substantial increases in revertant colony numbers with all strains confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

First Test (Range finding) - There were no substantial increases in revertant colony numbers over control counts in any of the tester strains following exposure to the test substance at any concentration in either the presence or absence of S9 mix.
Toxicity observed as a thinning of the background lawn of non revertant cells and reduced revertant colony counts, ocurred in all strains following exposure to the the test substance at concentrations of 500µg/plate and above.
A top concentration of 500µg/plate was therefore selected for use in the second test.

Second Test - There were no substantial increases in revertant colony numbers over control counts in any of the tester strains following exposure to the test substance at any concentration in either the presence or absence of S9 mix.
Toxicity observed as a thinning of the background lawn of non revertant cells and reduced revertant colony counts, ocurred in all strains following exposure to the the test substance at concentrations of 500µg/plate and above.

Conclusions:
Interpretation of results: negative

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

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MITI, MHW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", EEC Annex to Directive 92/69/EEC Part B Method For Determination OfToxicity B13 and B14 and the USA, EPA (TSCA) OPPTS harmonized guidelines.

Methods

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA/pKM101 were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system. The dose range for the first test (range-finding) ranged between 5 and 5000µg/plate. The second test used a pre-incubation method and a dose range 5 to 500µg/plate.

 

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 item caused a visible reduction in the growth of the bacterial background lawns and/or a substantial reduction in the frequency of revertant colonies in the absence and presenc of S9-mix at concentrations of 500µg/plate and above.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method.

Conclusion

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

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine for Salmonella.
Species / strain / cell type:
other: Salmonella Typhimurium TA 97, TA98, TA100, TA102 & TA1535
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 homogenate
Test concentrations with justification for top dose:
5, 1.6, 0.5, 0.16 and 0.05 µL/plate. The concentrations were based on OECD 471 recommended concentration for non cytotoxic materials.
Vehicle / solvent:
purified water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Vehicle
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
mitomycin C
other: 4-Nitro-O-phenylene-diamie and 2 aminofluorene
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Agar plate incorporation and spot test method.
Plates were incubated at 37 ± 2°C for 48 - 72 hours

NUMBER OF REPLICATIONS:
Plate incorporation - Triplicate plating.
Spot test method - Single plating

STRAIN GENOTYPE VERIFICATION
The strains used were checked or presence of appropriate strain genotype chracteristics. These tests included verification of :
Presence of uvrB mutation
Presence or absence of R-factor plasmid
Presence of rfa mutation
Requirement for histidine
Evaluation criteria:
Evaluation Criteria:
The mutagenic activity was assessed by applying the following criteria:
1. A two fold increase over the spontaneous reversion rate (percent of control >200%).
2.Demonstration of a clear dose related response when dilutions are tested.
Key result
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid

See attached background information

All five tester strains showed the appropriate results in the genotype verification assay.

The highest test concentration 5µl/plate showed signs of bacterial toxicity evident by a reduced reversion rate. The four lower test concentrations did not produce a two fold increase in the number of revertant or produce a clear dose related response in any of the 5 tester strains. The test item spot plates did not show a zone of increased reversion at the inoculation site but did show a small zone of bacterial toxicity.

Conclusions:
Interpretation of results: negative

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

Methods

Salmonella typhimuriumstrains TA97, TA98, TA100 TA102 and TA1535 were treated with the test item using both the plate incorporation and spot test methods both with and without the addition of a rat liver homogenate metabolising system. The plate incorporation method dose ranged between 0.5 and 5 µl/plate. The spot test method test used a dose concentration of 5 µl/plate.

 

Results

The highest test concentration 5µl/plate showed signs of bacterial toxicity evident by a reduced reversion rate. The four lower test concentrations did not produce a two fold increase in the number of revertant or produce a clear dose related response in any of the 5 tester strains. The test item spot plates did not show a zone of increased reversion at the inoculation site but did show a small zone of bacterial toxicity.

Conclusion

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

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

Based on negative results in two bacterial mutation studies, the substance is not classified for mutagencity.