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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May-June 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

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)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Colour: colourless
- Odour: odourless
- CAS-Number: 89-32-7
- Molecular formula: C10 H2 O6
- Molecular weight: 218.12
Specific details on test material used for the study:
- Sponsor's identification: LZ6119
- Description: white powder
- Batch number: CSA 0901038
- Purity: >= 99.5% w/w
- Date received: 01 May 2009
- Storage conditions: room temperature in the dark

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
CELLS USED
- Suitability of cells: characterisation checks were carried out
- Source of cells: University of California, Berkeley, USA on 4 August 1995 (Salm. typh.) and British Industrial Biological Research Association on 17 August 1987 (E. coli)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: dimethylsulfoxide (DMSO)
- Properly maintained: yes
Additional strain / cell type characteristics:
other: incapable of synthesising histidine
Metabolic activation:
with and without
Metabolic activation system:
S9 was prepared in-house from livers of male rats and induced with phenobarbitone/beta-naphthoflavone
Test concentrations with justification for top dose:
Preliminary Toxicity Test:
In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate. The test material was toxic to TA100 at and above 1500 ug/plate in both the presence and absence of S9. The test material was also toxic to WP2uvrA (absence of S9) at 5000 ug/plate but non-toxic to the same strain in the presence of S9.

Mutation Test- Experiment 1:
Seven concentrations of the test material (5, 15, 50, 150, 500, 1500 and 5000 ug/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method. Additional dose levels were included to allow for test material induced toxicity, ensuring that at least four non-toxic doses were achieved.

Mutation Test - Experiment 2:
The second experiment was performed using methodology as described for Experiment 1, using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as Experiment 1 (5 to 5000 ug/plate).
Vehicle / solvent:
Dimethylsulfoxide (DMSO)
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- In agar (plate incorporation)

DURATION
- Exposure duration: 48 hours
Rationale for test conditions:
In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate.
Evaluation criteria:
The test material will generally be considered mutagenic to bacteria if the following criteria are achieved. If the following criteria are not achieved then the test material will be considered non-mutagenic to bacteria:
1) In strain TA98, TA100 or WP2uvrA-pKM101, a two-fold increase in the mean number of revertants per plate compared to the mean value of the concurrent vehicle control.
2) In strain TA1535 or TA1537, a three-fold increase in the mean number of revertants per plate compared to the mean value of the concurrent vehicle control.
3) Increases in revertant numbers for all strains must be related to increases in test material concentration.
4) A positive response in one tester strain either with or without exogenous metabolic activation is sufficient to designate the test material as a bacterial mutagen.
Statistics:
none

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative 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
Remarks:
initially from 1500 ug/plate in both the presence and absence of S9 (500 ug/plate for TA98 in the absence of S9 in Experiment 2 only).
Vehicle controls validity:
valid
Untreated negative 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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The sensitivity of the tester strains to the toxicity of the test material varied slightly between strain type, exposures with or without S9-mix and experiment number. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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

The study was performed in conformity to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities, OECD no. 471 and EU-methods B.13/14. Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA pKM101 were tested with the test material using the Ames plate incorporation method at seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (S9). The dose range was determined in a preliminary toxicity assay and was 5 to 5000 ug/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. Additional dose levels were included to allow for test material induced toxicity, ensuring that at least four non-toxic doses were achieved. The test material caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 1500 ug/plate in both the presence and absence of S9 (500 ug/plate for TA98 in the absence of S9 in Experiment 2 only). The test material was 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 89-mix. No increases in the frequency of revertant colonies, in excess of two or three fold (depending on the tester strain type) greater than the concurrent solvent controls, were recorded for any of the bacterial strains, for any dose level of the test material, either with or without metabolic activation. The test material was considered to be non-mutagenic under the conditions of this test.