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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:
08 January 2003 to 17 January 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
See principles of method if other than guideline
Qualifier:
equivalent or similar to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
not applicable
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
not applicable
Principles of method if other than guideline:
Deviations from the protocol:
M. van der Kaaden was involved in this study instead of Ms. G.C.D.M. Bruyntjes-Rozier and Ms. M.J.M. van den Wijngaard.
This deviation is considered not to have influenced the integrity and outcome of the study.
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: colourless liquid
- Storage condition of test material: at < -18 °C, in the absence of light

Method

Target gene:
Histidine is the target gene for salmonela
Tryptophan is the target gene for E.coli
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
In total six concentrations were tested: 0.1, 0.3, 0.8, 2.3, 7 and 21 µg/plate.
The actual concentrations of the test material in the test solutions were not determined. Therefore, the concentrations quoted in this report are nominal concentrations.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
For TA1535, TA98, TA100 and E.coli WP2 uvrA in the presence of S9-mix.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
For TA1537 in the presence of S9-mix.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
For TA1535 and TA100 in the absence of S9-mix.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
For TA1537 in the absence of S9-mix.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
For TA98 in the absence of S9-mix.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylnitrosurea
Remarks:
For E.coli WP2 uvrA in the absence of S9-mix.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: The plates were incubated at ca. 37 °C for 48-72 hours.
- Exposure duration: 48-72 hours.

NUMBER OF REPLICATIONS:
All determinations were made in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is defined as a reduction in the number of revertant colonies andlor a clearing of the background lawn of bacterial growth.

Evaluation criteria:
The mutagenicity study is considered valid if the mean colony counts of the control values of the strains are within acceptable ranges, if the results of the positive controls meet the criteria for a positive response, and if no more than 5 % of the plates are lost through contamination or other unforeseen events.

A test material is considered to be positive in the bacterial gene mutation test if the mean number of revertant colonies on the test plates is concentration-related increased or if a reproducible two-fold or more increase is observed compared to that on the negative control plates.

A test material is considered to be negative in the bacterial gene mutation test if it produces neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the test points.

In case of an inconclusive first assay, a second independent assay was conducted. The first mutagenicity assay is regarded inconclusive if a positive or equivocal response at only one concentration is observed or if a positive or equivocal responses at several concentrations without a concentration-related increase are observed.

Omission of the second assay under these conditions is acceptable as a single assay does not or hardly results in false negative conclusions.

Positive results from the bacterial reverse mutation test indicate that a substance induces point mutations by base substitutions or frameshifts in the genome of either Salmonella typhimurium and/or Escherichia coli. Negative results indicate that under the test conditions, the test substance is not mutagenic in the tested strains.
Statistics:
No statistical analysis was performed.

Both numerical significance and biological relevance are considered together in the evaluation.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98 & TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
other: TA 1535, TA 98 and TA 100
Metabolic activation:
with
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
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
A dose range finding test to assess the toxicity of the test material to the bacteria was performed with TA98 both in the absence and presence of S9-mix. DMSO was chosen as vehicle. Just before use, the test material was diluted in the vehicle at 50 mg/mL. A transparent colourless solution was obtained. Serial 3-fold dilutions in DMSO were prepared and in total ten dilutions were tested, ranging from 0.3 to 5000 µg/plate.

Any other information on results incl. tables

The test material was diluted in DMSO. A dose range finding test was performed with TA98 in both the absence and the presence of S9-mix with ten different concentrations of the test substance, ranging from 0.3 - 5000 µg/plate. The test material was toxic at concentrations above 7 µg/plate.

In the main bacterial reverse mutation test six different concentrations were tested ranging from 0.1 - 21 µg/plate. Negative controls (solvent) and positive controls were run simultaneously with the test material.

In the absence of S9-mix, the test material was toxic to all Salmonella typhimurium strains at 7 and 21 µg/plate and in the presence of S9-mix to strain TA 1537 at 21 µg/plate, as was evidenced by the presence of pinpoints or a decrease in the mean number of revertant colonies compared to the negative controls.

In both the absence and the presence of S9-mix and in all strains, the test material did not cause a more than two-fold increase in the mean number of revertant colonies appearing in the test plates compared to the background spontaneous reversion rate observed with the negative control.

The mean number of his+ and trp+ revertant colonies of the negative controls were within the acceptable range, and the positive controls gave the expected increase in the mean number of revertant colonies.

It is concluded that the results obtained with the test material in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the Escherichia coli strain WP2 uvrA, in both the absence and the presence of the S9-mix, indicate that the test material was not mutagenic under the conditions employed in this study.

Applicant's summary and conclusion

Conclusions:
It is concluded that the results obtained with the test material in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the Escherichia coli strain WP2 uvrA, in both the absence and the presence of the S9-mix, indicate that the test material was not mutagenic under the conditions employed in this study.
Executive summary:

In a Bacterial reverse mutation test, the results obtained with the test material in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and in the Escherichia coli strain WP2 uvrA, in both the absence and the presence of the S9-mix, indicate that the test material was not mutagenic under the conditions employed in this study.

In the absence of S9-mix, the test material was toxic to all Salmonella typhimurium strains at 7 and 21 µg/plate and in the presence of S9-mix to strain TA 1537 at 21 µg/plate, as was evidenced by the presence of pinpoints or a decrease in the mean number of revertant colonies compared to the negative controls.

The study was conducted to the OECD Guideline no. 471.