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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:
The experimental phase of this study was performed between 10 January 2014 and 28 March 2014.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with GLP and OECD guideline, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
other: US Food and Drug Administration, Center for Food Safety& Applied Nutrition, Redbook 2000 Toxicological Principles for the safety of Food Ingredients. IV.C.1.a. Bacterial Reverse Mutation Test, July 2000
GLP compliance:
yes (incl. certificate)
Remarks:
TNO Triskelion, Utrechtseweg 48, 3704 HE Zeist, The Netherlands
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): FRET 10-0367
- Description: clear colourless liquid
- Storage conditions : 2-10 °C, protected from light
- Molecular Formula : C12H24O
- Molecular weight : 184.18 g/mol


Method

Target gene:
Salmonella typhimurium: histidine
Escherichia coli: tryptophan
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):
Frozen stocks of each strain were checked for histidine requirement and for sensitivity to ampicillin, crystal violet and UV radiation.
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Frozen stocks of each strain were checked for tryptophan requirement and for sensitivity to ampicillin, crystal violet and UV radiation.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 mix
Test concentrations with justification for top dose:
- First test: 5 concentrations, 62 to 5000 μg/plate
- Repeated first test: 6 concentrations, 4.7 to 150 μg/plate (Salmonella); 5 concentrations,313 to 5000 μg/plate (E. Coli)
- Second test: 5 concentrations, 11 to 900 μg/plate (Salmonella); 5 concentrations, 62 to 5000 μg/plate (E. Coli)
- Repeated second test: 5 concentrations, 1.9 to 150 μg/plate (Salmonella); 5 concentrations, 62 to 5000 μg/plate (E. Coli)
Vehicle / solvent:
- DMSO
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Strain 1537, with metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
Strain TA 1535, TA 98, TA 100 and WP 2 uvrA, with metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-ethyl-N-nitrosourea
Remarks:
Strain WP 2 uvrA, without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Strain TA 98, without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Strain TA1537, without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Strain TA 1535 and TA 100, without metabolic activation
Details on test system and experimental conditions:
METHODS
- Plate-incorporation method used in the first, repeated first and repeated second test: Molten top agar, bacteria, the test substance (vehicle or positive control) and when applicable S9-mix were thoroughly mixed and the mix was immediately poured onto minimal glucose agar plates. All determinations were made in triplicate. The plates were incubated for 48-72 hours at ca. 37°C. Subsequently, the his+ and trp+ revertants were counted.
- Treat and plate method, used in the second test: Bacteria, the test substance (vehicle or positive control) and when applicable S9-mix were mixed and incubated for ca. 3 hours at ca. 37°C while shaking. Thereafter, the mixtures were centrifuged and bacteria, after washing, were added to molten top agar, and the mixtures were poured onto minimal glucose agar plates. All determinations were made in triplicate. The plates were incubated for 48-72 hours at ca. 37°C. Subsequently, the his+ and trp+ revertants were counted.

DETERMINATION OF CYTOTOXICITY
- Toxicity was defined as a reduction (by at least 50%) in the number of revertant colonies and/or a clearing of the background lawn of bacterial growth as compared to the negative (vehicle) control and/or the occurrence of pinpoint colonies.
Evaluation criteria:
- The study was considered valid if the mean colony counts of the vehicle control values of the strains are within acceptable ranges, if the results of the positive controls meet the criteria for a positive response, if no more than 5 % of the plates are lost through contamination or other unforeseen events and if at least three doses are non-toxic.
- A test substance was considered to be positive in the bacterial gene mutation test if the mean number of revertant colonies on the test plates was increased in a dose-related manner or if a two-fold or greater increase was observed compared to the negative control plates. A clear positive response does not need to be verified. Marginally or weakly positive results should be verified by additional testing.
- A test substance was 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 minimal two-fold increase in the mean number of revertants at any of the test concentrations.
- Both numerical significance and biological relevance are considered together in the evaluation.
Statistics:
No statistical analysis is performed.

Results and discussion

Test resultsopen allclose all
Key result
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:
not examined
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON GENOTOXICITY:
- Four bacterial reverse mutation tests were performed, of which two were acceptable. In the first test, less than three non-toxic concentrations were tested, therefore the test was considered not acceptable. The test was repeated. The repeated first test was valid as three non-toxic concentrations were tested and the positive and negative controls showed the expected results. The second test was performed according to the treat and plate method. Unexpectedly the onset of cytotoxicity was within the three hours incubation. As a result, the treat and plate method did not allow exposure to higher concentrations. Therefore the test was considered not acceptable and the test was repeated according to the plate incorporation method.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In all tests, a dose related precipitation of the test substance in the final treatment mix (top agar) was observed. Precipitation was observed in all mixtures above 185 μg/plate. No precipitation was observed on the agar plates.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- In the first test, the test substance was considered toxic both in the absence and presence of S9-mix to all strains. Toxicity was observed at and above 185 μg/plate for strains TA 1535, TA 98 and TA 100, at and above 62 μg/plate for strain TA 1537 both in the absence and presence of S9-mix. For strain WP2 uvrA at 5000 μg/plate in the absence of S9-mix, and at and above 2500 μg/plate in the presence of S9-mix toxicity was observed.
- In the repeated first test, the test substance was considered toxic both in the absence and presence of S9-mix to all strains. Toxicity was observed at 150 μg/plate for all Salmonella strains in both the absence and presence S9-mix. For strain WP2 uvrA at 5000 μg/plate in the absence of S9-mix, and at and above 2500 μg/plate in the presence of S9-mix toxicity was observed.
- In the second test (treat and plate method), the test substance was considered toxic both in the absence and presence of S9-mix to all strains. Toxicity was observed at and above 33 μg/mL for strains TA 1535 and TA 98 and at and above 11 μg/mL for strains TA 1537 and TA 100 in both the absence and presence of S9-mix. For strain WP2 uvrA at and above 62 μg/mL in the absence of S9-mix, and at and above 185 μg/mL in the presence of S9-mix toxicity was observed.
- In the repeated second test, the test substance was considered toxic both in the absence and presence of S9-mix to all strains. Toxicity was observed at 150 μg/plate for strains TA 1535, TA 98 and TA 100, at and above 50 μg/plate for strain TA 1537 and for strain WP2 uvrA at 5000 μg/plate in the absence of S9-mix, and at and above 1667 μg/plate in the presence of S9-mix.
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 test material was non-mutagenic, in the absence and presence of metabolic activation, under the conditions of this bacterial reverse mutation test.
Executive summary:

In a reverse mutation assay, performed according to a OECD Guideline 471 and GLP, salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test substance in the absence and presence of a liver fraction of Aroclor 1254-induced rats (S9-mix). Four bacterial reverse mutation tests were performed of which two were acceptable. These two acceptable experiments were performed in triplicate using the plate incorporation method. In the first test, Salmonella strains were exposed to concentrations of the test substance ranging from 4.7 to 150 μg/plate and E. coli strain to concentrations ranging from 313 to 5000 μg/plate. In the second test, the Salmonella strains were exposed to concentrations of the test substance ranging from 1.9 to 150 μg/plate and the E. coli strain to concentrations ranging from 62 to 5000 μg/plate. The vehicle control plates gave counts of revertant colonies within the acceptable range. In all strains the positive controls gave the expected increase in the mean numbers of revertant colonies. In the first and second accepted test, the test substance was considered toxic both in the absence and presence of S9-mix to all strains. In the first test, for all Salmonella strains toxicity was observed at 150 μg/plate and for the E. coli strain at 5000 μg/plate in the absence of S9-mix and at and above 2500 μg/plate in the presence of S9-mix. In the second test, for strains TA 1535, TA 98 and TA 100 toxicity was observed at 150 μg/plate, for strain TA 1537 at and above 50 μg/plate, both in the absence and presence of S9-mix. For the E. coli strain at 5000 μg/plate in the absence of S9-mix and at and above 1667 μg/plate in the presence of S9-mix toxicity was observed. Toxicity was evidenced by a decrease in the mean number of revertants and/or a clearing of the background lawn of bacterial growth compared to the negative controls. In both accepted tests, in all strains tested, in both the absence and presence of S9-mix, the test substance did not induce a more than 2-fold and/or dose related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed with the negative control. The test material was therefore considered to be non-mutagenic under the conditions of this test.