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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:
4 April to 18 June 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study carried out in compliance with an internationally recognised guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Substance name in the test report: FRET 08-0338
- Empirical formula: C13H22O
- MW: 194.32
- Physical state: liquid
- Storage conditions: room temperature in the dark

Method

Target gene:
Histidine dependency S. typhimurium
Tryptophan dependency Escherichia coli
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):
- Type and identity of media: nutrient broth cultures.
- Properly maintained: yes
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Details on mammalian cell type (if applicable):
- Type and identity of media: nutrient broth cultures.
- Properly maintained: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver
Test concentrations with justification for top dose:
Test 1 5, 15, 50, 150, 500, 1500, 5000 µg per plate
Test 2 15, 50, 150, 500, 1500 µg per plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: recommended by test substance supplier
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
used for 2 µg/plate for TA98 without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
used for 2 µg/plate for TA100 and TA1535 without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
used for 50 µg/plate for TA1537 without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
used for 2 µg/plate for E. coli WP2 uvrA pKM101 without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
used for 5 µg/plate for TA100 and TA1535 and 10 µg/plate for WP2 uvrA (pKM101) with S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
used for 5 µg/plate TA98 and TA1537 with S9
Details on test system and experimental conditions:
Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first test was a standard plate incorporation assay; the second included a pre-incubation stage.


Test 1
Aliquots of 0.1 mL of the test substance solutions (seven concentrations up to 5000 µg/plate), positive control or vehicle control were placed in glass tubes. The vehicle control was DMSO. S9 mix (0.5 mL) or 0.1 M pH 7.4 phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a
10-hour bacterial culture and 2 mL of agar containing histidine (0.05 mM), biotin (0.05 mM) and tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar. Each Petri dish was individually labelled with a unique code, identifying the contents of the dish. Three Petri dishes were used for each treatment. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9 mix and sodium phosphate buffer. All plates were incubated at approximately 37°C for ca 72 hours. After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter.

Test 2
As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The variation used was the pre-incubation assay in which the tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 37°C for 30 minutes with shaking before the addition of the agar overlay. The maximum concentration chosen was 1500 µg/plate, and only five concentrations were used
Evaluation criteria:
If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system.

If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
Statistics:
If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are Dunnett’s test followed, if appropriate, by trend analysis.

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:
not applicable
Positive controls validity:
valid
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:
Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first test was a standard plate incorporation assay; the second included a pre-incubation stage.

First test - toxicity was seen in all strains following exposure to FRET 08-0338 at 5000 μg/plate (observed as a thin background lawn of non-revertant colonies, together with an absence of revertant colonies) and at 1500 μg/plate (observed as a slightly thin background lawn of nonrevertant colonies, together with a reduction in revertant colony numbers). A maximum exposure concentration of 1500 μg/plate was, therefore, selected for use in the second test. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to FRET 08-0338 at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

Second test - Toxicity was seen in all strains following exposure to FRET 08-0338 at 1500 μg/plate (observed as a slightly thin background lawn of non-revertant colonies, together with a reduction in revertant colony numbers). No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to FRET 08-0338 at any concentration up to and including 1500 μg/plate in either the presence or absence of S9 mix.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The absence of colonies on sterility check plates confirmed the absence of microbial contamination of the S9 mix, buffer and test substance formulation.

The viability counts confirmed that the viable cell density of the cultures of the individual organisms exceeded 109/mL

in all cases, and therefore met the acceptance criteria.

The mean revertant colony counts for the vehicle controls were within or close to the current historical control range of the laboratory.

Appropriate positive control chemicals (with S9 mix where required) induced substantial increases in revertant colony numbers with all

strains in all reported tests, confirming sensitivity of the cultures and activity of the S9 mix.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that FRET 08-0338 showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.
Executive summary:

FRET 08-0338 was determined to be not mutagenic in a bacterial assay conducted according to OECD TG 471.