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Diss Factsheets

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 study was conducted between 26 May 2010 and 22 June 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 471 using a Bacterial Reverse Mutation method and in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
4-(heptyloxy)-3-methylbutanal
EC Number:
802-100-7
Cas Number:
1093653-57-6
Molecular formula:
C12H24O2
IUPAC Name:
4-(heptyloxy)-3-methylbutanal
Test material form:
other: Liquid
Details on test material:
Identity: TM 09-217
Chemical name: Butanal, 4-(heptyloxy)-3-methyl
Appearance: Clear liquid
Storage conditions: Room temperature (ca 20°C) in the dark
Date received: 18 February 2010

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Test 1: 15, 50, 150, 500, 1500 and 5000 µg / plate
Test 2: 50, 150, 500, 1500, 5000 µg / plate
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental conditions:
Bacterial strains
The strains of S. typhimurium were obtained from the National Collection of Type Cultures, London, England.
The strain of E. coli was obtained from the National Collections of Industrial and Marine Bacteria, Aberdeen, Scotland.
Batches of the strains were obtained from master stocks held in liquid nitrogen. The test batches were stored as aliquots of nutrient broth cultures at approximately -80ºC. Dimethyl sulphoxide (DMSO) was added to the cultures at 8% v/v as a cryopreservative. Each batch of frozen strain was tested for amino acid requirement and, where applicable, for cell membrane permeability (rfa mutation), sensitivity to UV light, and the pKM101 plasmid, which confers resistance to ampicillin. The responses of the strains to a series of reference mutagens were also assessed.
For use in tests, an aliquot of frozen culture was added to 25 mL of nutrient broth and incubated, with shaking, at 37C for 10 hours. These cultures were intended to provide a viable cell density of at least 109 per mL, which was confirmed by performing viability counts, in which aliquots (0.1 mL) of a 10 6 dilution of the 10-hour cultures were spread on the surface of plates of nutrient agar. After incubation at 37°C for 24 hours, the total number of resultant colonies was counted.

S9 metabolizing system
Preparation of S9 fraction
S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver, was purchased from a commercial source and stored at approximately -80°C.

Preparation of S9 mix
The S9 mix contained: S9 fraction (10% v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADPH (4 mM) and NADH (4 mM) in water. All the cofactors were filter-sterilised before use.

Selection of vehicle and formulation of test substance
Information received from the sponsor indicated that aqueous solubility of TM-09-217 was low. Its solubility was assessed at 50 mg/mL in dimethyl sulphoxide (DMSO), in which it was soluble. DMSO (ACS spectrophotometric grade) was, therefore, used as the vehicle for this study.
The highest concentration of TM-09-217 tested in this study was 50 mg/mL in the chosen vehicle, which provided a final concentration of 5000 µg/plate. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. The highest concentration in each test was diluted with DMSO to produce a series of lower concentrations, separated by approximately half-log10 intervals.
All concentrations cited in this report are expressed in terms of the TM-09-217 sample as received.

Mutation test procedure
First test
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 vessels. 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 (Perceptive Instruments Sorcerer).
Any toxic effects of the test substance would be detected by a substantial reduction in mean revertant colony counts or by a sparse or absent background bacterial lawn. In the absence of any toxic effects, the maximum concentration selected for use in the second test would be the same as that used in the first. If toxic effects were observed, a lower concentration might be chosen, ensuring that signs of bacterial inhibition were present at this maximum concentration. Ideally, a minimum of four non-toxic concentrations should be obtained. If precipitate were observed on the plates at the end of the incubation period, at least four non-precipitating concentrations should be obtained, unless otherwise justified by the Study Director.

Second test
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 again 5000 µg/plate (except for strain TA1537, which was tested up to 1500 µg/plate), but only five concentrations were used.

Analysis of data
The mean number and standard deviation of revertant colonies were calculated for all groups. The “fold-increases” relative to the vehicle controls were calculated in order to compare the means for all treatment groups with those obtained for the vehicle control groups.

Criteria for assessing mutagenic potential
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) the concurrent vehicle controls, with some evidence of a positive dose-response relationship, it is considered to exhibit mutagenic activity in this test system. No statistical analysis is performed.
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.
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 usually Dunnett’s test followed, if appropriate, by trend analysis. Biological importance should always be considered along with statistical significance. In general, treatment-associated increases in revertant colony numbers below two or three times the vehicle controls (as described above) are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.
Occasionally, these criteria may not be appropriate to the test data and, in such cases, the Study Director would use his/her scientific judgement.
Evaluation criteria:
For a test to be considered valid, the mean of the vehicle control revertant colony numbers for each strain should lie within or close to the 99% confidence limits of the current historical control range of the laboratory unless otherwise justified by the Study Director. The historical range is maintained as a rolling record over a maximum of five years. Also, the positive control compounds must induce an increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls. Mean viable cell counts in the 10-hour bacterial cultures must be at least 109/mL.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Toxicity was seen in all strains following exposure at 5000 µg/plate in both tests.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
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 99% confidence limits of 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.

First test
Toxicity, observed as thinning of the background lawn of non-revertant colonies, together with a reduction in revertant colony numbers, was seen in all strains following exposure to TM-09-217 at 5000 µg/plate, and in strain TA1537 following exposure to TM-09-217 at 1500 µg/plate. A maximum exposure concentration of 5000 µg/plate was, therefore, selected for use in the second test with all strains except TA1537, and a maximum exposure concentration of 1500 µg/plate was selected for use with TA1537.
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to TM-09-217 at any concentration up to and including 5000 µg/plate in either the presence or absence of S9 mix.

Second test
Toxicity, observed as thinning of the background lawn of non-revertant colonies, together with a reduction in revertant colony numbers, was seen in all strains except TA1537 following exposure to TM-09-217 at 5000 µg/plate and in strain TA1537 following exposure to TM-09-217 at 1500 µg/plate.
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to TM-09-217 at any concentration tested in either the presence or absence 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

It is concluded that TM-09-217 showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.
Executive summary:

The mutagenic potential of the test substance, TM 09 -217, was assessed as negative according to OECD Test Guideline 471.