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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:
22nd May to 20th June 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no deviations from standard test guidelines and no methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1-bromohexadecane
EC Number:
204-008-7
EC Name:
1-bromohexadecane
Cas Number:
112-82-3
Molecular formula:
C16H33Br
IUPAC Name:
1-bromohexadecane
Details on test material:
- Substance type: White semi-solid block on arrival which melts into a clear colourless viscous liquid.
- Date received: 12th April 2002
- Storage condition of test material: Room temperature in a dark room

Method

Target gene:
S. typhimurium - histidine
E. coli - tryptophan
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 applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
10% Liver S9 in standard co-factors
Test concentrations with justification for top dose:
S. typhimurium - 15, 50, 150, 500, 1500 and 5000 µg/plate (15 µg/plate concentration was omitted in experiement 2).
E. coli - 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: Positive controls without S9-mix: N-ethyl-N'-nitro-N-nitrosoguanidine (TA100, TA1535, WP2uvrA); 9-Aminoacridine (TA1537) and 4-Nitroquinoline-1-oxide (TA98). Positive controls with S9-mix: 2-Aminoanthracene (all other strains) and Benzo(a)pyrene (TA98)
Details on test system and experimental conditions:
BACTERIA SOURCE AND MAINTENANCE
- Salmonella strains were obtained from the University of California at Berkeley on 4th August 1995; E. coli strain was obtained from the British Industrial Biological Research Association on 17th August 1987.
- Stored at -196ºC in a Statebourne liquid nitrogen freezer, model SXR 34
- Characterisation checks were carried out to confirm the amino-acid requirement, presence of rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate.

BACTERIAL PREPERATION
- Cultures were prepared overnight in nutrient broth (Oxoid Limited; lot no. 250177 05/06 and 257111 10/06) and incubated for approximately 10 hours at 37ºC.

TEST MATERIAL PREPERATION
Concentrations were weighed and mixed with acetone in a vortex mixer and sonication for 15 minutes at 40ºC on the day of the experiment. Prior to use the preparation was dried using molecular sieves.

METHOD OF APPLICATION
Direct plate incorporation method.

AGAR PREPERATION
Prepared with 0.6% Difco Bacto agar and 0.5% sodium chloride with 5 ml of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 ml of top agar.

NUMBER OF REPLICATIONS
For each experiment the plates were replicated in triplicate for each bacterial strain and each concentration.

DURATION
All of the plates were incubated at 37ºC for approximately 48 hours.

COLONIES
Colonies counted using a Domino colony counter.
Evaluation criteria:
The test material may be considered positive if the following criteria are met:

The test material should have induced a reproducible, dose-related and statistically significant increase in the revertant count in a least one strain of bacteria.
Statistics:
Dunnett's method of linear regression.

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:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
PRELIMINARY TOXICITY STUDY
Weakened lawns were recorded in TA100 without metabolic activation (S9) at > 1500 µg/plate. This observation was considered to not be valid, as no other evidence of toxicity was noted for any other strain.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 2. Mean Colony Counts for experiments 1 and 2 including control results

 Experiment 1
   

 Mean number of revertant colonies per plate

     Base-pair substitution type  Frameshift type
 With or without S9 mix  Test material concentration (µg/plate)  TA100  TA1535  WP2uvrA  TA98  TA1537
 -  0  75  20  23  17  16
 -  15  69  17  Not tested  20  9
 -  50  75  17  19  16  11
 -  150  69  17  19  15  7
 -  500  76  17  22  16  10
 -  1500  79  18  20  15  9
 -  5000  83  18  19  14  11
 Positive controls    497  339  745  139  1319
 +  0  103  16  27  33  19
 +  15  91  12  Not tested  28  18
 +  50  70  14  22  30  18
 +  150  74  12  18  27  15
 +  500  91  14  21  26  11
 +  1500  77  14  24  27  13
 +  5000  93  11  18  23  8
 Positive controls    1847  273  1350  221  347
 Experiment 2
 -  0  93  21  20  23  18
 -  50  85  20  20  19  13
 -  150  100  19  19  21  17
 -  500  93  22  22  17  18
 -  1500  93  23  20  18  14
 -  5000  89  17  18  22  16
 Positive controls    677  698  1136  123  2632
 +  0  99  16  23  30  19
 +  50  97  17  21  28  18
 +  150  87  14  19  25  19
 +  500  99  16  19  29  17
 +  1500  104  15  18  31  18
 +  5000  101  16  18  30  19
 Positive controls    1750  107  880  305  1365

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative All strains with and without metabolic activation.

Under the conditions of this assay, the test material gave a negative i.e. a non-mutagenic response in all strains, with or without metabolic activation. The test material is therefore considered to be non-mutagenic.
Executive summary:

The potential of the test material to cause gene mutation in bacteria was assessed in accordance with the following guidelines: OECD 471, EU Method B.13/14 and EPA OPPTS 870.5100. Salmonella typhimurium (strains; TA100, TA1537, TA 1535 and TA98) and Escherichia coli (strain: WP2uvrA) were exposed to concentrations of the test material ranging from 15 – 5000µg/plate with and without metabolic activation (S9-mix derived of rats liver). The test material gave a negative response where none of the assays showed a significant change in revertant colony numbers.

Under the conditions of the test the test material is therefore considered not to cause genetic mutation in bacteria.