Registration Dossier

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:
26th August to 9th October 2003
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 deficiences, 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:
2003

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Description: colorless liquid
Purity: 99.449%
Water: 0.0131%
Storage conditions: at room temperature and protected from light
Expiry date: May 2004

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 mix in standard co-factors
Test concentrations with justification for top dose:
The test item was freely soluble in the vehicule (DMSO) at 50 mg/mL.
Consequently, with a treatment volume of 100 µl/plate, the dose-levels were 10, 100, 500, 1000, 2500 and 5000 µg/plate.
Vehicle / solvent:
Dimethylsulfoxide (DMSO), batch K31602850 305 from Merck Eurolab
Controls
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Remarks:
See details field
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
Remarks:
2-Anthramine was used with S9 mix
Details on test system and experimental conditions:
TEST SYSTEM
1) Bacterial strains
The five strains of Salmonella typhimurium were supplied by B.N. Ames Laboratory. They are stored in a cryoprotective medium (1 mL nutrient broth and 0.09 ml dimethylsulfoxide) in liquid nitrogen.
The day before treatment, cultures were inoculated from frozen permanents: a scrape was taken under sterile conditions and put into approximately 6 mL of nutient broth. The nutrient broth was placed under agitation in an incubator at 37°C for about 14 hours, to produce bacterial suspensions.
Each strain derived from Salmonella typhimurium LT2 contains one mutation in the histidine operon, resulting in a requirement for histidine.
In addition, to increase their sensitivity to mutagenic items, further mutations have been added:
- the rfa mutation causes partial loss of the lipopolysaccharide barrier that coats the surface of the bacteria and increases permeability to large molecules that do not penetrate the normal bacteria cell wall,
- the uvrB mutation is a deletion of e gene coding for the DNA excision repair system, which renders the bacteria unable to use this repair mechanism to remove the damaged DNA,
- the addition of the plasmid pKM 101 to strains TA 98, TA 100 and TA 102 enhances their sensitivity of detection to some mutagens,
- in case of TA 102 strain, the histidine mutation is located on the multicopy plasmid pAQ1.
The TA 1535, TA 100 and TA 102 strains are reverted by base-pair substitution mutagens and the TA 1537 and TA 98 strains by frameshift mutagens. In addition, the TA 102 stain detects oxidative mutagens.

2) Metabolic activation system
The S9 mix consists of induced enzymatic systems contained in rat liver post-mitochondrial fraction (S9 fraction) and the cofactors necessary for their function. S9 fraction was purchased from Moltox and obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by the intraperitoneal route.
The S9 fraction was preserved in sterile tubes at - 80°C, until use.

The S9 mix was prepared at + 4°C immedialtely before use and maintened at this temperature until added to the overlay agar.

EXPERIMENTAL DESIGN
1) Treatment
The test item was tested in a preliminary test and two mutagenecity experiments.
The preliminary test, both experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method. The second experiment with S9 mix was performed according to the preincubation method.
The direct plate incorporation method was performed as follows: test itemsolution (0.1 mL), S9 mix when required or phosphate buffer pH 7.4 (0.5 mL) and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 45°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.
The preincubation method was performed as follows: test item solution (0.1 mL), S9 mix (0.5 mL) and the bacterial suspension (0.1 mL) were incubated for 60 minutes at 37°C, under shaking, before adding the overlay agar and pouring onto the surface of a minimum agar plate.
After 48 to 72 hours of incubation at 37°C, revertants were scored with an automatic counter.

2) Preliminary toxicity test
To assess the toxicity of the test item to the bacteria, six dose-levels (one plate/dose-level) were tested in the TA 98, TA 100 and TA 102 strains, with and without S9 mix.
The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

3) Mutagenecity experiments
In two independant experiments, using three plates/dose-level, each strain was tested, with and without S9 mix, with:
- at least five dose-level of the test item,
- the vehicule control,
- the appropriate positive control.
The sterility of the S9 mix was checked before the beginning and at the end of each experiment and was found to be satisfactory.
Evaluation criteria:
A reproductible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and /or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken in account in the evaluation of the data obtained.
Statistics:
Not specified

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
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
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

PRELIMINARY TOXICITY TEST

The test item was freely soluble in the vehicule (DMSO) at 50 mg/mL.

Consequently, with a treatment volume of 100 µl/plate, the dose-levels were 10, 100, 500, 1000, 2500 and 5000 µg/plate.

A moderate emulsion was observed in the Petri plates when scoring the revertants at dose-levels > 2500 µg/plate.

A moderate to marked toxicity was noted at dose-levels > 100 µg/plate in the TA 100 strain without S9 mix, at dose-levels > 500 µg/plate in the TA 100 strain with S9 mix and at dose-levels > 2500 µg/plate in the TA 98 strain without S9 mix.

No noteworthy toxicity was observed in the TA 98 strain with S9 mix as well as in the TA 102 strain with and without S9 mix.

MUTAGENECITY EXPERIMENTS

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

Since the test item was freely soluble and not severely toxic, the highest dose-level for the first experiment was 5000 µg/plate, according to the criteria specified in the international guidelines. For the following treatments, this highest dose-level was decreased when it was judged necessary, on the basis of the toxicity observed.

Experiments without S9 mix:

The selected treatment-levels were as follows:

- 61.73, 185.2, 555.6, 1667 and 5000 µg/plate, for all the strains in the first experiment, and for the TA 1537 and TA 102 strains in the second experiment,

- 20.58, 61.73, 185.2, 555.6 and 1667 µg/plate, for the TA 1535 and TA 98 strains in the second experiment,

- 2.29, 6.86, 20.58, 61.73 and 185.2 µg/plate, for the TA 100 strain in the second experiment.

A moderate emulsion was observed in the Petri plates when scoring the revertants at dose-levels < 1667 µg/plate, with and without S9 mix.

Except for the TA 102 strain where no toxicity was noted, a moderate to marked toxicity was induced at dose-levels > 61.73 µg/plate in the TA 100 strain, > 185.2 µg/plate in the TA 1535 strain and > 555.6 µg/plate in the TA 98 and TA 1537 strains.

Experiments with S9 mix:

The selected treatment-levels were as follows:

- 61.73, 185.2, 555.6, 1667 and 5000µg/plate, for all the strains in the first experiment, and for the TA 1535, TA 1537, TA 98 and TA 102 strains in the second experiment,

- 20.58, 61.73, 185.2, 555.6 and 1667 µg/plate, for the TA 100 strains in the second experiment.

A moderate emulsion was observed in the Petri plates when scoring the revertants at dose-levels < 1667 µg/plate, with and without S9 mix.

Except for the TA 102 strain where no toxicity was noted, a moderate to marked toxicity was induced generally at dose-levels > 555.6 µg/plate.

The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains.

Applicant's summary and conclusion

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

Under experimental conditions, 1-bromodecane did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.
Executive summary:

The potential of the test material to cause gene mutation in bacteria was assessed in accordance with the following guidelines: OECD 471 and EU Method B. 13/14. Salmonella typhimurium (strains TA 1535, TA 1537, TA 98, TA 100 and TA 102) were exposed to concentrations of the test material ranging from 15 to 5000 µg/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, test material is therefore considered not to cause genetic mutation in bacteria.