Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 Dec 2012 to 22 Feb 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

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)
Version / remarks:
30 May 2008
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
METI, MHLW and MAFF
GLP compliance:
yes (incl. QA statement)
Remarks:
Harlan Laboratories LTD, Shardlow Business Park, London Road, Shardlow, Derby, DE72 2GD
Type of assay:
bacterial reverse mutation assay

Test material

1
Chemical structure
Reference substance name:
3-(p-cumenyl)propionaldehyde
EC Number:
231-885-3
EC Name:
3-(p-cumenyl)propionaldehyde
Cas Number:
7775-00-0
Molecular formula:
C12H16O
IUPAC Name:
3-[4-(propan-2-yl)phenyl]propanal
Test material form:
liquid

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and ß-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
PRELIMINARY TOXICITY TEST
In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test item. The concentrations for WP2uvrA and TA100 (with S9-mix) were 0, 50, 150, 500, 1500 and 5000 µg/plate. However, due to an extremely toxic response, the dose range for TA100 and WP2uvrA (without S9-mix) was amended to 0, 0.5, 1.5, 5 15 and 50 pµg/plate.

Based on the results, the following dose range was selected for experiment 1 and 2 (according to the pre-incubation method):
- All tester strains (presence of S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate.
- All tester strains (absence of S9-mix): 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate.
Additional dose levels and an expanded dose range were selected in order to achieve both four non-toxic dose levels and the toxic limit of the test item.
Vehicle / solvent:
- Solvent used: DMSO
- The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible dimethyl sulphoxide at the same concentration in solubility checks performed in-house.
Controls
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see section "Any other information on materials and methods incl. tables"
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- Experiment 1 and 2: pre-incubation method
- The untreated controls were dosed using the standard plate incorporation method

DURATION
- Exposure duration: 20 minutes at 37°C with shaking at approximately 130 rpm

NUMBER OF REPLICATIONS:
- Triplicates in 2 independent experiments.

DETERMINATION OF REVERTANT COLONIES
- After the 20 min exposure, 2 mL of molten, trace histidine or tryptophan supplemented, top agar was added. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates (one tube per plate). After setting, the plates were placed in anaerobic jars or bags (one jar/bag for each concentration of test item/vehicle) during the incubation procedure. All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies was assessed using an automated colony counter.
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby (1979)).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 100, TA 1537 and TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
- Other confounding effects: The test item formulation and S9-mix used in this experiment were both shown to be sterile.

RANGE-FINDING/SCREENING STUDIES:
- The test item was toxic to TA100 and WP2uvrA from 50 and 150 pg/plate in the absence and presence of S9-mix respectively.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Although the revertant colony frequency for TA100 dosed in the absence of S9-mix was well below the historical control value for the strain the data was still considered acceptable as the purpose of the test was simply to ascertain the toxic limit of the test item.

CYTOTOXICITY
The test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 50 µg/plate in the absence of S9-mix and 150 µg/plate in the presence of S9-mix. The test item was, therefore, tested up to the toxic limit.

CONTROLS
- Results for the negative controls were considered to be acceptable.
- All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Applicant's summary and conclusion

Conclusions:
The test substance had, under the present test conditions, no ability to induce mutations in the bacterial reverse mutation assay according to OECD TG 471.
Executive summary:

The mutagenic activity of the test substance was evaluated in a study according to OECD TG 471 and GLP principles. The test was performed according to the pre-incubation method in triplicate in 2 independent experiments, in the absence and presence of S9-mix. The dose levels were selected based on a preliminary toxicity screen performed in all tested strains (S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2uvrA). Based on these results, All strains were tested at 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate in presence of S9 mix and 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate in absence of S9. Adequate positive, negative and vehicle controls were valid. The substance did not induce a significant increase in the number of revertant colonies in each of the five tester strains, both in the absence and presence of metabolic activation. Based on the results of this study it is concluded that the test substance is not mutagenic in the bacterial reverse mutation assay.