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Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9-28 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

1
Chemical structure
Reference substance name:
2-(1-ethylpentyl)-1,3-dioxolane
EC Number:
224-436-8
EC Name:
2-(1-ethylpentyl)-1,3-dioxolane
Cas Number:
4359-47-1
Molecular formula:
C10H20O2
IUPAC Name:
2-(1-ethylpentyl)-1,3-dioxolane

Method

Target gene:
- S. typhimurium: Histidine gene
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
- Dose range finding test:
TA 1535, TA 1537, TA 98, TA 100 and E.coli WP2uvrA (without and with S9): doses of 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

- Experiment 1:
TA 1535, TA 1537, TA 98, TA 100 and E.coli WP2uvrA (without and with S9): doses of 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

- Experiment 2:
E.coli strain WP2uvrA (with and without S9-mix): 15, 50, 150, 500, 1500 and 5000 µg/plate.
All Salmonella strains (with and without S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate.
One bacterial strain (WP2uvrA dosed in the absence of S9-mix) exhibited excessive toxicity and required a repeat experiment using an amended dose range of:
E.coli strain WP2uvrA (without S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate.
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: the test substance was found to be soluble in DMSO up to 5000 µg/plate
Controls
Untreated negative controls:
yes
Remarks:
(untreated plates)
Negative solvent / vehicle controls:
yes
Remarks:
(50 or 100 µL/plate DMSO)
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: plate incorporation method (0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel Bonner agar plate. In case of treatment with metabolic activation, 0.5 mL of S9 mix was added to the molten instead of phosphate buffer.)
- Experiment 2: pre-incubation method (0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel Bonner plates. In case of treatment with metabolic activation, 0.5 mL of S9 mix was added to the tube instead of phosphate buffer.)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain, in all experiments

DETERMINATION OF CYTOTOXICITY
- Method: on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation
Evaluation criteria:
For the test substance to be considered mutagenic:

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 (Cariello and Piegorsch, 1996)).

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

Results and discussion

Test results
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100 and E.coli WP2uvrA
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 precipitation was observed up to and including the top dose of 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES AND EXPERIMENTS 1 AND 2:
- In all strains toxicity was observed at at leat one concentration, both in the absence and presence of S9 in the experiments. No toxicity was noted to Escherichia coli strain WP2uvrA at any test item dose level.
The test item induced a stronger toxic response in the second mutation test (pre-incubation method), with weakened bacterial background lawns noted in the absence of S9-mix from 50 µg/plate (TA100 and TA1537) and 150 µg/plate (TA1535, TA98 and WP2uvrA). In the presence of S9 mix weakened bacterial background lawns were noted from 150 µg/plate (TA100 and TA1537), 500 µg/plate (TA1535 and TA98) and 1500 µg/plate (WP2uvrA).

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Small, statistically significant increases in revertant colony frequency were observed in the first mutation test at 1.5 and 50 µg/plate (TA98) and 1.5 µg/plate (TA1537) in the presence of S9-mix only. Further statistically significant increases in revertant colony frequency were observed in the second mutation test at 50 µg/plate (TA98) in the presence of S9-mix only. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the maximum fold increase was only 1.8 times the concurrent vehicle controls.
Further statistically significant increases in revertant colony frequency were observed in the first mutation test at 1500 and 5000 µg/plate (TA1535 in the absence of S9-mix) and 500, 1500 and 5000 µg/plate (TA1535 in the presence of S9-mix) and 500 and 5000 µg/plate (TA98 in the presence of S9-mix). Further statistically significant increases in revertant colony frequency were also observed in the second mutation test at 150 and 500 µg/plate (TA1535 dosed in the absence and presence of S9-mix respectively). These increases were also considered to have no biological relevance because weakened bacterial background lawns were also noted. Therefore these responses would be due to additional histidine being available to His- bacteria allowing these cells to undergo several additional cell divisions and presenting as non-revertant colonies.

Applicant's summary and conclusion

Conclusions:
The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay performed according to OECD 471.
Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 and according to GLP principles. The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay, both in the absence and presence of S9-mix. The dose levels were selected based on the dose finder study, up to the maximum recommended dose level of 5000 µg/plate. Adequate negative and positive controls were included. There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains (TA1535, TA1537, TA98, TA100 and E.coli WP2uvrA)

, with any dose of the test item, either with or without metabolic activation. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.

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