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Genetic toxicity: in vitro

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

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Dioctyl ether
EC Number:
EC Name:
Dioctyl ether
Cas Number:
Molecular formula:
Specific details on test material used for the study:
- Name of test material (as cited in study report): Cetiol OE
- Physical state: liquid
- Lot/batch No.: DENNECKE00090 Charge 0011189321


Species / strain
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Test concentrations with justification for top dose:
33µg-5000 µg/plate (standard plate test), 33µg-5000 µg/plate (preincubation test)
Vehicle / solvent:
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
With S9 mix: 2-aminoanthracene (2-AA) (Sigma-Aldrich; 96%) (60 μg/plate, dissolved in DMSO); Without S9 mix: 4-nitroquinoline-N-oxide (4-NQO) (Sigma-Aldrich; 98%) (5 μg/plate, dissolved in DMSO)
Details on test system and experimental conditions:
For testing, a deep-frozen (-70°C to -80°C) bacterial culture (E. coli WP2 uvrA) is thawed at room temperature, and 0.1 mL of this bacterial suspension is inoculated in nutrient broth solution (8 g/L Difco nutrient broth + 5 g/L NaCl) and incubated in the shaking water bath at 37°C for about 12 - 16 hours. Fresh cultures of bacteria should be grown up to late exponential or early stationary phase of growth (approximately 109 dells per mL). This culture grown overnight is kept in iced water from the beginning of the experiment until the end in order to prevent further growth. The use of the strain mentioned is in accordance with the current scientific recommendations for the conduct of this assay. The Escherichia coli strain was obtained from Merck KGaA, Darmstadt, Germany (09 Sep 1991).

Escherichia coli WP2 uvrA which has an AT base pair at the primary reversion site is a derivative of E. coli WP2 with a deficient excision repair and is used to detect substances which induce base pair substitutions. The rate of induced back mutations from tryptophan auxotrophy (trp-) to tryptophan independence (trp+) is determined.

E. coli WP2 uvrA is checked for UV sensitivity. Tryptophan auxotrophy is checked in each experiment via the spontaneous rate.

The S9 fraction was prepared according to Ames et al. at BASF SE in an AAALACapproved laboratory in accordance with the German Animal Welfare Act and the effective European Council Directive. At least 5 male Wistar rats [Crl:WI(Han)] (200 - 300 g; Charles River Laboratories Germany GmbH) received 80 mg/kg b.w. phenobarbital i.p. and -naphthoflavone orally (both supplied by Sigma-Aldrich, 82024 Taufkirchen, Germany) each on three consecutive days.
During this time, the animals were housed in Makrolon cages: central air conditioning with a fixed range of temperature of 20 - 24°C and a fixed relative humidity of 30 - 70%. The day/night rhythm was 12 hours (light period from 6.00 - 18.00 hours and dark period from 18.00 - 6.00 hours). Standardized pelleted feed and drinking water from bottles were available ad libitum. 24 hours after the last administration, the rats were sacrificed, and the livers were prepared using sterile solvents and glassware at a temperature of +4°C. The livers were weighed and washed in a weight-equivalent volume of a 150 mM KCl solution and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9 000 x g for 10 minutes at +4°C, 5 mL portions of the supernatant (so-called S9 fraction) were stored at -70°C to -80°C.

The S9 mix was prepared freshly prior to each experiment (1, 2). For this purpose, a sufficient amount of S9 fraction was thawed at room temperature and 1 volume of S9 fraction is mixed with 9 volumes of S9 supplement (cofactors). This preparation, the so-called S9 mix, was kept on ice until used.
To demonstrate the efficacy of the S9 mix in this assay, the S9 batch was characterized with
Evaluation criteria:
Acceptance criteria
Generally, the experiment was considered valid if the following criteria were met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for the tester strain
• The sterility controls revealed no indication of bacterial contamination
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above
• Fresh bacterial culture containing approximately 109 cells per mL were used.

Assessment criteria
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling of the spontaneous mutation rate in the tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for the tester strain was within the historical negative control data range under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
Untreated negative controls validity:
Positive controls validity:
Additional information on results:
In the Standard plate test and in the 2nd-PIT, test substance precipitation was found from about 2500 μg/plate onward with and without S9 mix.

Any other information on results incl. tables

According to the results of the present study, the test substance did not lead to a biologically relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in several experiments carried out independently of each other (standard plate test and preincubation assay). However, the slight increase in the number of trp+ revertants observed in the preincubation test without S9 mix was not reproduced in a repeat experiment, and so these findings have to be regarded as biological irrelevant. Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study.

In this study with and without S9 mix, the number of revertant colonies in the negative control was within or marginally below the range of the historical negative control data for the tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control


Applicant's summary and conclusion

Dioctyl ether is not considered to be mutagenic in this test.