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Toxicological information

Genetic toxicity: in vitro

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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:
2002-09-11 to 2002-10-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
equivalent or similar to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Principles of method if other than guideline:
Note: Both direct plate assay and preincubation assay methods applied.
GLP compliance:
yes
Remarks:
No certificate
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
impurity
Type:
impurity
Test material form:
liquid
Specific details on test material used for the study:
- Name of test material (as cited in study report): Lemonile
- Substance type: Colourless to pale yellow liquid
- Physical state: Liquid
- Analytical purity: 99.4 %
- Impurities (identity and concentrations): No data
- Composition of test material, percentage of components: No data
- Isomers composition: No data
- Purity test date: No data
- Batch No: 9000477843
- Expiration date of the batch: 2004-07-11
- Stability under test conditions (in vehicle DMSO): "not indicated"
- Storage condition of test material: At room temperature in the dark
- Stability under storage conditions: Stable
- Specific gravity: 0.8647

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
other: See text
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Dose range-finding test 1 (direct plate assay): 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate.
Dose range-finding test 2 (preincubation assay): 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate.
Direct plate assay 1: 3, 10, 33, 100, 333, 1000, 3330 and 666 µg/plate.
Direct plate assay 2: 100, 333, 1000 and 3330 µg/plate.
Preincubation assay 1: 10, 33, 100, 333, 1000 and 2000 µg/plate.
Preincubation assay 2: 1, 3, 10, 33, 100, and 333 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: No data
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
Without metabolic activation. Strain - TA1535. Solvent - saline. Concentration/plate - 5 µg.
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without metabolic activation. Strain - TA1537. Solvent - saline. Concentration/plate - 60 µg. Direct plate assay only.
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Without metabolic activation. Strain - TA1537. Solvent - saline. Concentration/plate - 15 µg. Preincubation assay only.
Positive controls:
yes
Positive control substance:
other: Daunorubicin
Remarks:
Without metabolic activation. Strain - TA98. Solvent - saline. Concentration/plate - 4 µg.
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without metabolic activation. Strain - TA100. Solvent - DMSO. Concentration/plate - 650 µg.
Positive controls:
yes
Positive control substance:
cumene hydroperoxide
Remarks:
Without metabolic activation. Strain - TA102. Solvent - DMSO. Concentration/plate - 0.1 µg.
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene [EC name: 2-anthrylamine]
Remarks:
With metabolic activation. Strains - TA1535, TA1537, TA98, TA100. Solvent - DMSO. Concentrations/plate - 1 µg (TA1535, TA98, TA100), 2.5 µg (TA1537).
Positive controls:
yes
Positive control substance:
other: 1,8-Dihydroxyanthraquinone [EC name: dantron]
Remarks:
With metabolic activation. Strain - TA102. Solvent - DMSO. Concentration/plate - 30 µg.
Details on test system and experimental conditions:
METHODS OF APPLICATION: In agar (plate incorporation); preincubation

STRAINS
- Strains were obtained from Dr Bruce N. Ames, University of California at Berkeley, United States. For details on the strains used, see the "Any other information materials and methods incl. tables" section below.
- Strains were regularly checked to confirm their histidine requirement, crystal violet sensitivity, ampicillin resistance (TA98, TA100 and TA102), tetracycline resistance (TA102), UV-sensitivity and the number of spontaneous revertants.
- Each tester strain contained the following additional mutations:
-- rfa deep rough (defective lipopolysaccharide cellcoat)
-- gal mutation in the galactose metabolism
-- chl mutation in nitrate reductase
-- bio defective biotin synthesis
-- uvrB loss of the excision repair system (deletion of the ultraviolet repair B gene)
- Stock cultures of tester strains were stored in liquid nitrogen (-196 °C)

CELL CULTURE
- Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid No. 2) and incubated in a shaking incubator (37 °C, 150 spm) until the cultures reached an optical density of 1 ± 0.1 at 700 nm (10⁹ cells/mL). Freshly grown cultures of each strain were used for a test.
- Agar plates (9 cm diameter) contained 25 mL glucose agar medium. Glucose agar medium contained, per litre, 18 g purified agar (oxoid code L28) in Vogel-Bonner medium E, 20 g glucose, 0.5 mg biotin and 0.6 mg histidine
- Top agar medium contained 0.6 % (w/v) agar and 0.5 % (w/v) NaCl, heated to dissolve the agar. Samples of 3 mL top agar were transferred into 10 mL glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 1 °C.
- Incubations performed in dark at 37 ± 1 °C. Temperature monitored.

METABOLIC ACTIVATION SYSTEM
Preparation of S9 Fraction:
- Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles River, Sulzfeld, Germany
- Animals housed at NOTOX in a special room under standard laboratory conditions, as described in the SOP.
- The rats were injected intraperitoneally with a solution of 20% (w/v) Aroclor 1254 (500 mg/kg bw) in corn oil. 5 days later, the rats were sacrificed by decapitation, having been denied food for at least 12 hrs.
- The livers of the rats were removed aseptically and washed in cold (0 °C) sterile 0.1 M sodium phosphate buffer (pH 7.1) containing 0.1 mM Na₂-EDTA. Subsequently the livers were minced in a blender and homogenised in 3 volumes of phosphate buffer with a Potter homogeniser. The homogenate was centrifuged for 15 mins at 9000 G. The supernatant (S9) was transferred into sterile ampules, which were stored in liquid nitrogen (-196 °C) and identified by the day of preparation.
- Before use, all S9-batches were characterised with the metabolic activation requiring positive control: benzo[a]pyrene (Sigma)

Preparation of S9-Mix:
- S9 mix was prepared immediately before use and kept on ice. S9-mix contained, per 10 mL, 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 mL Milli-Q water, 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl₂ solution, 1 mL of 0.33 M KCl solution.
- The above solution was filtered (0.22 µm)-sterilised. To 9.5 mL of S9-mix components, 0.5 mL S9-fraction (batches 0-26 and 0-27) was added 5 % (v/v) S9-fraction to complete the S9-mix.

TEST PROCEDURE
Dose range-finding tests:
- Dose range-finding tests were performed to both methods (direct plate and preincubation)
- Selection of a range of doses was based on dose-range finding tests both with and without S9-mix. 8 concentrations were tested in triplicate, and the results formed part of the complete dataset.
- The highest concentration of test material used in the subsequent mutation assay was the level at which the test substance inhibited bacterial growth or the test substance exhibited limited solubility.

Mutation assay:
- ≥ 5 different doses, at approx. half-log steps, were tested in triplicate at each strain.
- Test substance was tested both with and without S9-mix.
- 2 additional experiments were performed with TA1535, TA1537, T98 and TA102; one direct plate assay and one preincubation assay.

Direct plate assay:
- Top agar in top agar tubes was molten and heated to 45 °C. The following solutions were successively added to 3 mL molten agar: 1 mL of a fresh bacterial culture 10⁹ cells/mL of one of the tester strains; 0.1 mL of a dilution of the test substance in DMSO; and either 0.5 mL S9-mix or 0.5 mL 0.1 M phosphate buffer (as appropriate). The ingredients were mixed on a Vortex and the contents of the top agar tube were poured onto a selective agar plate. After solidification of the top agar, the plates were turned and incubated in the dark at 37 °C for 48 hr. After this period the revertant colonies were counted.

Preincubation assay:
- Top agar in top agar tubes was molten and heated to 45 °C. The following solutions were preincubated for 30 mins by 70 rpm at 37 °C, either 0.5 mL S9-mix or 0.1 M phosphate buffer (as appropriate), 0.1 mL of a fresh bacterial culture (10⁹ cells/mL) of one of the tester strains, 0.1 mL of test material in DMSO.
- After preincubation period the solutions were added to 3 mL molten top agar. The ingredients were mixed using a vortex and the contents of the top agar tube were poured onto a selective agar plate. After solidification of the top agar, the plates were turned and incubated in the dark at 37 °C for 48 hr. After this period the revertant colonies were counted.

Evaluation
- Revertant colonies automatically counted using a Protos model 50000 colony counter or manually if fewer than 40 colonies per plate were present. Plates with sufficient test article precipitate to interfere with automatic colony counting were counted manually.
- Further details on evaluation are included in Appendix 1 (attached).
Evaluation criteria:
A substance is considered negative (not mutagenic) in the test if:
1. The number of revertants in any tester strain at any concentration is not greater than 2 times the solvent control value, with or without metabolic activation.
2. The negative response should be reproducible in at least one repeat experiment.

A test substance was considered positive (mutagenic) in the test if:
1. It induces at least a twofold dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, with or without metabolic activation. However, any plate count of less than 20 is considered not to be significant.
2. The positive response should be reproducible in at least one repeated experiment.

The above criteria were not absolute and other modifying factors could enter into the final evaluation decision.

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:
cytotoxicity
Remarks:
≥333 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA:
- Historical control data are presented in the additional materials attached.
- The negative and strain-specific positive values were within laboratory background historical control data ranges, indicating that the test conditions were adequate and the metabolic activation system functioned properly.
Remarks on result:
other: all strains/cell types tested

Any other information on results incl. tables

For results tables, please see the attached supporting information.

Applicant's summary and conclusion

Conclusions:
The test material was found not to be mutagenic in an Ames test with Salmonella typhimurium strains TA100, TA1535, TA1537, TA98 and TA102.