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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:
2009-06-08 to 2009-08-26
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:
Bis(2-ethylhexyl) cyclohexane-1,4-dicarboxylate
EC Number:
EC Name:
Bis(2-ethylhexyl) cyclohexane-1,4-dicarboxylate
Cas Number:
Molecular formula:
bis(2-ethylhexyl) cyclohexane-1,4-dicarboxylate
Test material form:
Specific details on test material used for the study:
Batch No.: 20090226
Purity: 99.38%


Target gene:
S. typhimurium: Histidine
E. coli: Trp operon
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:
S9 mix
Test concentrations with justification for top dose:
Preliminary test: 0, 4, 20, 100, 500, 1500, 2500 µg/plate.
Main test: 0 (vehicle), 15.63, 31.25, 62.5, 125, 250, 500 µg/plate.
Vehicle / solvent:
- Vehicle/solvent used: Ethanol
- Justification for choice of solvent/vehicle: Sponsor indicated that test substance was insoluble in water. Solubility tests were conducted in DMSO, distilled water, minimum essential medium (MEM) and ethanol. The test material was soluble in ethanol, up to the highest tested concentration of 25 mg/mL, but not in any of the other solvents. Therefore, ethanol was chosen as the solvent/vehicle.
Controlsopen allclose all
Negative solvent / vehicle controls:
Positive controls:
Positive control substance:
sodium azide
TA100, TA1535, without metabolic activation only
Positive controls:
Positive control substance:
TA98 without metabolic activation only
Positive controls:
Positive control substance:
TA1537 without metabolic activation only
Positive controls:
Positive control substance:
E. coli WP2uvrA without metabolic activation only
Positive controls:
Positive control substance:
other: 2-aminoanthracene
TA1535, E. coli WP2uvrA, with metabolic activation only
Positive controls:
Positive control substance:
TA100, TA98, TA1537 without metabolic activation only
Details on test system and experimental conditions:
- All strains were supplied by Molecular Toxicology, Inc. Boone, North Carolina, United States

- Each 25 mL of oxoid broth was innoculated with tester strain from master plates and then incubated in a gyrotary incubator (200 rpm) at 37 ± 1 °C for approx. 10 hrs
- Minimal glucose agar plates were prepared using 1.5 % Bacto agar (Difco), Vogel-Bonner medium E and 2 % glucose per L, and then 25 mL of agar placed in 100 × 15 mm petri dishes.
- The agar plates for the E. coli strain were supplemented with 0.25 mL of 0.1 % tryphtophan solution per L of agar.
- Top agar was prepared using 0.6 % agar and 0.5 % NaCl. Top agar for the Salmonella TA strains was supplemented with 0.05 mM L-histidine.HCl and biotin.

- To each mL of suspension, 0.09 mL of DMSO was added. The mixture was stored at approx. -80 °C.
- After thawing, master plates of each strain were made and kept in a refrigerator to be used in the experiments.

- The genotypes of the tester strains were confirmed according to the methods of Maron and Ames (1983), prior to the start of experiments:
-- Auxotrophy: The S. typhimurium tester strains were histidine auxotrophs while E. coli WP2uvrA tester strain was a tryptophan auxotroph.
-- UV sensitivity (uvrB and uvrA): Tester strains TA98, TA100, TA1535, TA1537 and WP2uvrA exhibited sensitivity to UV light.
-- rfa mutation: All S. typhimurium strains exhibited resistance to crystal violet.
-- R-factor: Tester strains TA98 and TA100 exhibited resistance to ampicillin.
-- Each tester strain exhibited a characteristic number of spontaneous revertant colonies when plated.

- S-9 was supplied by Molecular Toxicology, Inc. Boone, North Carolina, United States.
- Type: S-9 Aroclor 1254-induced Sprague-Dawley male rat liver
- Protein content: 42.5 mg/L
- Date of receipt: 2008-10-16
- Expiry date: 2010-07-03
- Storage conditions: Frozen (approx -20 °C)

- Cofactor-1 was supplied by Wako Pure Chem. Ind., Ltd, Japan
- Manufactured by Oriental Yeast Co., Ltd
- Date of receipt: 2009-04-30
- Expiry date: 2011-04-29

- Content of 1 mL S9-mix: 8 µmol MgCl2.6H2O, 33 µmol KCl, 5 µmol G-6-P, 4 µmol NADPH, 4 µmol NADH, 100 µmol sodium phosphate buffer (pH 7.4) and 50 µL S-9.
- Prepared S-9 mix was kept in an ice bath until use and used at 0.5 mL/plate.

- DEHCH was dissolved in ethanol to yield a 20-fold stock solution of the target concentrations. The highest test item solution was diluted with the same vehicle control.
- To determine test item concentration, 10 mL of each concentration was analysed. As a consequence, the first formulation of test material was rejected and a second batch prepared. This second batch was within ± 10 % of the nominal concentrations and were therefore considered valid and used.
- Positive control items were dissolved either in distilled water (sodium azide), or in DMSO (2-nitrofluorene, 9-aminoacridine, 4-nitroquinoline-N-oxide, 2-aminoanthracene, benzo(a)pyrene).
- Positive control substances were stored at -20 °C for up to 10 months and were thawed prior to use.
- Positive control substance concentrations were as follows:
-- TA 100 -S9: Sodium azide 0.5 µg/plate
-- TA 100 +S9: Benzo(a)pyrene 2 µg/plate
-- TA 1535 -S9: Sodium azide 0.5 µg/plate, 2-aminoanthracene 2 µg/plate
-- TA 1535 +S9: 2-aminoanthracene 2 µg/plate
-- TA 98 -S9: 2-nitrofluorene 2 µg/plate, benzo(a)pyrene, 2 µg/plate
-- TA98 +S9: Benzo(a)pyrene 2 µg/plate
-- TA1537 -S9: 9-aminoacridine 50 µg/plate
-- TA1537 +S9: Benzo(a)pyrene 2 µg/plate
-- WP2uvrA -S9: 4-nitroquinoline-N-oxide 0.5 µg/plate
-- WP2uvrA +S9: 4-nitroquinoline-N-oxide 4µg/plate

- A preliminary dose range-finding study was performed to determine the concentration range for the confirmatory mutagenicity study.
- The highest concentration in the preliminary study was 2500 µg/plate.
- 500 µg/plate was chosen as the highest concentration for the main study.

- 2 experiments were performed under identical conditions, each including vehicle and positive controls.
- Triplicate treatements were conducted for each concentration in the presence and absence of S-9 mix.

- Autoclaved top agar (2 mL) was dispensed to 12×75 mm tubes at 45 °C in a heating block. 0.05 mL of test item, 0.5 mL of S-9 mix or sodium phosphate buffer as appropriate, and 0.1 mL of bacterial culture were added to each tube. The mixture was stirred gently for 2-3 s using a Vortex mixer and was then poured onto minimal glucose agar plate.
- Vehicle control groups were treated with 0.05 mL of vehicle control and each of the positive control groups was treated with strain-specific controls (0.1 mL) instead of test item.
- Samples of the highest concentration tested (0.05 mL) and S-9 mix (0.5 mL) were also plated withouth bacterial culture to verify sterility.
- After the top agar was solidified, the plates were inverted, and incubated at 37 ± 1 °C for approx. 48 hrs. Revertants were then counted with a hand counter. Each concentration of test item was assayed in triplicate.

Each plate was evaluated for contamination by observation for any formation of a background lawn or any other abnormality.

- Results were expressed as the mean number of revertant colonies from each triplicate of plates per dose with standard deviations.
Evaluation criteria:
A result was considered positive if there was a concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation. In addition, any increases in revertant colonies were required to have occurred in the absence of toxicity (antibacterial effect) as defined as a clearing or diminution of background lawn, the appearance of microcolonies and/or a decrease of more than 50 % in the number of colonies compared to that of vehicle control.

Results and discussion

Test results
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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:
- Precipitation of test substance was observed at 250 µg/plate when the test item was added to top agar.
- Precipitation of test substance was observed at 500 µg/plate in all studies after 48 hr incubation.

- No antibacterial effects (cytogenicity) was observed at any concentration in the presence and absence of S-9 mix.
- Precipitation of test item was observed at concentrations of ≥ 500 µg/plate when test item solutions were mixed with top agar and incubated for 48 hrs.

- The number of revertant colonies in the vehicle controls was within the range of KIT historical control data.

Any other information on results incl. tables

Sterility results:

No contaminant colonies were observed on the sterility plates for the highest concentration of the test substance or the S9 mix.

Confirmatory mutagenicity study:

There was no increase in the number of revertant colonies compared to the vehicle control at any concentration of the test substance either in the presence or absence of metabolic activation system in any strain tested. The precipitation of test item was observed at the concentration more than 250µg/plate in all strains used, when test item solutions were mixed with top agar. Precipitation was observed at the 500µg/plate concentration after 48 h incubation. The number of viable cells in the overnight cultures as determined by optical density was within acceptable limits: 1.2 to 2.3E09 cells/mL (S. typhimurium strains) and 2.3E09 cells/mL (E. coli strain); 1.1 to 2.2E09 cells/mL (S. typhimurium strains) and 2.1E09 cells/mL (E. coli strain) in the 1st and 2nd experiments respectively. The positive controls induced a significant increase in the number of revertant colonies indicating the assay was valid.

Dose Analysis:

Dosing solutions were analysed. The concentration analyses indicate that the actual concentration of the dosing solutions used were 101.81 % to 103.82 % and 94.47 % to 103.81 % of target concentration in the 1st and 2nd experiments, respectively.

Applicant's summary and conclusion

The test substance was assessed for genotoxicity according to OECD Guideline 471. The test substance was concluded to be negative in four test strains of Salmonella typhimurium and one test strain of Escherichia coli.