diethyl 1,4-cyclohexanedicarboxylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

between 03 November 2010 and 08 February 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No. 440/2008 and the United Kingdom Environmental Mutagen Society (Cole et al, 1990).

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

417-310-0

EC Name:

-

Cas Number:

72903-27-6

Molecular formula:

C12H20O4

IUPAC Name:

1,4-diethyl cyclohexane-1,4-dicarboxylate

Test material form:

liquid

Details on test material:

- Substance type: pure active substance
- Physical state: colorless liquid
- Stability under test conditions: stable
- Storage condition of test material: Refrigerator (between 0 and 10°C under nitrogen)

Method

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells.

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination:yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Cell Line
The Chinese hamster ovary (CHO-K1) cell line was obtained from ECACC, Salisbury, Wiltshire.

Cell Culture
The stocks of cells were stored in liquid nitrogen at approximately -196°C. Cells were routinely cultured in Ham's F12 medium, supplemented with 5% foetal calf serum and antibiotics (Penicillin/Streptomycin at 100 units/100 µg per ml) at 37°C with 5% CO2 in air.

Cell Cleansing
Cell stocks spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen down they were cleansed of HPRT- mutants by culturing in HAT medium for 4 days. This is Ham's F12 growth medium supplemented with Hypoxanthine (13.6 µg/ml, 100 µM), Aminopterin (0.0178 µg/ml, 0.4 µM) and Thymidine (3.85 µg/ml, 16 µM). After 4 days in medium containing HAT, the cells were passaged into HAT-free medium and grown for 4 to 7 days. Bulk frozen stocks of HAT cleansed cells were frozen down, with fresh cultures being recovered from frozen before each experiment.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 was prepared in house from the livers of male Sprague-Dawley CD strain rats. These had received three daily oral doses of a mixture of phenobarbitone (80 mg/kg) and beta-naphthoflavone (100 mg/kg), prior to S9 preparation on the fourth day.

Test concentrations with justification for top dose:

Preliminary Cytotoxicity Test: dose ranges of 35.63 to 1140 µg/mL and 35.63 to 2280 µg/mL were used for the 4-hour without and with S9 exposure groups respectively. A dose range of 8.91 to 2280 µg/mL was used for the 24-hour exposure group.

4-hour without S9: 35.63, 71.25, 142.5, 285, 427.5, 570, 712.5, 855 µg/mL
4-hour with S9 (2%): 71.25, 142.5, 285, 570, 1140, 2280 µg/mL (up to 10 mM)
24-hour without S9: 8.91, 17.81, 35.63, 71.25, 142.5, 285, 570, 855 µg/mL
4-hour with S9 (1%): 71.25, 142.5, 285, 570, 1140, 2280 µg/mL (up to 10 mM)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle:The test material formed a solution with the solvent suitable for dosing.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Dimethyl benzanthracene (DMBA) (Sigma Batch Numbers 105K1312 and 040H1231 were used for Experiment 1 and Experiment 2 respectively at 0.5 and 1.0 µg/mL as the positive controls in cultures with S9.

Remarks:

With metabolic activation.

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation Migrated to IUCLID6: Sigma Batch Number 0001423147 at 500 and 750 µg/mL was used in the 4-hour cultures without S9 and at 200 and 300 µg/mL for the 24-hour cultures without S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Experiment 1: 4 hours with S9 (2%) and without S9
Experiment 2: 4 hours with S9 (1%) and 24 hours without S9
- Expression time (cells in growth medium): 6 to 7 days
- Selection time (if incubation with a selection agent): 14 days

SELECTION AGENT (mutation assays): 10 μg/mL 6-Thioguanine

NUMBER OF REPLICATIONS: duplicate

NUMBER OF CELLS EVALUATED: 2 x 10E5 cells/75 cm² flask (5 replicates per group) in Ham's F12 growth media (5% serum), supplemented with 6-TG

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency (triplicate at 200 cells/25 cm² flask in 5 mL of growth medium)

Evaluation criteria:

Significant or dose-related increases in mutant frequency per survivor (see Calculations in "Any other information on materials and methods incl. Tables"

Statistics:

none

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no significant changes
- Effects of osmolality: the osmolality did not increase by more than 50 mOsm
- Evaporation from medium: no data in the report but not expected due to the low vapor pressure of the test material
- Water solubility: NA as the test material was dissolved in DMSO
- Precipitation: none observed

RANGE-FINDING/SCREENING STUDIES:
Preliminary Cytotoxicity Test
Dose ranges of 35.63 to 1140 µg/ml and 35.63 to 2280 µg/ml were used for the 4-hour without and with S9 exposure groups respectively. A dose range of 8.91 to 2280 µg/ml was used for the 24-hour exposure group. A greasy/oily precipitate of the test item was observed at the end of exposure at and above 285 µg/ml in the 24-hour exposure group. In the 4-hour exposure groups a greasy/oily precipitate was seen at and above 570 µg/ml and 285 µg/ml in the absence and presence of S9 respectively at the end of exposure. The results of the individual flask counts and their analysis are presented in the attached Table 1. It can be seen that there was a very steep toxicity curve exhibited in the 4-hour exposure group in the absence of S9 between 285 µg/ml and 570 µg/ml. The 4-hour exposure group in the presence of S9 demonstrated no marked reduction in the cloning efficiency (CE) up to and including the maximum recommended dose. The 24-hour exposure group demonstrated a more gradual dose related reduction in cloning efficiency and was completely toxic at 1140 µg/ml.
Mutagenicity Test - Experiment 1
The Day 0 and Day 7 cloning efficiencies are presented in the attached Table 2 and Table 3. As was seen in the preliminary toxicity test there was a steep toxicity curve in the 4-hour group in the absence of metabolic activation (S9) with the dose levels of 570 µg/ml and above being too toxic for plating and the dose level of 427.5 µg/ml showing a 33% reduction in cloning efficiency at Day 0 only. The Day 7 cloning efficiencies demonstrated a very steep toxicity curve between 427.5 and 570 µg/ml. The 4-hour exposure group in the presence of metabolic activation (S9) demonstrated only a slight reduction in cloning efficiency at the maximum dose tested when compared to the vehicle control.
The Day 7 vehicle control cloning efficiencies for the 4-hour exposure group in the absence of S9 were marginally less than 70% but were considered to be acceptable. In the presence of S9 the Day 0 and Day 7 vehicle control cloning efficiencies achieved acceptable levels.
The mutation frequency counts and mean mutation frequency per survivor values are presented in the attached Table 2 and Table 3. Both of the vehicle control mutant frequency per survivor values were within the maximum upper limit of 25 x 10E6 mutants per viable cell, and all positive control dose levels produced marked increases in mutant frequency per survivor values over the vehicle controls. This was taken to indicate that the test method and metabolic action system were functioning adequately.
There were no marked increases in mutation frequency per survivor which exceeded the vehicle control value by 20 x 10E-6 in either the absence or presence of S9.
Mutagenicity Test - Experiment 2
The Day 0 and Day 7 cloning efficiencies are presented in the attached Tables 4 and 5. It can be seen that in the 4-hour exposure group in the presence of S9 there was a reduction in the cloning efficiency at the maximum dose of 2280 µg/ml at Day 0 although this was not continued onto Day 7. This increased toxicity when compared to Experiment 1 is in line with the reduction in S9 concentration in Experiment 2 and confirms the increased toxicity of the test item in the absence of S9. The 24-hour exposure group demonstrated a very sharp toxicity curve and was slightly more toxic than in the preliminary toxicity test. The dose levels of 570 µg/ml and 855 µg/ml were both too toxic for plating and the dose levels up to and including 285 µg/ml showed no reduction in cloning efficiency when compared to the vehicle control groups.
The vehicle control cloning efficiencies for the Day 0 and Day 7 4-hour and 24 hour exposure groups were less than 70% but were considered to be acceptable as they achieved over the 50% minimum requirement and there was no indication of an increase in mutation frequency for the test item.
The mutation frequency counts and mean mutation frequency per survivor per 10E6 cells values are presented in Tables 4 and 5. Both of the vehicle control mutant frequency per survivor values were within the maximum upper limit of 25 x 10E6 mutants per viable cell, and all positive control dose levels produced marked increases in mutant frequency per survivor values over the vehicle controls. This was taken to indicate that the test method and metabolic action system were functioning adequately.
In the absence and presence of metabolic activation there were no increases in mutation frequency per survivor which exceeded the vehicle control value by 20 x 10E-6. The test item was tested to the maximum recommended dose in the presence of S9. In the absence of S9 the steepness of the toxicity curve made it difficult to achieve a dose response curve, however it is considered that the test item has been adequately tested

All tables are attached.

COMPARISON WITH HISTORICAL CONTROL DATA: All values withing historical range

Any other information on results incl. tables

Due to the quantity and or format of the tables please see attached tables.

 

REFERENCES

Cole J, Fox M, Garner R C, McGregor D B, and Thacker J (1990) Gene Mutation in Cultured Mammalian Cells. In'Basic Mutagenicity Tests: UKEMS Recommended Procedures', (Kirkland D J, Ed),Press, Cambridge University Press, New York.

Hsie A W, Brimer P A, Mitchell T J, and Gosslee D G (1975) The dose response relationship for ethylmethane sulfonate induced mutations at the HPRT locus in Chinese hamster ovary cells,Somatic Cell Genetics,1, 247-261.

Hsie A W, Casciano D A, Couch D B, Krahn D F, O'Neill J P, and Whitfield B L (1981) The use of Chinese hamster ovary cells to quantify specific locus mutation and to determine mutageniety of chemicals. A report of the Gene-tox program,Mut. Res.,86, 193-214.

Scott, D., Galloway, S.M.,, R.R., Ishidate, M. Jr, Brusick, D., Ashby, J. and Myhr, B.C. (1991). Genotoxicity under Extreme Culture Conditions. A report from ICPEMC task Group 9. Mutation Res.,257, 147 – 205.

Applicant's summary and conclusion

Conclusions:

The test item did not induce significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation in either of the two experiments. The test item was therefore considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of this test.

Executive summary:

Introduction.

The study was conducted to assess the potential mutagenicity of the test item on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of Chinese hamster ovary (CHO) cells. The test method used was designed to be compatible with the OECD Guidelines for Testing of Chemicals No. 476 'In Vitro Mammalian Cell Gene Mutation Tests', Commission Regulation (EC) No 440/2008 and United Kingdom Environmental Mutagen Society (Cole et al, 1990). The technique used is a plate assay using tissue culture flasks and 6-thioguanine (6­TG) as the selective agent.

Methods.

Chinese hamster ovary (CHO) CHO-K1 cells were treated with the test item at a minimum of six dose levels, in duplicate, together with vehicle (solvent) and positive controls. Four treatment conditions were used for the test, i.e. In Experiment 1, a 4‑hour exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration and a 4-hour exposure in the absence of metabolic activation (S9). In Experiment 2, the 4-hour exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

The dose ranges selected for Experiment 1 and Experiment 2 were based on the results of the preliminary cytotoxicity test and were as follows:-

Exposure Group	Final concentration oftest item(µg/ml)
4-hour without S9	35.63, 71.25, 142.5, 285, 427.5, 570, 712.5, 855
4-hour with S9 (2%)	71.25, 142.5, 285, 570, 1140, 2280
24-hour without S9	8.91, 17.81, 35.63, 71.25, 142.5, 285, 570, 855
4-hour with S9 (1%)	71.25, 142.5, 285, 570, 1140, 2280

 

Results.

The vehicle (solvent) controls gave mutant frequencies within the range expected of CHO cells at the HPRT locus.

The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolising system.

The test item demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.

Conclusion. 

The test item was considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of the test.

This study is considered as acceptable and satisfies the requirement for the mammalian cell gene mutation endpoint.