Cyclohex-1,4-ylenedimethanol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

CHDM is oxidized in vivo to CHDM (see "toxicokinetics") which is valid for read-x justification

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Cyclohexane-1,4-dicarboxylic acid

EC Number:

214-068-6

EC Name:

Cyclohexane-1,4-dicarboxylic acid

Cas Number:

1076-97-7

Molecular formula:

C8H12O4

IUPAC Name:

cyclohexane-1,4-dicarboxylic acid

Details on test material:

lot no. 3038678

Method

Target gene:

HGPRT

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Test concentrations with justification for top dose:

1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 ug/ml

Vehicle / solvent:

1% DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

5-bromo-2'deoxyuridine (BrdU) = + control without S9 activation Migrated to IUCLID6: with S9 activation

Details on test system and experimental conditions:

A. Dosing Procedure
EC 94-0212, CHDA formed a white suspension in water at 50.0 mgfml but was a clear colorless liquid in DMSO at about 200 mg/ml and a
cloudy white suspension at about 300 mgfml. The vehicle of choice for the cytotoxicity assay was therefore DMSO. The test material
was suspended in DMSO at 100 times the highest desired treatment concentration. Primary IOOX test material stock solutions at lower
concentrations were then prepared by serial dilution with DMSO. Final IX dosing stocks were prepared by making 1:100 dilutions of
the primary stocks into culture medium containing eight percent fetal calf serum for nonactivation studies and five percent serum
for activation studies. Based on the cytotoxicity assay, concentrations up to 5000 ~g/ml were chosen for the mutation assays.
Since high concentrations were to be tested and the test material was acidic and required neutralization, the vehicle was changed to
Fl2 culture medium. For each trial of the mutation assay, a stock was prepared at twice the concentration needed for the highest
concentration and neutralized. Serial 2X stocks were then prepared from the neutralized stock and diluted 1:2 with Fl2 medium to
prepare the different concentrations of dosing medium. Treatments were initiated by replacing the medium on the cells with the
appropriate concentration of test material. The volume of culture medium diluent in the activation studies was reduced to compensate
for the volume of S9 reaction mix used. Preparations of test material in the vehicle were prepared fresh
each day for biological testing. Treatments were initiated by replacing the culture medium on the cell cultures with the treatment
medium containing the test material at the desired concentrations. The stability of the test material under the conditions of treatment
is the responsibility of the sponsor.
B. Rangefinding Cytotoxicity Testing
A wide range of test article concentrations was tested for cytotoxicity both with and without S9 metabolic activation. Ten
concentrations that spanned a 3-log concentration range were used. The applied doses ranged from 1.95 ~g/ml to 1000 ~/ml. In
addition, three negative (media) controls and three vehicle controls containing 1% DMSO were used in each cytotoxicity assay.
The cells were seeded at 200 cells per dish, allowed to attach overnight (16 to 18 hours) and exposed to the test or control
article for four hours at 37" ± 1.5"C in a humidified atmosphere containing about 5% C02 • The cells were then washed twice with
Dulbecco's phosphate buffered saline (PBS) and incubated in Fl2 culture medium for six additional days to allow colony development.
Colonies were then fixed in alcohol, stained with Giemsa and counted by eye, excluding those with approximately 50 cells or less.
Cytotoxicity was expressed as a percentage of colony counts in treated cultures versus control cultures. The preliminary cytotoxicity
information was used to select eight doses for the mutation assay that covered a range from approximately 0% to 90% reduction in
colony-forming ability. Treatment conditions chosen for both the nonactivation and activation portions of the mutation assay ranged
from 125 ~g/ml to 5000 ~g/ml.
C. Mutagenicity Testing (Nonactivation Assay)
The assay procedure was based on that reported by Hsie, et At. (1975), and reviewed by Hsie, et al. (1981), with modifications suggested by Myhr and DiPaolo (1978). The cleansed cells were plated at about 3 x 10" cells per T-75 (75 cm2) tissue culture flask on the day before dosing. The time between plating and treatment was about 18 hours. Cell cultures were treated with test or control material for four hours at 37" ± 1.5"C in a
humidified atmosphere with about 5% C02 • Cell cultures normally contain at least 4 x 10" cells by the time of treatment termination. After treatment, the cell monolayers were washed twice with phosphate buffered saline, trypsinized, and suspended in culture medium. The cell suspension from each dose level was counted using a Coulter Counter and replated at 1.5 x 10" cells into each of two 150-mm dishes and at 200 cells into each of three 60-mm dishes. The small dishes were incubated for seven days to permit colony development and the determination of the cytotoxicity associated with each treatment. The large dishes were incubated for seven days to permit growth and expression of induced mutations. The mass cultures were subcultured every two or three days during the expression period to maintain logarithmic cell growth. At each subculture the cells from the two
150-mm dishes from each dose level were combined and reseeded at about 1.5 x 10" cells into each of two 150-mm dishes. At the end of the expression period (seven days), each culture was reseeded at 2 x 105 cells per 100-mm dish (12 dishes total) in mutant selection medium. Also, three 60-mm dishes were seeded at 200 cells each in culture medium to determine the cloning efficiency of each culture. After incubation for seven to ten days, at 37"C ± 1.5"C in a humidified atmosphere with about 5% C02 , the colonies were fixed with alcohol, stained with Giemsa and counted to determine the number of TG-resistant colonies in mutant selection dishes and the number of colonies in the cloning efficiency dishes. The colonies were counted by eye, excluding those with approximately 50 cells or less.
D. Mutagenicity Testing (Activation Assay)
The activation assay was performed independently with its own set of negative and positive controls. The procedure was
identical to the nonactivation assay except for the addition of the S9 fraction of rat liver homogenate and necessary cofactors
during the four-hour treatment period. The fetal bovine serum content of the medium used for dosing was reduced to 5% by
volume. The cofactors consisted of nicotinamide adenine dinucleotide phosphate (NADP, sodium salt), glucose-6-phosphate,
calcium chloride, potassium chloride, and magnesium chloride, all of which were in a pH 7.8 sodium phosphate buffer.

Evaluation criteria:

I. Relative Survival to Treatment: The percentage of the cells that were able to formcolonies after the treatment period in both the rangefinding cytotoxicity assays and the mutation assays relative to the concurrent negative controls. The average number of colonies in three dishes (seeded at 200 cells each) was determined for each treatment condition.
Relative Survival (%) = (Average no. of colonies per treated culture/Average no. of colonies per negative control dish) x 100%

2. Relative Population Growth: The cumulative growth of the treated cell population, relative to the negative control growth, over the entire expression period and prior to mutant selection. In general, highly toxic treatments will reduce the growth rate as well as the survival. Values less than 100% indicate growth inhibition. Treated populations that are more than 2 or 3 doublings behind the control might not achieve maximum expression of the TG-resistant phenotype. The relative population growth is calculated from cell count data not presented in this report and is intended to provide only an approximate indication of growth during the expression period, since cells are easily lost or not completely released by trypsin during the subculture procedures. Relative Population Growth (%) = (Treated culture population increase over the expression period/negative control population increase over the expression period) x 100%
·3. Absolute Cloning Efficiency (CE): The ability of the cells to form colonies at the time of mutant selection. The observed number of mutant colonies can be converted to the frequency of mutant cells in the treated population. Absolute CE (%) = (Average no. of viable colonies per dish/200) x 100%
4. Mutant Frequency
The mutant frequency is the endpoint of the assay, calculated as the ratio of colonies found in thioguanineselection medium to the total number of cells seeded, adjusted by the absolute C.E. (expressed as the number of mutants per one million cells).

Results and discussion

Test results

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:

valid

Positive controls validity:

valid

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

The test material, EC 94-0212, CHDA, is considered negative for inducing forward mutations at the HGPRT locus in Chinese hamster ovary cells
under both the 59 metabolic activation and nonactivation conditions of the assay.

Executive summary:

The objective of this in vitro assay was to evaluate the ability of EC 94-0212, CHDA to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary cells under conditions with and without metabolic activation. The test material was soluble in dimethylsulfoxide (DMSO) at 200 ug/ml. For the cytotoxicity assay, the test material was prepared in DMSO and lower concentrations were prepared by dilution with DMSO. Treatment media were prepared by making 1:100 dilutions of the primary stocks into F12 culture medium. The cytotoxicity assay was initiated with concentrations from 1.95 ug/ml to 1000 ug/ml and the pH of treatments from 125 ug/ml to 1000 ug/ml were acidic and required neutralization. Preliminary cytotoxicity testing showed the test material to be noncytotoxic to CHO cells without activation up to 1000 ug/ml and, at most, moderately cytotoxic up to 1000 ug/ml in the presence of metabolic activation. The mutation assays were initiated with concentrations up to 5000 ug/ml because of the lack of appreciable cytotoxicity at 1000 ug/ml. Since the test material was insoluble in DMSO at 300 mg/ml and stocks up to 500 mg/ml would be needed for the mutation assay, the vehicle was changed to F12 culture medium. In addition, the use of media would aid in neutralization required at the higher concentrations. A stock was prepared for each mutation assay at twice the concentration needed for the highest concentration and neutralized. Serial 2X stocks were then prepared from the neutralized stock and then diluted 1:2 with F12 medium to prepare the dosing stocks. EC 94-0212, CHDA was soluble in all stocks prepared in F12 medium (up to 10,000 ug/ml).

Two trials of the nonactivation assay were initiated but the first trial had incomplete data because of loss due to contamination. The data is presented in Table 3 but a second trial was performed to confirm the results. In both of the nonactivation assays presented in this report, six treatments from 500 ug/ml to 5000 ug/ml were analyzed for mutant induction. No toxicity was induced at any of the concentrations analyzed. The mutant frequencies of treated cultures varied randomly with dose. All treatments in both trials were within the range acceptable for background mutant frequencies which is 0 to 15 x 10^-6. EC 94-0212, CHDA was therefore considered negative without activation in this assay.

In the presence of metabolic activation, two trials were also performed. The second trial was initiated because the two negative controls and three dose levels including the high dose had excessive contamination in Trial 1. Six treatments from 500 ug/ml to 5000 ug/ml were analyzed in both trials and low to moderate cytotoxicities were observed. Sporadic small increases were observed, but the increases were not dose related and were still within the acceptable range for mutant frequency variation in the negative controls. The test material was therefore nonmutagenic with activation in this assay.