Dichlorodifluoromethane

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:

01 March 1979 to 03 May 1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Remarks:

Study report, not conducted to GLP but well documented with methodology and results included.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1979

Report date:

1979

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

Not specified

Deviations:

no

Principles of method if other than guideline:

See below under "details of test system and conditions"

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

Dichlorodifluoromethane

EC Number:

200-893-9

EC Name:

Dichlorodifluoromethane

Cas Number:

75-71-8

Molecular formula:

CCl2F2

IUPAC Name:

dichlorodifluoromethane

Test material form:

aerosol dispenser: not specified

Remarks:

migrated information: aerosol

Details on test material:

Test Material: Methane, dichlorodifluoro-
Haskell No.: 12,830
Other Code: OCNB 4915-23
Composition: 99.9+% Dichlorodifluoromethane (FC-12)
11 ppm Chlorodifluoraaethane (FC-22)
20 ppm Trichlorofluoromethane (FC-11)
Other Names:
Freon 12
FC-12
Fluorocarbon 12

Method

Target gene:

The Chinese Hamster Ovary (CHO) cell line is used to detect mutations in a gene coding for the enzyme hypoxanthine-guanine phosphoribosyl transderase (HGPRT).

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

The cells were shown to be free of mycoplasma.

Additional strain / cell type characteristics:

other: performed with the BH4 clone of the CHO-K1 cell line.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver, S-9 fraction

Test concentrations with justification for top dose:

0 - 51 %

Vehicle / solvent:

Air for test substance

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Air, and respective solvents for positive controls

True negative controls:

yes

Remarks:

Air

Positive controls:

yes

Positive control substance:

other: FC-31, chlorofluoromethane; VC1, chloroethane

Details on test system and experimental conditions:

Cell Culture
This study was performed with the BH4 clone of the CHO-K1 cell line. Cells were routinely cultured as monolayers in Han’s F12 medium without hypoxanthine (F12 (w/o hx)) containing 10% dialyzed heat-inactivated fetal calf serum (DHIFCS). In experiments, the culture medium also contained penicillin (50 untis/ml) and streptomycin (50 micrograms/ml). Culture dishes were incubated at 37°C in an atmosphere containing 5% CO2 and 90+% relative humidity. Prior to subculturing, the cells were removed from the dishes with 0.05% trypsin. The cells were shown to be free of mycoplasma.
Activation System
Source of Liver: Livers were obtained from 8 to 9 week old male rats (Charles River CD) given Aroclor 1254 (500 mg/kg) 5 days before sacrifice. The rats were starved for the 24hr preceding sacrifice.
Preparation of Liver Homogenate: Rats were decapitated and bled. Livers from at least 6 rats were homogenized (for 5 sec) in a Waring Blender with 1.5 volumes of 0.115 M KCI at 4°C and then with a Dounce homogenizer (5 strokes). The homogenate was centrifuged under the same conditions and the resulting S-9 was stored in liquid nitrogen vapour. The concentration of protein in the S-9 was determined.
Treatment Medium
Without Activation: The treatment medium (3 ml) contained F12 (w/o hx), 10% DHIFCS, penicillin (50 units/ml), streptomycin (50 micrograms/ml), and HEPES buffer, pH 7.2 (2.5 x 10E-2M).
With Activation: The treatment medium (3 ml) contained F12 (w/o hx), S-9 (3 mg protein, unless otherwise specified), penicillin (50 units/ml), streptomycin (50 micrograms/ml), HEPES buffer, pH 7.2 (2.5 x 10-2M) MgCL2 (5.5 x 10-3M), glucose-6-phosphate (5.0 x 10E-3M), NADP (1.5 x 10E-3M), and NADH (1 x 10E-3M).
Experimental Design
Preliminary cytotoxicity experiments, with and without activation, were conducted to establish appropriate test sample concentrations for the mutagenesis experiments. Ideally, the highest concentration of test chemical should give about 10% of control survival. In each mutagenesis experiment duplicate treatments were made for each test concentration. Two or more experiments were performed with and without activation. Each experiment included a positive and solvent control.
Mutagenesis Assays
Five x 10E-5 cells were plated per 25 cm2 flask in 5 ml of culture medium. The next day the culture medium was removed and the treatment medium was added. The treatment flasks were flushed with different test gas-air mixtures. An amount of test gas-air mixture equivalent to 10 volumes of the treatment flask was flushed through each flask at a rate of about 500 ml/min. Control flasks were flushed with air. A pair of flow meters was used to generate the test gas-air mixtures. The cells were incubated for 18-19 hr (without activation) and 5 hr (with activation). Treatment flasks were incubated on a rocker panel to maximize the contact between cells and test chemical. After incubation, the flasks were flushed with air, the treatment medium was removed and the flasks were washed with culture medium. Cells treated with activation were subcultured immediately after treatment and the cells treated with activation were incubated in culture medium for 21-26 hr before subculturing.
At the time of subculturing, one portion of the cells was plated tyo assess cytotoxicity and another portion plated to allow expression of mutants resistamt to 6-thioguanine (6-TG). To assess cytotoxicity, 200 cells were plated per 60 mm dish (6 dishes) from each flask of cells in the experiment. These dishes were incubated for 7 days and the colonies were stained and counted. Cell survival was determined by dividing the total number of colonies by the total number of cells plated and was expressed both as the percent plated and as the percent of the solvent control survival.
To allow expression of the 6-TG resistant phenotype, cells from each treatment flask were plated at 1 x 10E-6 / 100 mm dish (1 dish). These cells were maintained in exponential growth for 7 days by subculturing twice (2 days after the initial subculturing and then three days later of vice versa). On the 7 th day the cells were plated to assess the cell survival and the frequency of 6-TG-resitant cells, 2 x 10E5 cells were plated per 100 mm dish (5 dishes) in culture medium containing 1 x 10-5M 6-TG. The cells were incubated for 7 days and the colonies stained and counted. The mutation frequency was expressed as the number of mutant colonies/10-6 surviving cells at the time of mutant selection.

Evaluation criteria:

The following guidelines are used to classify a test sample as a mutagen or nonmutagen in the assay.
A test sample is classified as a nonmutagen when:
A. The probability is greater than 0.05 that the numbers of mutants at each of the test sample concentrations studies are not greater than the number of mutants in the solvent control.
AND
B. The probability is greater than 0.05 that there is not a positive correlation between the numbers of mutants and increasing concentrations of the test sample.
A sample is classified as a mutagen when:
A. The probability is less than 0.01 that the numbers of mutants at one or more of the test sample concentrations studied are not greater than the number of mutants in the solvent control.
AND
B. The probability is less than 0.01 that there is not a positive correlations between the number of mutants and increasing concentrations of the test sample.
The samples that cannot be classified by the above criteria are dealt with on a case-by-case basis.

Statistics:

Because of the complex nature of the experimental errors in this assay, mutation frequency data were transformed prior to analysis. The formula Y = (mutation frequency + 1)E+0.15 was used because this power transformation provided data which satisfied assumptions required for performing parametric statistical analyses. The analysis used a two variable (dose and experiment). Analysis of Variance (ANOVA) Model that allows for unequal numbers of dose points and unequal numbers of test results in each trial. In one analysis, the effect of each chemical dose was compared to the solvent control by a t-test of significance to determine whether a dose caused a significant increase in the mutation frequency. The second analysis was an ANOVA to evaluate a dose-response relationship. Linear, quadratic and higher order effects were tested by an F-test of significance.

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:

valid

Positive controls validity:

valid

Additional information on results:

The test chemical, dichlorodifluoromethane, was introduced into the plastic treatment flasks in the gaseous phase. An R-2-15A flow meter with a stainless steel float was used to deliver the test chemical into the flasks at a flow rate of 600 ml/min for one minute. Samples of the headspace of the flasks were taken and analysed by gas chromoatography (HY-FI, Model 600-D) using an SE-30 column at 70-80°C at the start of the treatment. The carrier gas, N2, was set at 30 psig and the flow rate of H2 was 23 ml/min. The peak heights obtained from the analysis of samples taken from each flask at the beginning of treatment were compared to the peak height of a standard vessel containing 20% test chemical and 80% air to give the concentrations (%) reported.
Two trials were performed without activation using 2.0 ml of treatment medium. The cells were exposed to the undiluted test chemical for one, five or eighteen hours. A cytoxicity response was not observed at any of these exposure times. The statistical analysis revealed no mutagenic activity according to the guidelines stated.
Two trials using an activation system containing 2.0 mg S-9 protein per ml of treatment medium (2.0 ml) were also performed. The exposure times to the undiluted test chemical were one, three and five hours. Again, no cytoxicity responses were observed. The statistical analysis using hours of exposure as the concentration showed no points to have a significant increase in the number of mutants over the solvent control and a linear dose response was not observed. The Dose X Trial Interaction suggested that the dose-response relationship in the two trials was significantly different ( p = 0.0221). When each trial was analyzed separately no significant points or linear dose responses were observed.

Any other information on results incl. tables

TABLE I – TOXICITY AND MUTAGENCITY OF H-12,830 IN CHINESE HAMSTER OVARY CELLS WITHOUT ACTIVATION – TRIAL ONE

Compound	Concentration (%)	Cells/flasks** (x 10^6)	TOXICITY		MUTAGENICITY
			Cell Survival		Cell Survival (% Plated)	Mutants
			(% Plated)	(% Control)		Total	Frequency*
12,830	0 (a)	3.93	59.8	100	77.8	7	9.0
“	42 (a) 44 (a)	3.86 3.61	58.2 59.8	97.3 100.0	77.4 80.7	4 1	5.2 1.2
“	0 (b) 0 (b)	3.95 3.74	65.1 66.9	100+	79.8 75.0	0 5	0 6.7
“	40 (b) 42 (b)	3.55 3.63	58.2 67.8	88.2 102.7	68.1 77.4	3 0	4.4 0
“	0 (c) 0 (c)	2.88 2.95	0 60.6	100+	64.8 72.2	5 1	7.7 1.4
“	42 (c) 40 (c)	2.95 2.88	63.7 61.6	104.6 101.1	67.8 70.0	4 4	5.9 5.7
FC-31	++ (a)	3.42	50.2	83.9	67.8	42	61.9

(a) One hour exposure

(b) Five hour exposure

(c) Eighteen hour exposure

12,830 = Dichlorodifluoromethane

 

TABLE II – TOXICITY AND MUTAGENICITY OF H-12,830 IN CHINESE HAMSTER OVARY CELLS WITHOUT ACTIVATION – TRIAL TWO

Compound	Concentration (%)	Cells/flasks** (x 10^6)	TOXICITY		MUTAGENICITY
			Cell Survival		Cell Survival (% Plated)	Mutants
			(% Plated)	(% Control)		Total	Frequency*
12,830	0 (a)	3.57	65.6	100	78.8	6	7.6
“	49 (a) 49 (a)	3.71 3.77	71.8 60.1	109.5 91.6	85.2 89.0	2 3	2.3 3.4
“	0 (b) 0 (b)	3.92 3.74	63.2 70.7	100+	89.5 85.6	6 7	6.7 8.2
“	49 (b) 49 (b)	3.79 3.78	55.9 60.9	83.4 90.9	89.3 89.0	21 1	23.5 1.1
“	0 (c) 0 (c)	3.09 2.92	60.2 61.2	100+	91.2 88.8	3 4	3.3 4.5
“	46 (c) 43 (c)	2.49 2.63	66.3 72.9	109.2 120.1	76.9 72.2	11 4	14.3 5.5
FC-31	++ (a)	2.81	39.4	60.1	59.8	21	35.1

(a) One hour exposure

(b) Five hour exposure

(c) Eighteen hour expousre

12,830 = Dichlorodifluoromethane

 

TABLE III – TOXICITY AND MUTAGENICITY OF H-12,830 IN CHINESE HAMSTER OVARY CELLS WITH ACTIVATION – TRIAL ONE

Compound	Concentration (%)	Cells/flasks** (x 10^6)	TOXICITY		MUTAGENICITY
			Cell Survival		Cell Survival (% Plated)	Mutants
			(% Plated)	(% Control)		Total	Frequency*
12,830	0 (a) 0 (a)	2.49 2.84	62.5 55.1	100+	83.7 79.0	7 8	8.4 10.1
“	44 (a) 44 (a)	2.83 2.80	64.9 63.4	110.4 107.4	79.1 73.8	3 5	3.8 6.8
“	0 (b) 0 (b)	2.10 2.18	61.2 56.4	100+	80.1 77.6	3 19	3.7 24.5
“	36 (b) 40 (b)	2.39 2.05	54.8 56.3	93.2 95.7	72.7 70.8	4 10	5.5 14.1
“	0 (c) 0 (c)	1.83 1.45	57.5 59.1	100+	59.4 74.3	7 14	11.8 18.8
“	44 (c) 42 (c)	2.43 2.02	67.4 62.8	115.6 107.7	78.2 78.2	1 0	1.3 0
VC1	†† (a) †† (a)	1.34 1.46	34.0 30.2	57.8 51.4	50.4 45.2	195 118	386.9 261.1

(a) One hour exposure

(b) Three hour exposure

(c) Five hour exposure

12,830 = Dichlorodifluoromethane

 

TABLE IV – TOXICITY AND MUTAGENICITY OF H-12,830 IN CHINESE HAMSTER OVARY CELLS WITH ACTIVATION – TRIAL TWO

Compound	Concentration (%)	Cells/flask** (x 10^6)	TOXICITY		MUTAGENICITY
			Cell Survival		Cell Survival (% Plated)	Mutants
			(% Plated)	(% control)		Total	Frequency*
12,830	0 (a)	2.42	74.2	100	94.2	20	21.2
“	51 (a) 51 (a)	2.61 2.72	67.2 57.1	90.6 77.0	90.2 94.3	11 0	12.2 0
“	0 (b) 0 (b)	2.08 2.00	68.6 67.3	100+	89.4 92.4	16 17	17.9 18.4
“	44 (b) 48 (b)	2.41 2.46	64.2 62.1	94.4 91.3	84.7 89.7	3 2	3.5 2.2
“	0 (c) 0 (c)	1.68 1.57	66.3 68.9	100+	100.7 91.5	2 2	2.0 (d) 2.2
“	47 (c) 48 (c)	2.16 2.34	64.5 69.8	95.4 103.3	90.5 80.3	18 8	19.9 10.0
VC1	†† (a)	1.41	43.6	58.8	84.2	179	212.6

 (a) One hour exposure

(b) Three hour exposure

(c) Five hour exposure

(d) A cell survival of 100% was used to calculate mutant frequency

12,830 = Dichlorodifluoromethane

Symbols:

(*) Mutants per 10⁶surviving cells

(**) Cells per flask at the time of the initial subculturing

(+) The average of the duplicate plating efficiencies was set equal to 100%

(†) Computed from the average transformed mutation frequency, Y.

(††) 100% VC1 in the gaseous phase was introduced into the treatment flasks for one minute.

(NF) None found

(++) A mixture of two thirds FC-31 and one third air was introduced into the treatment flasks for one minute.

Applicant's summary and conclusion

Conclusions:

negative with and without metabolic activation
Dichlorodifluoromethane was tested for mutagenic activity in the Chinese Hamster Ovary Cell Assay with and without activation system. The chemical did not exhibit mutagenic activity.

Executive summary:

The Chinese Hamster Ovary (CHO) cell line is used to detect mutations in a gene coding for the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT). A chemical is tested for mutagenic activity on both the presence and absence of an activation system. The activation system contains rat liver homogenate and is a necessary treatment condition because CHO cells lack the ability to metabolize many carcinogens and mutagens to an active form. After treatment, the cells go through a recovery period called expression time. HGPRT deficient mutants among the surviving cells are identified by using culture medium that only allows the mutant cells to grow. The number of HGPRT deficient mutants in the treated sample compared to the number in the solvent control is the measure of mutagenic activity. A dose-response relationship is sought for chemicals displaying mutagenic activity.

RESULTS

The test chemical, dichlorodifluoromethane, was introduced into the plastic treatment flasks in the gaseous phase. An R-2-15A flow meter with a stainless steel float was used to deliver the test chemical into the flasks at a flow rate of 600 ml/min for one minute. Samples of the headspace of the flasks were taken and analysed by gas chromoatography (HY-FI, Model 600-D) using an SE-30 column at 70-80°C at the start of the treatment. The carrier gas, N2, was set at 30 psig and the flow rate of H2 was 23 ml/min. The peak heights obtained from the analysis of samples taken from each flask at the beginning of treatment were compared to the peak height of a standard vessel containing 20% test chemical and 80% air to give the concentrations (%) reported.

Two trials were performed without activation using 2.0 ml of treatment medium. The cells were exposed to the undiluted test chemical for one, five or eighteen hours. A cytoxicity response was not observed at any of these exposure times. The statistical analysis revealed no mutagenic activity according to the guidelines stated.

Two trials using an activation system containing 2.0 mg S-9 protein per ml of treatment medium (2.0 ml) were also performed. The exposure times to the undiluted test chemical were one, three and five hours. Again, no cytoxicity responses were observed. The statistical analysis using hours of exposure as the concentration showed no points to have a significant increase in the number of mutants over the solvent control and a linear dose response was not observed. The Dose X Trial Interaction suggested that the dose-response relationship in the two trials was significantly different ( p = 0.0221). When each trial was analyzed separately no significant points or linear dose responses were observed.

CONCLUSIONS

Dichlorodifluoromethane was tested for mutagenic activity in the Chinese Hamster Ovary Cell Assay with and without activation system. The chemical did not exhibit mutagenic activity.