Chlorodifluoromethane

Genetic toxicity: in vitro

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:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Basic data given: study comparable to guideline/standards

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

publication

Title:

Mutagenicity of a halocarbon refrigerant mono-chlorfluoromethane (E-22) in Salmonella typhimurium

Author:

Longstaff E and McGregor D

Year:

1978

Bibliographic source:

Toxicol Lett 2(1): 1-4

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Principles of method if other than guideline:

none

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

R-22

IUPAC Name:

R-22

Constituent 2

Reference substance name:

Chlorodifluoromethane

EC Number:

200-871-9

EC Name:

Chlorodifluoromethane

Cas Number:

75-45-6

Molecular formula:

CHClF2

IUPAC Name:

chlorodifluoromethane

Details on test material:

Commercially available chlorodifluoromethane (Arcton 22, ICI Ltd); Impurities: R-12: 3552 ppm; R21: <15 ppm; R-23: <6 ppm; R-31: <6 ppm; R-32: 43 ppm; R-142b: <2 ppm; R143a: 2 ppm; methyl chloride: <5 ppm; unknown: 2 ppm.

Method

Target gene:

histidine requirement and ampicillin resistance

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not specified

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not specified

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat liver post-mitochondrial supernatant diluted 1 to 3 with co-factor (S-9 mix) prepared from rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Gas concentrations: 0% / 1% / 33% / 50% R-22 and 50% vinyl chloride (positive control)

Vehicle / solvent:

none

Controls

Untreated negative controls:

yes

Remarks:

0% R-22

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: vinyl chloride / EC 200-831-0 / CAS 75-01-4

Remarks:

Concentration of vinyl chloride: 50%

Details on test system and experimental conditions:

Test system modified for testing gases. Petri dishes containing Vogel-Bonner basal medium were overlain with "top-agar" containing the bacterial tester trains TA1535, TA1538, TA98 or TA100, with or without liver post-mitochondrial supernatant diluted 1 to three with co-factor (S-9 mix) prepared from rats induced with Aroclor 1254. The tester strains wer obtained directly from Professor Ames' laboratory and conformed to be described characteristics of deep-rough mutation, histidine requirements and amicilline resistance, although the batch of TA100 used in the table II experiment had a very low spontaneous mutation frequency. The seeded dishes ware exposed to the test gas by incubation at 37°C inside gas-tight 5-litre glass reaction vessels into which were metered the various nominal gas/air mixtures through calibrated rotameters. In an initial experiment, exposure time to the test gas/air mixtures was 24 h at 37°C followed by a further 24 h incubation in air after which time the number of histidine-revertant colonies per dish was counted using an automatic colony counter. In later experiments, exposure to the gas was for the intire incubation period of 2-3 days.

Evaluation criteria:

not available

Statistics:

not available

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

not available

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The initial reverse mutation test with commercially available R-22 using an exposure time of 24 h showed a mutagenic response in strains TA1535 and TA 100, and this respose was dose related (Table II).

Table II

 

Number of revertant colonies per plate obtained after exposure of S. typhimurium TA1535 and TA100 to crude R-22 for 24 h followed by a further 24 h incubation in air

 

Nominal gas concentration R-22 in air %	TA1535			TA100
	With S9	Without S9		With S9	Without S9
0	17	83		16	17
1	28	65		17	30
33	79	124		54	62
50	146	156		126	99

 

 There was no effect on strains TA1538 or TA98 with or without hepatic post-mitochondrial S-9 mix. These results were confirmed in further experiments employing longer incubation periods with the test compounds. The mean results of three trials (total of five plates per datum point) using commercially available crude R-22 and vinyl chloride as the test control gas, all at the nominal 50 % gas/air mixture concentration are presented in table III.

Table III

 

Number of revertant colonies per plate obtained after exposure of S. typhimurium TA1535 and TA100 to crude R-22 and VCM for 24-72 h

 

(Mean results ± standard deviation from three experiments)

Nominal gas concentration	TA 1535			TA100
	With S9	Without S9		With S9	Without S9
0	12 ± 4	19 ± 7		81 ± 15	70 ± 7
50% R-22	70 ± 12	80 ± 21		123 ± 32	238 ± 347
50% vinyl chloride	114 ± 21	105 ± 19		277 ± 252	361 ± 492

 

The results clearly indicated a mutagenic effect of R-22 in the TA100 strain were less convincing. This strain gave a positive response in one one of the three experiments. The vinyl chloride (VCM) system positive control gas was mutagenic in both TA1535 and TA100 with and without microsomal S-9 mix. It is perhaps noteworthy that under these conditions VCM was positive without S-9 activation. It has been assumed that VCM required activation by microsomal or post-mitochondrial supernatant enzymes to show a posive response in the test system. Also, because of the protracted exposure time employed, an artefact of this plate test system is that when the presence of mammalian-type metabolism is not an absolute requirement for mutagenic activity, it is difficult to identify a detoxifying effect of the liver preparation. The microsomal enzymes are inactivated within a few hours but the test substance persits and can continue its direct effect on the bacteria. Detoxification can therefore be swamped by subsequent direct activity.

The mean results from further two experiments comparing crude and purified R-22 (total of 4 plates per datum point) are presented in table VI.

Table IV

 

Comparision of the mean numbers (± standard deviations) of revertant colonies per plate of S. typhimurium strains TA1535 and TA 100 obtained after incubation with crude and purified R-22, and vinyl chloride

 

Nominal gas concentration	TA 1535
	With S9	Without S9		With S9	Without S9
0%	11 ± 3	18 ± 7		80 ± 17	71 ± 7
50% crude R-22	72 ± 13	87 ± 17		110 ± 14	83 ± 13
50% purified R-22	69 ± 19	70 ± 13		100 ± 18	86 ± 13
50% vinyl chloride	119 ± 20	113 ± 12		165 ± 30	141 ± 5

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

This work was able to demonstrate a positive response in any of the four tester strains with or without S-9 activation, and it was forced to conclude, therefore, that R-22 is a bacterial mutagen per se under the test conditions. The response in the test system was relatively weak and in order to have a realistic assessment of the risk to man from compounds like R-22 it is necessary to await the results of conventional animal studies.

Executive summary:

Chlorodifluoromethane (R-22) in the commercial available grade was found to reproducably respond positive in the Salmonella typhimurium reverse mutation assay (Ames' test) modifid for gaseous substances. This positive response was shown to be dependent neither on the presence of rat post-mitochondrial supernatant in the incubation medium nor in the presence of known mutagenic gases contaminating the commercial grade substance.