Norflurane

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Norflurane

EC Number:

212-377-0

EC Name:

Norflurane

Cas Number:

811-97-2

Molecular formula:

C2H2F4

IUPAC Name:

1,1,1,2-tetrafluoroethane

Details on test material:

The original sample of HFC 134a (Batch ref 13679/16/1) was supplied in two cylinders (samples 1&2) by ICI Chemicals & Polymers Limited on behalf of PAFT as a colourless gas with a boiling point of -26.5° C. Analysis of this batch gave a purity of 99.98% HFC 134a.
Three further batches (samples 3 -5) were subsequently supplied on behalf of PAFT with the following purities: Batch ref 13679/16/1/5 (99.98%) and Batch ref 13960/72/1-3 (99.84%) (both supplied by ICI Chemicals & Polymers Limited); Batch ref DACAW83856A (99.988%) supplied by Dupont.
The sponsor supplied information that the samples were stable under normal storage conditions and under the test conditions of this study. The test compound was assayed as received.

Method

Species / strain

Species / strain / cell type:

other: S. Typhimurium: TA1535, TA1537, TA1538, TA98, TA100

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

up to 100% (417 g/m3)

Details on test system and experimental conditions:

The mutagenicity assays were conducted using the Salmonella plate incorporation assay as described by Maron and Ames (1983), with certain minor modifications to allow for testing of a gaseous compound.
In phase I the original sample of HFC 134a was assayed at least twice both in the presence and absence of a liver S-9 mix prepared from AROCLOR 1254-induced Sprague Dawley rats, using six dose levels designed to give a range of exposure concentrations. The five Salmonella strains tested in this assay were TA1535, TA1537, TA1538, TA98 and TA100 and have been fully decribed in the literature (Ames et al 1975).
In phase II, the second sample of the original batch was assayed twice in strain TA1535 only, under a range of exposure and metabolic activation conditions. The test plates were exposed to the sample in the presence of a liver S9-mix prepared from AROCLOR 1254-induced Alpk:APfSD rats for the entire 3-day incubation period, the initial 24-hour incubation period only, and for the entire 3-day incubation period in the presence of S9 without cofactors.
In phase III the three further batches were assayed concurrently using TA1535 in the presence of S9-mix (Alpk:ApfSD rats) only using base plates and top agar prepared from standard agar and from purified agar. Sample 3 and 5 were tested twice, but due to a limited amount, sample 4 was tested only once.
For each experiment, the appropriate positive control compounds were tested to validate the dosing regime used, the baterial strains and where appropriate to confirm the activity of the S9-mix used. For each experiment the incubation period was 3 days at 37°C.
In addition to the negative controls, 'absolute' negative controls were included in each test. These confirmed the mutation rates for the tester strains. Revertant colonies were counted using an automatic electronic colony counter (AMS 40-10 Image Analyser fitted with appropriate software, Analytical Measuring Systems Ltd.)

Evaluation criteria:

Test data from individual experiments are considered valid if:
a) the concurrent negative control data are acceptable
b) the "assay" positive control data shows unequivocal positve, dose-related responses.
c) at least the lower test compound dose shows no evidence of toxicity, and at least three test doses show no significant toxicity (i.e significant loss of background growth and/or reductions in colony numbers).
Failure of one or more tester strain/S9 combinations does not invalidate the data for the remainder of a concurrent experiment.
A positve response in a valid individual experiment is achieved when one or both of the following criteria are met:
a) a statistically significant dose-related increase in the number of revertant colonies is obtained;
b) a two-fold or greater increase in the mean number of revertant colonies (over that observed for the negative control plates) which is statistically significant, is observed at at least one dose level.
A negative result in a valid experiment is achieved when:
a) there is no statistically significant dose-related increase in the mean number of revertant colonies per plate observed for the test compound; and
b) in the absence of any such dose response, no increase in colony numbers is observed (at any test dose) which exceeds 2x the current negaive control.
For a positive result in an individual experiment to be considered indicative of an unequivocal positive result for that strain/S9 combination, then the observed effects must be consistently reproducible.
For plates showing microbial contamination the results were not included in the calculation of mean colony count and standard deviation.

Statistics:

The assessment of statistical significance was carried out using a one-tailed Student's t-test (Ehrenberg 1984). The corresponding probability for each dose level was derived by computer using the appropriate degrees of freedom. Values of p<0.01 were treated as significant, with values of 0.01

Results and discussion

Test results

Species / strain:

other: S. Typhimurium: TA1535, TA1537, TA1538, TA98, TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Experimental Phase I.

In replicate experiments, the original sample of HFC 134a did not induce any significant, reproducible increases in the observed numbers of revertant colonies in any strain in the absence of an auxiliary metabolising system (S9) or in strains TA1537, TA1538, TA98 and TA100 in the presence of S9. Due to microbial contamination observed in strains TA1538 and TA100 (+S9) in experiment 3, the compound was retested in these strains with S9 in the final experiment. In each experiment, the toxic effects observed at the highest exposure level (thinning of background level and/or reductions in colony numbers) confirms that the compound was tested to an effective maximum dose.

With strain of TA1535 in the presence of S9, the sample of HFC 134a gave a reproducible, dose-related significant response, reaching maxima of 3.2x and 2.9x background mutation rates. The apparent negative result obtained in the first experiment can be discounted due to the excessive toxic effects observed at all test doses (which may in turn be an artefact of the high negative control values observed). Based on these results, it was originally concluded that HFC 134a was mutagenic to S.typhimurium strain TA1535, and that this mutagenicity was S9 -dependent.

Experimental Phase II.

In an attempt to investigate the response observed in phase I, a further sample of HFC 134a (from the same batch of material as the original sample) was tested in strain TA1535 under a series of exposure conditions. The sample was exposed, in the presence of a standard S9 mix, to the test plates for the entire 3 -day incubation period (as normal), and also for only the initial 24 hour part of this incubation period. This reduced exposure phase, whilst assumed to be sufficient to induce any mutations, may reduce any toxic and/or growth-inhibitory effects of prolonged exposure to (high doses) of the test chemical, and therefore has the potential or more clearly revealing any apparent mutagenic effect that is produced.

The sample of HFC 134a was also exposed to strain TA1535 (for the entire 3-day incubation period) in the presence of a "depleted" S9 mix , where the cofactors required for normal functioning of the S9 -mix were replaced by buffer. These conditions were designed to determine whether the mutagenic response observed in Phase I was actually dependent upon metabolic activation, or whether by simply modifying the specific environmental conditions on the test plates in such a way that an inherent (bacterial) mutagenicity of HFC 134a could be expressed.

In replicate experiments, the sample of HFC 134a gave unequivocal negative results under all three exposure conditions. The slight toxicity observed at the highest exposure concentration in each case demonstrated by a slight reduction in observed colony numbers and/or reduction in the background lawn on test plates, indicates that the sample of HFC 134a was tested to and effective maximum dose.

Experimental Phase III

In an attempt to determine whether the positive response observed with sample 1 of HFC 134a was due to an agar effect rather than to compound-induced mutations (see de Raat et al, 1984), three samples of HFC 134a (samples 3 -5) were tested in strain TA1535 (in the presence of S9 only) using top and base agars prepared using purified agar as well as normal comercial agar. Due to the limited amount of sample 4, this sample was only tested once with each agar: the assays with samples 3 and 5 were repeated.

In each experiment, all three HFC 134a samples gave unequivocal negative results when assayed in both agar media.

Control chemicals

With the exception of strain TA1535, +S9 experiment 1 where an abnormally high value was observed for the negative control, the negative control data for each strain were within acceptable limits in each experiment when compared to published literature values (Kier et al, 1986). The lack of variability in the TA1535 data shows that the "agar effects" discussed above were not observed during this experimental work.

The positive controls for each experiment induced the expected responses indicating the strains were rresponding satisfactorily in each case. The apparent failure of 2AA in experiment 5b and the reduced 2AA response in experiment 6b is as expected under the experimental conditions used (+S9, -cofactor). Similarly, the responses observed for vinyl chloride in each experiment (where appropriate) confirmed the effectiveness of the dosing regime used for assaying gaseous compounds.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

Under the initial experimental conditions used in this assay, the original sample of HFC 134a gave a positive response in S.typhimurium strain TA1535 in the presence of an auxiliary metabolising system (S9). The compound gave an unequivocal negative, i.e. non-mutagenic, response in strain TA1535 (-S9), and in strains TA1537, TA1538, TA98 and TA100 in both the presence and absence of S9, when tested to an exposure level which produced toxic effectws in each case.
Following this initial finding, four further samples of HFC 134a (including one from the same batch as the original sample) gave unequivocal negative, i.e. non-mutagenic, response in strains TA1535 (+S9) when tested under a range of experimental conditions. The non-reproducibility of the original observation indicates that this effect was not due to HFC 134a-induced mutations.

Executive summary:

HFC 134a was not genotoxic to Salmonella bacteria strains TA1535, TA1537, TA1538, TA98, TA100 with or without S9 metabolic activation.