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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12/16/1987 to 1/15/1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test procedure in accordance with national standar methods.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
The test sample of tetrafluoroethylene (TFE) was supplied by ICI plc, Chemicals and Polymers Group, Runcorn, Cheshire UK. The test sample was contained in a cylinder with a terpene stabiliser and assigned the CTL reference no. Y02066/004/001. The purity of TFE was greater thatn 99.0% v/v.

The positive control vinyl chloride (with a purity of 100%) was supplied by Cambrian Gases, Croydon UK. This was given the CTL reference no. Y02066/004/001.

Test animals

Species:
mouse
Strain:
other: C57BL/6jfC-1/Alpk mice
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Male and female C57BL/6JfCD-1/Alpk mice were supplied by the Animal Breeding Unit ICI plc, Pharmaceuticals Division, Alderley Park, Macclesfield Cheshire, UK.
- Age at study initiation: Phase I in the range 8-13 weeks; Phase II in the range 8 - 10 weeks
- Weight at study initiation:
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study:
- Housing: wire mesh cages (up to 15 animals per cage)
- Diet (e.g. ad libitum): On arrival given food (Porton Combined Diet)
- Water (e.g. ad libitum): On arrival fitered tap water (via an automatic system) ad libitum.
- Acclimation period:


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 45-55
- Air changes (per hr):
- Photoperiod (12 hrs dark / 12 hrs light):


IN-LIFE DATES: From: 16 December 1987 To: 15 January 1988

Administration / exposure

Route of administration:
inhalation
Vehicle:
TFE was mixed with air to provide the exposure concentrations.
Details on exposure:
For the TFE exposures, each exposure chamber consisted of a round glass jar with a plastic screw top. The diameter of the jar was approximately 15 cm and the height 20 cm giving a volume of approximately 3.5 litres. The chambers were used for each dose level. In Phase I each two chambers per dose level contained 5 male and 5 female mice. For Phase II 15 mice were divided such that 7 or 8 mice were in each of the of the two chambers per dose level. The male and female mice were exposed on separate days for Phase II.
For the vinyl chloride (positve control) exposures. Each chamber consisted of a 17.5 litre all-glass desicator fitted with a perforated aluminium base. Again, the males and females were exposed in separate chambers.

Generations systems
Tetrafluoroethylene: the system for TFE was designed to allow the dilution of the top dose level to the lower levels using cleaned and conditioned air such that the consumption of test material was kept to a minimum for safety reasons. Non-return valves were incorporated into each gas line to ensure that pressurised TFE did not come into contact with pressurised oxygen. As supplied the TFE contained a terpene stabiliser to prevent spontaneous polymerisation which can occur under certain conditions. This stabiliser was removed using an activated charcoal trap which was made inert by the flow of nitrogen. Oxygen was then added at 1 litre/minute together with 4 litres / minute nitrogen to achieve a normal (air) ratio of oxygen / nitrogen. The temperature of the charcoal bed was monitored to guard against and uncontrolled exothermic reaction which can occur at temperatures greater than 40°C.
Extra air was added between each dose level to achieve the dilution necessary for the next, lower concentration. The whole system was pressure balanced by applying a vacuum to the exhaust equal to the total flow of gas through the system. All gas flows were monitored using in-line variable area low meters (Rotameter).
Vinyl chloride: Gaseous vinyl chloride was taken from the cylinder and mixed with compressed air at such flow rates as to achieve 50 000 ppm v/v. Both air and vinyl chloride were monitored by in-line Rotameters.
During the exposure period the temperature within the chambers was 20.7 - 21.6° for male mice, and 21.1 - 22.3°C for female mice; the relative humidity was maintained in the range 57 - 82% for male mice and 59 - 89% for female mice.
Atmosphere analysis.
The TFE atmospheres were sampled using a gas tight syringe at least 3 times an hour and analysed by gas chromatography. The vinyl chloride atmospheres were sampled in a similar manner and analysed by infra-infra red gas analyser.
Duration of treatment / exposure:
6 hours
Frequency of treatment:
Single expouse
Post exposure period:
Bone marror samples taken at 24, 48 and 72 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
Males: 0, 5000, 12000 or 19000 ppm; Females: 0, 7000, 17000, 28000ppm
Basis:
analytical conc.
No. of animals per sex per dose:
15
Control animals:
yes, concurrent no treatment
Positive control(s):
Vinyl chloride administered to groups of five male and five female mice at a target dose level of 50 000 ppm.

Examinations

Tissues and cell types examined:
Bone marrow, erythrocytes
Details of tissue and slide preparation:
Bone marror smears were prepared at 24, 48 and 72 hours after dosing. The preparations were stained with polychrome methylene blue and eosin to visualise the various cell types.
Evaluation criteria:
1000 polychromatic erythrocytes (PE) per slide were evaluated for the presence of micronuclei, and 1000 erythrocytes were counted to determine the percentage of PE in the total erythrocyte population, and thus obtain an indication of cytotoxicity.
Statistics:
The incidence of micronucleated polychromatic erythrocytes (MPE) and percentage PE were considered using analysis of variance, regarding each combination of sampling time, dose level, and sex as a separate group. The results were examined to determine whether any differences between control adn treated groups were consistent between the sexes and across sampling times. Where extended MPE counts were taken, these data were also considered by analysis of variance. First, jointly with the relevant original counts to determine whether any differences between control and treated groups were consistent between original and extended counts. Second, if the results were inconsistent between original and extended counts, as an independent set of data. The data for the incidence of MPE was transformed using a natural logarithmic transformation to stabilise variability, prior to analysis.
All analysis were carried out using the GLM procedure in SAS (1985). For the original Micronuclei and the % Polychromatic Erythrocytes, each treatment group was compared with the control group mean at the corresponding sampling time using a one-sided Student's t-test, based on the error mean square in the analysis. These comparisons were made for separate sex group means. For the analysis of the extended Micronuclei counts only, each treatment group mean was compared with the control group mean using a one-sided Student's t-test based on the mean square which estimates between animal variation. Unbiased estimates of the group means were provided by the least square means (LSMEANS option in SAS) but for simplicity standard means are presented.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Results

Atmopsheric Analysis

The TFE and vinyl chloride atmosphere concentrations (for Phase II) were found to within 10% of the target concentrations, with the exception of Group 1 males (target concentrations 19 000 ppm) where the actual concentration was 12% higher (21 300) than the target concentration. Group mean data for Phase I and Phase II are shown in Tables 4 and 5 respectively (below).

Phase I - MLC Determination

The MLC was determined by probit analysis (Finney, 1971) to be 23 308 ppm for male mice and 34 419 for female mice. From the dose levels to represent 80%, 50% and 20% MLC levels were selected to be 19 000 ppm, 12 000 ppm and 5 000 ppm for male mice and 28 000 ppm, 17 000 ppm and 7 000 ppm for female mice. These levels were the target dose levels for Phase II.

Phase II - Micronucleus Test

Group data are shown in Tables 1, 2 and 3 (below). Vinyl chloride gave the expected statistically and biologically significant increase in MPE in both male and femal mice.

In animals exposed to TFE, a statistically significant increase in the frequency of MPE was observed only in male mice, and only at the 72 hour sampling time following exposure to TFE at target dose levels of 5 000 and 12 000 ppm. (Table 1). No such effect was noticed in males at the top dose level (19 000 ppm) at the 72 hour sampling time, or at any other dose level or time point. No statistically significant increases in MPE were observed in female mice at any dose level or sampling time.

In order to gain more confidence in these MPE values for the male control and 72 hour mid and low dose TFE groups, extended counts (3 000 additional PE assessed) were carried out for the male control mice and TFE treated mice from the 5 000 and 12 000 ppm dose levels at the 72 hour sampling time. Following the extended counts the MPE incidence in control animals increased, and while the TFE treated mice from both dose levels (5 000 and 12 000) still showed a small increase in MPE over the control level, these increases were not statistically significant. When comparing percentage PE in control mice with those in TFE treated mice, statistically significant reductions were observed in both males and females at a number of dose levels and sampling times. This effect was most marked at the 48 hour and 72 hour sampling times (see Table 3).

Table 1: Mean Incidence of Micronuclei/1000 Polychromatic Erythrocytes at Three Sampling Times

 Group  Compound  Target Dose (ppm)  Sex        Incidence of Micronuclei/1000PE
         24 hours  48 hours  72 hours
 1  Tetrafluoroethylene  19 000  Male  3.8  3.2  1.6
   80% MLC  28 000  Female  1.6  0.8 (4)  0.8
 2  Tetrafluoroethylene  12 000  Male  2.4  3.0  3.4*
   50% MLC  17 000  Female  1.8  0.6  2.4
 3  Tetrafluoroethylene  5 000  Male  2.2  3.6  4.8**
   20% MLC  7 000  Female  1.0  0.6  0.6
 4  Control  Not applicable  Male  3.4  2.8  1.2
   (Laboratory Air)    Female  1.0  0.4  1.5 (4)
 5  Vinyl chloride  50 000  Male  15.6**    
       Female  10.0**    

* Statistically significant increase in micronucleated polychromatic erythrocytes at p<0.05 Student's 't' test (one-sided)

** Statistically significant increase in micronucleated polychromatic erythrocytes at p<0.01 Student's 't' test (one-sided)

All mean values based on 5 observations unless indicated in parenthesis

Table 2: Mean Incidence of Micronuclei/1000 Polychromatic Erythrocytes from Extended Counts (Means Based on the Assessment of an Extra 3,000 Polychromatic Erythrocytes per Animal.

Group Mean Animal Data (Males Only)

 Group  Compound  Target Dose (ppm)  Sex  Incidence of Micronuclei/1000PE (72 hours)
 2  TFE 50% MLC  12 000  Male  4.3
 3  TFE 20% MLC  5 000  Male 4.3 
 4  Control (Laboratory Air)  Not applicable  Male  2.8

All mean values based on 15 observations (3 observations at 1000 PE on each of 5 animals per group).

Table 3: Mean Percentage of Polychromatic Erythrocytes at Three Sampling Times

Group Mean Animal Data

 Group  Compound  Target Dose (ppm)  Sex        % Polychromatic Erythrocytes
         24 hours  48 hours  72 hours
 1  Tetrafluoroethylene  19 000  Male  27.0  22.4**  15.9**
   80% MLC  28 000  Female  31.0  28.9 **(4)  25.1**
 2  Tetrafluoroethylene  12 000  Male  25.9*  30.5  27.2*
   50% MLC  17 000  Female  29.9  31.7**  21.4**
 3  Tetrafluoroethylene  5 000  Male  28.2  28.8*  25.3*
   20% MLC  7 000  Female  36.2  43.4  34.2
 4  Control  Not applicable  Male  35.4  38.1  39.2
   (Laboratory Air)    Female  29.2  46.9  42.8 (4)
 5  Vinyl chloride  50 000  Male  27.3    
       Female  32.0    

* Statistically significant increase in micronucleated polychromatic erythrocytes at p<0.05 Student's 't' test (one-sided)

** Statistically significant increase in micronucleated polychromatic erythrocytes at p<0.01 Student's 't' test (one-sided)

All mean values based on 5 observations unless indicated in parenthesis

Table 4. Group Mean Results for Tetrafluoroethylene Atmosphere Analysis over the Six Hour Exposue Period (Phase I)

 Group  Target concentration of TFE (ppm)  Actual Concenration of TFE (ppm)
 1  30 000  34 500 ± 5 980
 2  23 000  22 800 ± 6680
 3  20 000  17 900 ± 4840

Table 5 Group Mean Result for Tetrafluoroethylene and Vinyl Chloride Atmosphere Analysis over the Six Hour Exposure Period

 Group  Sex  Compound /Target (ppm) Concentration  Actual Concentration (ppm)
 1  Male  TFE 19 000  21 300 ± 2390
   Female  TFE 28 000  28 300 ± 2970
 2  Male  TFE 12 000  13 100 ± 1540
   Female  TFE 17 000  18 100 ± 1770
 3  Male  TFE 5 000  5330 ± 718
   Female  TFE 7 000  7210 ± 789
 5  Male  VC 50 000  51 800 ± 5360
   Female  VC 50 000  52 000 ± 3960

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
No statistically significant increase in micronucleated polychromatic erythrocytes (MPE) in females. In males, numerically small increases in MPE at 72 hours sampling time of the 5000 and 12000 ppm groups but not in the 19000 ppm group or in any of the dose levels at 24 and 48 hour sampling times. These increases were judged to be of no biological significance.
Executive summary:

Tetrafluoroethylene (TFE) has been evaluated for its ability to induce micronucleated polychromatic erythrocytes (MPE) in the bond marrow of treated mice. Groups of five male and five female C57BL/6JfCD-1Alpk mice were exposed to TFE for a six hour period by inhalation, at target dose levels of 19 000, 12 000 or 5 000 ppm in male mice, and 28 000, 17 000 or 7 000 in female mice; these dose levels being equivalent to 80%, 50% and 20% of the median lethal concentration (MLC) for the respective sexes. These dose levels were confirmed as appropriate by the statistically significant reductions in the percentage of polychromatic erythrocytes observed at the 48 and 72 hour sampling times in both male and female mice, thus indicating that either TFE or a metabolite had reached the bone marrow.

Bone marrow samples from negative controls and animals exposed to 80%, 50% or 20% MLC dose levels were taken at 24, 48 and 72 hours after the end of the exposure period.

Bone marrow samples from animals exposed to the positive control substance (vinyl chloride, administered for a six hour exposure period by the inhalation route, at a target dose level of 50 000 ppm) were taken 24 hours after the end of the exposure period and gave significantly increased frequencies of MPE, thus demonstrating the sensitivity of the test system to be a known clastogen.

In female mice exposed to TFE, no statistically significant increases in MPE above control values were observed at any dose level at any of the sampling times investigated. In male mice exposed to TFE, no statistically significant increases above control levels were observed at any dose level at the 24 and 48 hour sampling time. However, numerically small but statistically significant increases were observed at both the 5 000 and 12 000 ppm dose levels at the 72 hour sampling time. No such increases were observed at the top (19 000 ppm) dose level of TFE. Following further investigations and a consideration of the responses in the context of the historical control database, these apparent effects were concluded to be of no biological significance.

It is therefore concluded that TFE is not clastogenic in this assay.