Hexafluoropropene, oxidized, oligomers, reduced, fluorinated

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1999-04-15 to 1999-11-01

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Few missing details compared to current guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1992

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

1998

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Tetrafluoroethylene, oxidized, oligomers, reduced, decarboxylated

EC Number:

500-539-6

EC Name:

Tetrafluoroethylene, oxidized, oligomers, reduced, decarboxylated

Cas Number:

161075-02-1

Molecular formula:

CF2H-O-(CF2-CF2-O)m-(CF2-O)n-CF2H

Test material form:

liquid

Details on test material:

Purity: > 99%

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: not specified in the report.
- Expiration date of the lot/batch: no data, assumed to be stable for the in-life phase of the study
- Purity test date: no data

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Hsd/Ola outbred albino rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan UK Ltd, Bicester, Oxon, England
- Age at study initiation: no data
- Weight at study initiation: 140 - 149 g
- Fasting period before study: no
- Housing: 5 animals/cage. Grill cages. Cage size (cm):40.5 x 38.5 x 18
- Diet : RM1(E)SQC standard laboratory pelleted rodent diet. Available ad libitum
- Water : tap water ad libitum.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/-2 °C
- Humidity (%): 55 +/- 10%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12-hour cycle

IN-LIFE DATES: From: 26-May-1999 To: 29-Jul-1999

Administration / exposure

Route of administration:

inhalation: vapour

Vehicle:

- Vehicle used: clean air

Details on exposure:

TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 0.75 m3 exposure chamber connected to a vapour generator and equiped with a metering valve.
- Method of holding animals in test chamber: rats were placed in individual compartment of exposure cages (4 compartments/cage)
- Source and rate of air: heated compressed air supply, 75l/min
- Method of conditioning air: pressurised reservoirs of liquid HFPE supplied substance to copper oil vaporisers. For each group the test substance delivery rate was controlled using a metering valve. Fluid passing through each metering valve was delivered into a vaporisation coil immersef in a water batch maintained at a temperature of approx. 70°C. The generator air supply (75L/min) was first passed through a separate copper oil, which was also immersed in the generation water bath. The test substance vapour and air mixture was passed through fibre reinforced PVC piping to a glass and stainless steel mixing vessel where it was mixed with a further supply of diluent air (75 l/min). The test atmosphere was then delivered to the inlet manifold of theexposure chamber.
- Temperature, humidity, pressure in air chamber:

pressure: -5 mm H2O below ambient
- Air flow rate: 150 litres/min
- Air change rate: no data
- Treatment of exhaust air: extracted air was vented to an exhaust stack.

TEST ATMOSPHERE
- Brief description of analytical method used: Samples of chamber air were collected into 20 ml, gas tight, polypropylene syringes. The samples were injected directly into sample loop of the gas chromatograph.
- Samples taken from breathing zone: yes, via a sampling port at the animal exposure level. A syringe needle was inserted through a septum seal, flushed with a minimum of 2 volumes of chamber air before removing a sample for analysis.


POSITIVE CONTROL - PREPARATION OF DOSING SOLUTIONS:
Cyclophosphamide was dissolved in reverse osmosis purified water to obtain the concentration of 2 mg/ml.

Duration of treatment / exposure:

6-hour continuous exposure

Frequency of treatment:

One single exposure

Post exposure period:

- all groups: 24 hours after completion of exposure
- negative controls and high exposure group: 48 hours after completion of exposure

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

(clean air control)

Doses / concentrations 2

Dose / conc.:

5 000 ppm

Remarks:

Target concentration (0.5% v/v)

Doses / concentrations 3

Dose / conc.:

4 572 ppm (analytical)

Remarks:

6169 ppm (nominal)

Doses / concentrations 4

Dose / conc.:

10 000 ppm

Remarks:

Target concentration (1.0% v/v)

Doses / concentrations 5

Dose / conc.:

9 804 ppm (analytical)

Remarks:

9932 ppm (nominal)

Doses / concentrations 6

Dose / conc.:

20 000 ppm

Remarks:

Target concentration (2.0 % v/v)

Doses / concentrations 7

Dose / conc.:

19 814 ppm (analytical)

Remarks:

20103 ppm (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: intragastric
- Doses / concentrations: 20 mg/kg (10 ml/kg bw)
- preparation: prepared as a solution in reverse osmosis purified water, prepared in-house, at a concentration of 2 mg/ml just prior to administration.

The animals (5 males and 5 females) treated with cyclophosphamide were sacrified 24 hours after treatment.

Examinations

Tissues and cell types examined:

Bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The higher exposure level of 20000 ppm was based on a previous acute inhalation study in rats where it was well tolerated. The concentration exceeds the OECD limit dose for standard acute inhalation toxicity and approaches the maximum achievable atmosphere generation.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
- treatment: 6-hour continuous exposure, whole body, 5 males/5 females per group.
- First sampling time (24 hours)
Negative control (air-exposed animals)
Animals treated with the test article (5000, 10000 and 20000 ppm)
Positive control (cyclophosphamide-treated animals)

- Second sampling time (48 hours)
Negative control (air-exposed animals)
Animals treated with the test article (20000ppm)

DETAILS OF SLIDE PREPARATION:
After animal sacrifice, femurs were removed, the bone marrow was flushed out, and pooled in a total volume of 10 ml Hank's balanced salts solution by aspiration through a 21g needle fitted to a plastic syringe. The cell suspensions were spun at 1000 rpm (150 x g) for 5 minutes. The cell pellet was resuspended in 2 ml of filtered foetal calf serum before being sedimented out using the MSE centrifuge. The final cell pellet was resuspended in a small volume of foetal calf serum to facilitate smearing on glass microscope slides.
Several smears were prepared for each animal femur.

The slides were fixed and stained as follows:
- 10 min fixation in SLR grade methanol
- hydrolysed in Bouin's fluid at room temperature for 30 hours. Followed by 3 successive washes in purified water (5 min/wash)
- Stained in Schiff's reagent for 1 hour at room temperature, followed by 3 successive washes in purified water (5 min/wash)
- counter-stained for 10 min in very dilut (approx 0.06 g/l) aqueous eosin yellowish, followed by 5-min wash in purified water.
- Stained for 30 min in Mayer's Haemalum diluted 9:1 in purified water, immediately prior to use with aqueous 1 mg/ml acridine orange solution.
- rinsed briefly in purified water, then briefly in running tap water
- Washed for 5 min in purified water, air-dried.
- mounted slides were allowed to harden at approx 37°C.

The stained slides were coded and read by light microscopy.

METHOD OF ANALYSIS
2000 polychromatic erythrocytes per animal were counted and scored for the presence of micronuclei. Usually one smear per animal was examined. The remaining smears were held temporarily in reserved in case if technical problems with the first smear.
The proportion of immature erythrocytes for each animal was assessed by examination of at least 1000 erythrocytes.
The number of micronucleated mature erythrocytes was recorded.

IDENTIFICATION CRITERIA
Micronuclei were identified by the following criteria:
- large enough to discern morphological characteristics
- generally rounded shape with a clearly defined outline
- should be deeply stained and similar in colour to the nuclei of other cells - not black
- should lie in the same focal plane as the cell
- lack internal structure, i.e. they are pyknotic
- there should be no micronucleus-like debris in the area surrounding the cell

Evaluation criteria:

The test subtance is considered to induce micronuclei if a statistically significant increase in the micronucleus incidence (at p < 0.01) is observed in any treatment group compared to control group.
Individual and/or group mean values should exceed the laboratory historical control range.
The result is negative when individual and group mean incidences of micronucleated immature erythrocytes for the treated group are not significantly greater than incidences for the concurrent control group (p > 0.01) and when these values fall within the historical control range.

An equivocal response is obtained when the results do not meet the criteria specified for a positive or negative response.

Bone marrow cell toxicity (or depression) is normally indicated by a substantial and statistically significant dose-related decrease in the proportion of immature erythrocytes (p < 0.01). This decrease would normally be evident at the 48 hour sampling time.

Statistics:

The results were statistically evaluated by comparison of the micronucleus frequencies of the control group with the frequencies observed for the reference mutagen and with the frequencies observed for the group treated with the test article. Data were evaluated by a non-parametric method.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Remarks:

(air)

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY

- Induction of micronuclei : On the basis of the results and the statistical analysis there was no statistically significant difference (p > 0.01) between the micronucleus frequency in the treated group in comparison with the control group, at either sampling time. (see table)
The positive control Cyclophosphamide caused statistically significant increases (p < 0.001) in the frequency of micronucleated immature erythrocytes in comparison to the control group.

- Toxicity:
The proportion of immature erythrocytes was not decreased significantly (p > 0.01).
Cyclophosphamide did no cause any significant decrease in the proportion of immature erythrocytes at 24 hours. However even very cytotoxic compounds do not always produce a substantial decraes as early as 24 hours because of the lag caused by erythrocyte maturation.

Any other information on results incl. tables

Summary of the results:

Sampling time	Treatment	Exposure level	% of immature erythrocytes	incidence of micronucleated immature erythrocytes (mean)	Incidence of micronucleated mature erythrocytes (total)
24 hours	Negative control (air)	-	50	1.3	0.0
	HFPE (inhalation)	5000 ppm	47	1.5	0.0
	HFPE (inhalation)	10000 ppm	51	1.5	0.0
	HFPE (inhalation)	20000 ppm	52	0.6	0.0
	Cyclophosphamide (oral)	20 mg/kg bw	50	54.1**	0.0
48 hours	Negative control (air)	-	48	1.8	0.0
	HFPE (inhalation)	20000 ppm	53	1.3	0.0

** p< 0.001 (non-parametric analysis based on permutation, one sided probabilities)

Historical controls (data from September 1990 to May 1998):

	Treatment	dose	Mean micronucleated immature erythrocytes (mean value for individual animals)	Mean micronucleated immature erythrocytes (group mean)
Negative controls	-	-	1.84 per 2000 cells	1.85 per 2000 cells
Positive control	Cyclophosphamide (oral)	20 mg/kg bw	41.6 per 2000 cells	-

Applicant's summary and conclusion

Conclusions:

The test article H GALDEN did not induce any statistically significant increase in the frequency of micronucleated cells in the bone marrow of males and female rats exposed during a single continuous 6-hour inhalation exposure.

Executive summary:

The potential genotoxic activity of H GALDEN was assessed using the in vivo micronucleus assay in rats exposed by inhalation, according to OECD guideline 474 and in compliance with GLP standards.

Male and females Sprague Dawley rats (5/sex/group) were exposed to a single 6-hour whole body atmospheric exposure of the test substance at concentrations of 5000, 1000 and 20000 ppm (0.5, 1 and 2% v/v).

A concurrent negative control received clean air only. A positive control group was dosed orally with Cyclophosphamide at 20 mg/kg bw.

Groups of 5 males/5 females were sacrificed at two times after treatment: 24 and 48 hours.

- First sampling time (24 hours)

Negative control (air-exposed animals)

Animals treated with the test article (5000, 10000 and 20000 ppm)

Positive control (cyclophosphamide-treated animals)

- Second sampling time (48 hours)

Negative control (air-exposed animals)

Animals treated with the test article (20000 ppm)

After sacrifice the femurs were removed and the bone marrow was taken and processed. One bone marrow smear from each animal was scored for the presence of micronucleated cells in 2000 immature erythrocytes. The cytotoxicity was assessed by examining the proportion of immature erythrocytes in at least 1000 erythrocytes from each animal. The incidence of micronucleated mature erythrocytes was also recorded.

Data obtained were statistically evaluated by a non parametric method.

There was no statistically significant difference between the micronucleus frequency in the treated groups in comparison with the control group, at any sampling time and exposure concentrations. Furthermore the proportion of immature erythrocytes remained unaffected by the treatment with the test article, indicating no toxicity to bone marrow cells.

The negative and positive controls produced the expected responses, in line with the historical control data.

The test article H GALDEN did not induce any significant increase in the frequency of micronucleated cells in the bone marrow of Sprague-Dawley rats following a single inhalation exposure up to 20000 ppm. It is concluded that there is no evidence of chromosome damage or bone marrow cell toxicity caused by H GALDEN in the conditions of this study.