Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The registered substance was tested in an in vitro reverse mutation assay performed according to OECD test guideline 471, and in a chromosome aberration assay performed according to the OECD test guideline 473 both in compliance with GLP and adapted to assess gas exposure.

Hexafluorobutadiene did not induce gene mutation in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A, with or without metabolic activation, at exposure levels up to the toxic range.

Hexafluorobutadiene did not induce clastogenic effects in the human lymphocytes treated for 3hrs in the presence or absence of of metabolic activation. However, in the absence of metabolic activation a continuous treatment (19 hrs) caused a statistically significant increase in chromosome aberrations at the high concentration (40% v/v). An increase was also observed at 10% and 20% v/v but without concentration-dependence.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03-AUG-1992 to 22-OCT-1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: Ames et al. (1975)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine and tryptophan
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S9 microsomal fraction from Aroclor 1254-treated rat liver
Test concentrations with justification for top dose:
- 1st experiment: 0.3125, 0.625, 1.25, 2.5, 5 and 7.5% (v/v)
- 2nd experiment: 0.015, 0.05, 0.15, 0.5, 1.5 and 5% (v/v)
Vehicle / solvent:
- Vehicle used: air
- Justification for choice of vehicle: the test substance is a gas
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
air
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
methylmethanesulfonate
other: 4-acetylaminofluorene (4-AAF), vinyl chloride (gaseous positive control)
Remarks:
Methylmethanesulfonate (MMS) was dissolved in sterile, ultra-pure water. 9-aminoacridine (9-AA) and 4-acetylaminofluorene (4-AAF) were dissolved in dimethylsulphoxide (DMSO).
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
For use of S. typhimurium strains, top agar was prepared by adding 5 mL of 1 mM L-histidine and 1 mM biotin solution to 100 mL of dilute agar. For use of E. coli strain, top agar was prepared by adding 1 mL of 1.35 mM L-tryptophan to 100 mL dilute agar. Each top agar was thoroughly mixed prior to use. Agar preparations were kept in a water bath at a temperature not exceeding 45°C.
In the course of testing the substance, 2 mL of soft agar was dispensed to a small, plastic sterile tube. This was followed by 0.5 mL of S9 mix or 0.05 M phosphate buffer (pH 7.4) and, finally, the bacteria (0.1 mL). Continually cooling, the tube contents were mixed and then poured in minimal medium plates prepared in-house. These plates contained 25 mL 1.5% BBL purified agar in Vogel-Bonner Medium E with 2% glucose. When the soft agar had set, the plates were inverted and metal spacers inserted under the lids. The plates were then placed in wide-necked, straight-sided flasks of known volume (6.25 L). The greased lids were replaced and the openings filled with either Quickfit Dreschel tops (T-junctions) through which the gas passed into the jars, or suba-seal caps through which the gas was injected into the jars. The latter method was used when the concentrations to be tested were very low.
The method used for dosing the toxicity test and the gaseous positive control was to take, simultaneously, hydrocarbon-free air and test gas through separate calibrated rotameters and allowing the gases to mix before they passed into the incubation jars. Approximately 25 L of gas/air mixture were allowed to flush through each incubation jar before entrance and exit taps were switched off. In all cases, before plates were removed from the jars after 48-h incubation, at least 50 L of air were passed through the system to clear the test atmosphere.
The following control groups were established, duplicate plates being poured for each mean datum point:
- Air-only controls plated in duplicate with each strain used, both in the presence and absence of S9 mix.
- Gas control: vinyl chloride tested at 30% in air with S. typhimurium TA 1535 and TA 100 in the presence and absence of S9 mix.
- With S9 mix - non-gaseous control: 4-AAF tested at 1000 µg/plate with S. typhimurium TA 1538 and TA 98.
- Without S9 mix - non-gaseous control: 9-AA tested at 80 µg/plate with S. typhimurium TA 1537 and MMS at 200 µg/plate with E. coli.

DURATION
- Exposure duration: 48 h at 37°C
- Expression time (cells in growth medium): 24 h at 37°C following the initial 48-h incubation period

NUMBER OF REPLICATES: duplicate plates were prepared for each bacterial strain and dose level in both the presence and absence of S9 mix and for each of the two independent tests.

METHOD OF EVALUATION: colonies were counted using a Biotran III automated counter at a maximum sensitivity (i.e., colonies of 0.1 mm or more in diameter).

DETERMINATION OF CYTOTOXICITY
- Method: a toxicity test using strain TA 100 only was performed in the presence and absence of S9 mix to establish suitable dose levels for the mutation tests. One plate of 10, 50 and 100% (v/v) exposure levels each was used.

OTHER EXAMINATIONS: the plates were examined for precipitates and, microscopically, for microcolony growth.
Rationale for test conditions:
Standard test conditions were adapted to test the gaseous substance.
Evaluation criteria:
A test was considered acceptable if for each strain:
- The bacteria demonstrated their typical responses to crystal violet, ampicillin and UV light.
- At least one of the vehicle control plates were within the following ranges: TA 1535, 4-30; TA 1537, 1-20, TA98, 10-60, TA 100, 60-200, TA 1538, 10-35 and E. coli WP2urvA (pKM101), 10-100.
- On at least one of the positive control plates there were x 2 the mean vehicle control mutant numbers per plate. If the mean colony count on the vehicle control plates was less than 10, then a value of 10 was assumed for assessment purposes. In such cases a minimum count of 20 was required before a significant mutagenic response was identified.
- No toxicity or contamination was observed in at least 4 dose levels.
- In cases where a mutagenic response was observed, that no more than one dose level was discarded before the dose which gave the highest significant mean colony number.

Where these criteria were met, a significant mutagenic response was recorded if there was:
- For S. typhimurium strains TA 1535, TA 1537, TA 1538 and TA 98 and for E. coli, at least a doubling of the mean concurrent vehicle control values at some concentration of the test substances and, for S. typhimurium strains TA 100, a 1.5-fold increase over the control value. If the mean colony count on the vehicle control plates was less than 10, than a value of 10 was assumed for assessment purposes. In such cases a minimum count of 20 was required before a significant mutagenic response was identified.
- A reproducible effect in independent tests.
Statistics:
No data available
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At the highest dose level of 7.5% in test 1 (without S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from the dose level of 1.5% in the main tests, with and without S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from the dose level of 1.5% in the main tests, with and without S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from the dose level of 5% in the main test, with and without S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from the dose level of 1.5% in the main tests, with and without S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from the dose level of 1.5% in the main tests, with and without S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY: In the preliminary toxicity assay, high toxicity to S. typhimurium TA 100 strain was observed at all 3 concentrations of test gas used (i.e., 10, 50 and 100% (v/v)). At the 10% exposure level, some remnants of the background lawn of microcolonies were apparent although no revertant colonies were present. All subsequent exposures resulted in complete killing of the bacteria.
7.5% v/v was therefore selected as top dose for the test 1 of the main assay.
5% was selected as top dose in test 2 and 3 of the main assay.

Results are presented as mean number of revertants from duplicate plates in 3 separate tests

Conclusions:
The test substance was not mutagenic to Salmonella typhimurium or Escherichia coli when tested as a gas at exposure levels extending into the toxic range.
Executive summary:

The test substance was tested as gas for mutagenic activity in Salmonella typhimurium  and Escherichia coli according to a protocol equivalent to OECD guideline 471 (adapted for a gaseous exposure) and in compliance with good laboratory practices (GLP).

Five S. typhimurium  strains TA1535, TA1537, TA1538, TA98, TA100, and one E. coli strain WP2uvrA were used in two independent experiments performed in duplicate, using a plate incorporation method. The tests were conducted at exposure levels ranging from 0.015 to 7.5% v/v, on agar plates placed in 6.25 litres jars and exposed to the gas for 48 hours, in the presence and absence of an Aroclor 1254 induced rat liver preparation and co-factors (S9 mix) required for mixed-function oxidase activity.

A toxicity test using S. typhimurium  strain TA 100 only was performed in the presence and absence of S9 mix to establish suitable dose levels for the mutation tests. One plate of 10, 50 and 100% (v/v) exposure levels each was used. Toxicity was observed at all 3 concentrations of test gas used. At the 10% exposure level, some remnants of the background lawn of microcolonies were apparent although no revertant colonies were present. All subsequent exposures resulted in complete killing of the bacteria. 

No mutagenic activity was observed in any of the six bacterial strains used. The lowest exposure level where toxicity to the bacteria strains was recorded was 1.5%. The positive and negative controls were within respective normal ranges.

It was concluded that the test substance was not mutagenic to Salmonella typhimurium  or Escherichia coli when tested as a gas at exposure levels extending into the toxic range.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10-JUN-2002 to 13-FEB-2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: In vitro mammalian cells chromosome aberration
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
Blood taken aseptically from healthy male non-smoking donors was pooled and diluted with RPMI 1640 tissue culture medium supplemented with 10% foetal calf serum, 1 U/mL Heparin, 20 I.U/mL penicillin/20 µg/mL streptomycin and 2.0 mM glutamine. The cultures were incubated in the presence of phytohaemagglutinin (PHA) before being treated with the test substance. Aliquots (0.4 mL blood : 4.5 mL medium : 0.1 mL PHA) of the cell suspension were placed in sterile universal containers and incubated at 37°C for ca. 48 h. The cultures were gently shaken daily to resuspend the cells.
Metabolic activation:
with and without
Metabolic activation system:
supplemented liver fraction (S9 mix) prepared from rats previously treated with Aroclor 1254
Test concentrations with justification for top dose:
- 1st experiment:
1.25, 2.5, 5, 10, 20, 40 and 70% (v/v) - 3-h treatment and 16-h recovery period - with and without S9 mix

- 2nd experiment:
1.25, 2.5, 5, 10, 20, 40, 50, 60 and 70% (v/v) - 19-h continuous treatment - without S9 mix
5, 10, 20, 40, and 70% (v/v) - 3-h treatment and 16-h recovery period - with S9 mix

For these test conditions 70% (v/v) was the highest dose achievable.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: air
- Justification for choice of solvent/vehicle: the test substance is a gas
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
air
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
With S9 mix: mitomycin C (MMC) at 0.2 µg/mL (3-h treatment) and 0.1 µg/mL (continuous treatment) in sterile purified water; without S9 mix: cyclophosphamide (CP) at 10 µg/mL in sterile purified water
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
After ca. 48 h, the cultures were centrifuged and cells were resuspended in fresh culture medium. Atmospheres of test substance were established in sealed glass bottles with septum caps to give final concentrations of 1.25, 2.5, 5, 10, 20, 40 and 70% (v/v). Air was withdrawn from each bottle and then an appropriate volume of test item was introduced using syringe and needle, inserted in the septum cap. After evaporation of the test item and equilibration of the atmosphere at 37°C, the lymphocyte cultures were injected into the bottles. The glass bottles were then incubated on their sides at 37°C in a roller apparatus, which rotated the bottles once every 8 min approximately. The lymphocytes coated the inside of the bottles and were immersed in cultures medium once every revolution and exposed directly to the test substance for the rest of the revolution. The solvent controls (air) were established in duplicate cultures and contained an atmosphere of air. MMC at 0.2 µg/mL was added to duplicate cultures.
Immediately before treatment of the second set of cultures, 1 mL of medium was removed from each culture and discarded. This was replaced with 1 mL of S9 mix. The cultures were then added to the appropriate glass bottle giving the same series of final concentrations as above. The duplicate solvent control cultures were established under an atmosphere of air. CP was added to duplicate cultures at a final concentration of 10 µg/mL.
Three hours after dosing, the cultures were centrifuged at 500 g for 5 min. The cells were rinsed and resuspended in fresh medium in universal containers. They were then incubated for a further 16 hours.

DURATION
- Exposure duration: 3 or 19 h
- Expression time (cells in growth medium): up to 16 h
- Fixation time (start of exposure up to fixation or harvest of cells): 19 h

SPINDLE INHIBITOR: Colcemid (0.1 µg/mL) added 2 h before the cells were harvested.
STAIN: 10% Giemsa

NUMBER OF CELLS EVALUATED: the proportion of mitotic cells per 1000 cells was recorded in each culture. One hundred metaphase figures were examined, where possible, from each culture.
Aberrations were scored according to the classification of the ISCN (1985). The total number of cells containing aberrations both with and without gaps was calculated.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index calculated for all cultures treated with the test substance and the solvent control.

OTHER EXAMINATIONS:
- Determination of polyploidy and endoreplication: yes; polyploid and endoreduplicated cells were noted when seen. The incidence of polyploid metaphase cells, out of 500 metaphase cells, was determined quantitatively for all cultures used in the analysis for chromosomal aberrations.
Evaluation criteria:
An assay was considered to be acceptable if the negative and positive control values lied within the current historical control range.
The test substance was considered to cause a positive response if the following conditions were met:
- Statistically significant increases in the frequency of metaphases with aberrant chromosomes (excluding gaps) were observed at one or more test concentration.
- The increases exceeded the negative control range of this laboratory, taken at the 99% confidence limit.
- The increases were reproducible between replicate cultures.
- The increases were not associated with large changes in osmolality of the treatment medium or extreme toxicity.
- Evidence of a dose-relationship was considered to support the conclusion.

A negative response was claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies were observed, at any dose level.
Statistics:
The number of aberrant metaphase cells in each treatment group was compared with the solvent control value using Fisher's test.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
3-hour exposure; see below in "additional information on results"
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see below in "additional information on results"
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
19-hr exposure, clastogenic activity ;see below in "additional information on results"
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see below in "additional information on results"
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the first experiment, in both the absence and the presence of S9 mix, the test substance caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any dose level, when compared with the solvent control.

In the second experiment, in the absence of S9 mix after a continuous exposure (19 hrs), the test substance caused a statistically significant increase in the proportion of metaphase figures containing aberrations which consisted mainly in chromatid breaks and chromatid gaps at dose level of 40% (v/v) (both including and excluding gap type aberrations), when compared with the solvent control. Statistically significant increases were also observed at dose levels of 10% (v/v) (both including and excluding gaps) and 20% (v/v) (including gaps only), but values were not reproducible between cultures.
In the presence of S9 mix, the test substance caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any dose level, when compared with the solvent control.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- 1st experiment: in the absence of S9 mix, the test substance caused a reduction in the mitotic index to 52% of the solvent control value at 40% (v/v). The dose levels selected for the metaphase analysis were 10, 20 and 40% (v/v).
In the presence of S9 mix, the test substance caused a reduction in the mitotic index to 76% of the solvent control value at 70% (v/v). The dose levels selected for the metaphase analysis were 20, 40 and 70% (v/v).
The quantitative analysis of polyploidy showed no increases in the number of polyploid metaphase figures when compared to the solvent control.

- 2nd experiment: as not enough metaphase figures were scoreable at the highest dose level (70% (v/v)) in the absence of S9 mix, the continuous treatment was repeated.
In the absence of S9 mix, the test substance caused a reduction in the mitotic index to 46% of the solvent control value at 40% (v/v). The dose levels selected for the metaphase analysis were 10, 20 and 40% (v/v).
In the presence of S9 mix, the test substance caused no significant reduction in the mitotic index compared to the solvent control value. The dose levels selected for the metaphase analysis were 20, 40 and 70% (v/v).
The quantitative analysis of polyploidy showed no increases in the number of polyploid metaphase cells when compared to the solvent control.

Table 1: Summary of results (mean)

Exposure period (hrs) S9 mix 

Test substance concentration

(% v/v atmosphere) 

Cells with aberrations excluding gaps  Cells with aberrations including gaps  Relative mitotic index (%)  Polyploidy (%) 
Test 1            
0 (air)  0.5  0.5  100  0.1 
    10 2.0  2.0  96  0.1 
    20 0.5  1.0  68  0.0 
    40 2.0  2.0  52  0.1 
0.2 µg/mL MMC  17.5*  17.5*  92  0.1 
0 (air)  1.5  1.5  100  0.4 
    20 1.5  2.0  81  0.2 
    40  1.0  1.0  98  0.1 
    70  1.5  1.5  76  0.1 
    10 µg/mL CP  23.5*  23.5*  22  0.0 
Test 2             
19  0 (air)  1.0  1.0  100  0.1 
    10 7.5*  9.5*  86  0.5 
    20  4.5  8.5*  72  0.1 
    40 21.5*  29.5*  46  0.3 
    0.1 µg/mL MMC 38.5*  42.5*  49  0.2 
3 0 (air)  0.5  0.5  100  0.2 
    20 2.0  2.0  88  0.1 
    40 2.0  2.5  84  0.2 
    70 2.5  2.5  83  0.3 
    10 µg/mL CP 33.5*  33.5* 18  0.0 

* = statistically significant, p< 0.001

Conclusions:
The test substance showed evidence of clastogenic activity in this in vitro cytogenetic test system, in the absence of S9 mix after a continuous exposure only, under the experimental conditions described.
Executive summary:

This study was conducted to assess the ability of the test item to induce chromosomal aberrations in human lymphocytes cultured in vitro. The study was performed according to OECD 473, EPA OPPTS 870.5375, and EU B.10 guidelines, and in compliance with GLP.

Human lymphocytes were collected from healthy male non-smoking donors. The cells were stimulated to divide by addition of phytohaemagglutinin (PHA), and exposed to the test substance (gas) both in the presence and absence of S9 mix derived from rat livers. The following experimental conditions were applied:

- 1st experiment: 1.25, 2.5, 5, 10, 20, 40 and 70% (v/v) - 3-h treatment and 16-h recovery period - with and without S9 mix

- 2nd experiment:

* 1.25, 2.5, 5, 10, 20, 40, 50, 60 and 70% (v/v) - 19-h continuous treatment - without S9 mix

* 5, 10, 20, 40, and 70% (v/v) - 3-h treatment and 16-h recovery period - with S9 mix

Solvent and positive control cultures were also prepared. The positive controls selected were: mitomycin C (MMC) at 0.2 µg/mL in a 3-h treatment and at 0.1 µg/mL in a continuous treatment, both in the presence of S9 mix; cyclophosphamide (CP) at 10 µg/mL in the absence of S9 mix. Two hours before the end of the incubation period, cell division was arrested using Colcemid, the cells harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage. Cultures were carried out in duplicate.

In order to assess the toxicity of the test substance to cultured human lymphocytes, the mitotic index was calculated for all cultures treated with the test substance and the solvent control. On the basis of these data, the following concentrations were selected for metaphase analysis: 10, 20, 40 % (v/v) with and without S9 mix, and up to 70% (v/v) with S9 mix. One hundred metaphases were analysed per culture.

In the presence of S9 mix, the test item caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any dose level, when compared with the solvent control, in either test.

In the absence of S9 mix after a 3-h exposure at up to 70% (v/v) (the maximum achievable concentration), the test substance caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any dose level, when compared with the solvent control. After a 19-h exposure, the test item caused a statistically significant increase in the proportion of metaphase figures containing chromosomal aberrations at dose level of 40% (v/v) (both including and excluding gap type aberrations), when compared with the solvent control. Statistically significant increases were also observed at dose levels of 10% (v/v) (both including and excluding gaps) and 20% (v/v) (including gaps only), but values were not reproducible between cultures.

A quantitative analysis for polyploidy was made in all cultures used for the analysis of chromosomal aberrations. No increases in the proportion of polyploid cells were seen in either test.

All positive control compounds caused large, statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.

It was concluded that the test substance showed evidence of clastogenic activity in this in vitro cytogenetic test system, in the absence of S9 mix and after a continuous exposure only, under the experimental conditions described.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Following the clastogenic effects observed in vitro in conditions without S9, an in vivo micronucleus assay was integrated into the 28-day repeated dose toxicity assay by inhalation in males, as part of the OECD421 screening study required for the tonnage band. This is an acceptable method (OECD TG474, ECHA Endpoint specific guidance R7a, R7.7.6.3).

Bone marrow cells isolated from the femur of the male rats at the end of the 28-day treatment period were processed and micronucleated erythrocytes were analysed in three treatment groups, control animals and in a group of positive control treated rats.

The systemic availability of the test substance was demonstrated by slight effects on body weight change, food consumption, relative kidney weight in the high concentration group and by the blue pattern on the incisors indicating plasmatic exposure to the substance or fluoride derivative(s) resulting in enamel modification. In the bone marrow, there was a slight concentration-dependent decrease in immature erythrocytes/total (-2.92%, -7.6%, -16.95% compared to the control group), although not statistically significant.

Furthermore, the additional 28-day repeated dose toxicity study in Sprague-Dawley rats showed increases in urinary fluoride that were statistically significant in the high and mid-concentration groups, demonstrating a systemic exposure to the test substance (and possible metabolites) before urinary excretion.

No significant increase in the mean number of micronucleated polychromatic erythrocytes was observed in any of the treatment groups.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7-SEP-2016 to 21-NOV 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
SOURCE OF TEST MATERIAL
- batch No.of test material: SMF271SG08 to SMF271SG17 (same production batch supplied in 10 cylinders)
- Expiration date of the lot/batch: 11-08-2021
Reason / purpose:
reference to same study
Remarks:
satellite study of OECD421 screening
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
Not a stand-alone assay, but included as part of an OECD421 inhalation study with a 28-day treatment in males.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: mammalian cell cytogenetic assay
Species:
rat
Strain:
Wistar
Remarks:
Wistar Han IGS (Crl:WI(Han))
Details on species / strain selection:
This rat strain is routinely used at the test facility for this type of studies (available historical control database)
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS (mainly refering to males of the study)
- Source: Charles River Deutschland, Sulsfeld, Germany
- Age at study initiation: males were about 9 weeks old; males were 11 weeks old at the start of pre-treatment period, 13 weeks at the start of exposure.
- Weight at study initiation:
On day 0 of treatment, mean BW were:
controls: 342.17 +/- 14.54 g, low dose: 339.52 +/- 14.28 g, mid-dose: 335.67 +/- 14.67 g, high dose: 337.07 +/- 13.98 g

- Fasting period before study: not applicable
- Housing: Makrolon cages, after allocation, during pre-mating period, the animals were housed 4 or 5/cage (separated by sex). For mating, one male and one female were housed together. During exposure periods, the rats were individually housed in the exposure unit without access to food or water. Immediately after exposure, the animals were returned to their home cage.
- Diet : ad libitum. Cereal-based (closed formula) powder rodent diet (VRF1(FG)). Each batch of this diet is analyzed by the supplier for nutrients and contaminants.
- Water : ad libitum. domestic mains tap water suitable for human consumption. The water was given in polypropylene bottles, which were cleaned weekly and filled as needed. Results of the routine physical, chem ical and microbiological examination of drinking water as conducted by the supplier are made available to the test facility
- Acclimation period: at least 14 days prior to the start of exposure

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 45-65%
- Air changes (per hr): 10 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hrs dark/ 12 hrs light

IN-LIFE DATES: From: 7-SEP-2016 (arrival of the animals) To: 2-NOV-2016 (necropsy of males)
Route of administration:
inhalation: gas
Vehicle:
clean air
Details on exposure:
TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
The exposure conditions are reported under the OECD421 study.
Duration of treatment / exposure:
male rats: 28 days, 6hrs/day, 5 days/week
Frequency of treatment:
Exposure to the test substance during the different study phases:
- Pre exposure period: male were not exposed.
- Premating period: male were exposed during 2 weeks prior to mating to the test substance for 5 days/week (i.e. 10 exposure days in total)
- Mating period: male and female rats were exposed daily until confirmation of mating.
- Post-mating period: males were exposed daily until sacrifice for a total of 28 days.
Post exposure period:
The males were sacrificed the day following the last exposure
Dose / conc.:
0 ppm (nominal)
Dose / conc.:
5 ppm (nominal)
Dose / conc.:
4.95 ppm (analytical)
Remarks:
SD: +/- 0.24 ; average exposure
Dose / conc.:
15 ppm (nominal)
Dose / conc.:
15.04 ppm (analytical)
Remarks:
SD: +/- 1.05 ; average exposure
Dose / conc.:
50 ppm (nominal)
Dose / conc.:
50.69 ppm (analytical)
Remarks:
SD: +/- 2.16; average exposure
No. of animals per sex per dose:
5 males/concentration (one spare sample was collected from a sixth male rat in each group)
5 males in the positive control group
Control animals:
yes, concurrent vehicle
Positive control(s):
mitomycin C
- Justification for choice of positive control(s): one of the standard positive controls recommended by the OECD474 guideline (there is no specific standard for inhalation route).
- Route of administration: intra-peritoneal
- Doses / concentrations: 1.5 mg/kg bw in physiological saline as the vehicle, administered about 24-hrs before sacrifice.
It is acceptable that the positive control be administered by a route different from the test chemical, using a different treatment schedule, and for sampling to occur only at a single time point.
Tissues and cell types examined:
erythrocytes from the bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
- Dose selection rationale: The exposure levels were selected on the basis of the results of a 28-day inhalation study in rats. The micronucleus analysis is part of a reproduction/developmental screening study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
*Treatment:
Males were exposed for a total of 28 days.
For the micronucleus test, bone marrow was collected at scheduled necropsy, the day following the last treatment, from 5 males of each group (one spare sample was collected from a sixth male rat in each group; this sample was not evaluated).
In addition, bone marrow cells were collected from the 5 males in the positive control group (injected once with 1.5 mg/kg bw Mitomycin C, 24 hrs before sacrifice).

*Sampling:
Bone marrow cells from one of the femurs of the rats designated for the micronucleus test were immediately collected into foetal calf serum and processed into glassdrawn smears according to the method described by Schmid (1976).

DETAILS OF SLIDE PREPARATION:
Four bone marrow smears per animal were made, air-dried and fixed in methanol. Two fixed smears were stained with May-Grünwald Giemsa. The other two fixed smears were kept as reserve. If needed, these reserve slides were also stained with May-Grünwald Giemsa solution and used for microscopic examination.

METHOD OF ANALYSIS:
Randomly coded slides (two per animal) were read by moving from the beginning of the smear (label end) to the leading edge in horizontal lines taking care that areas selected for examination are evenly distributed over the whole smear.

The following criteria were used for the scoring of cells:
• A polychromatic erythrocyte (PE) is an immature erythrocyte that still contains ribosomes and can be distinguished from mature, normochromatic erythrocytes by a faint blue stain.
• A normochromatic erythrocyte (NE) is a mature erythrocyte that lacks ribosomes and can be distinguished from immature, polychromatic erythrocytes by a yellow stain.
• A micronucleus is a small, normally round, nucleus with a diameter of circa 1/20 to 1/5 of an erythrocyte, distinguished from the cytoplasm by a dark blue stain.
Number of erythrocytes scored: at least 500 erythrocytes (E) per animal. The numbers of PE and NE were recorded.
If micronuclei are observed, these were recorded as micronucleated polychromatic erythrocytes (MPE) or micronucleated normochromatic erythrocytes (MNE). Once a total of 500 E (PE + NE) have been scored, an additional number of PE were scored for the presence of micronuclei until a total of 4000 PE have been scored.
Evaluation criteria:
The micronucleus test was considered valid if the positive controls give a statistically significant increase in the mean number of MPE/4000 PE and the negative controls are within the historical range.
A substance was considered to cause chromosomal damage and/or damage to the mitotic apparatus if it showed a dose related positive response or a clear increase of micronucleated cells in a single dose group. A substance was considered to be negative in the micronucleus test if it produced no positive response at the dose level(s) analysed.
A response was considered to be positive if the mean number of MPE/4000 PE was statistically significantly higher compared to the negative control group.
Statistics:
In all tests a significance level of 5% was used (α = 0.05)
Data were analysed by one-way analysis of variance (ANOVA). Two ANOVA models were applied. In one of the ANOVA models it was tested if the positive control differed from the negative control (t test). In the other ANOVA model (including Dunnett’s test as post-hoc test) it was tested if the test substance (different doses) differed from the negative control. It was checked if the ANOVA assumptions were met (i.e. if variances are equal). In case assumptions were not met, square root transformation (sqrt(x+1)) was applied to 'normalise' the distribution of the counts. In case this would not be sufficient, non-parametric testing was
performed using the Mann-Whitney test (positive control compared with negative control) or Kruskal-Wallis analysis of variance (test material groups compared with negative control).
Key result
Sex:
male
Genotoxicity:
negative
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS
- Clinical signs of toxicity in test animals: There was no clinical signs reported during the study period.

- Evidence of cytotoxicity in tissue analyzed: there was a slight concentration-dependent decrease in immature erythrocytes/total erythrocytes(-2.92%, -7.6%, -16.95% compared to the control group), although not statistically significant.

- Rationale for exposure: Systemic availability of the test substance was demonstrated by slight effects on body weight gain and food consumption in males, as well as on the relative kidney weight in the high concentration group. In addition, the blue stripy pattern noticed on the teeth of all the high concentration males shows plasmatic exposure to the test substance and/or its metabolites (e.g. fluoride or related metabolites) and resulting effect on enamel development.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay):
In the negative control group, the group mean number of micronucleated polychromatic erythrocytes (MPE) per 4000 polychromatic erythrocytes (PE) of the negative control group (clean air treatment) and the positive control group (treated intraperitoneally with 1.5 mg/kg bw mitomycin C) showed the expected response. Therefore the study met the validity criteria.

- Ratio of PCE/NCE (for Micronucleus assay):
- Frequency of micronucleated immature erythrocytes (PE): there was no difference in the treated groups compared to control.
- Frequency of micronucleated mature erythrocytes (NE): 0 in all groups treated for 28 days, as well as in the negative and positive control groups.

- Appropriateness of dose levels and route: Inhalation is the most likely route of exposure to the gaseous substance. The repeated exposure design is consistent with potential exposure in the industrial setting.

- Statistical evaluation:
There was no statistically significant increase in the mean number of MPE/4000 E or in the mean number of PE/500 E in any of the groups treated with Hexafluorobutadiene when comparing to the concurrent negative control group.

Mean numbers of PE/500 erythrocytes and MPE/4000 polychromatic erythrocytes

   test substance  target concentration

 PE/500 erythrocytes

(mean + SD)

MPE/4000 PE

(mean + SD) 

 control  clean air  -  171 + 32 4.6 + 2.2 
 low concentration Hexafluorobutadiene   5 ppm  166 + 26 4.0 + 1.4 
mid concentration Hexafluorobutadiene  15 ppm  158 + 43  5.8 + 2.4
high concentration Hexafluorobutadiene  50 ppm  142 + 21  4.0 + 1.9
 positive control MN  Mitomycin C  1.5 mg/kg bw i.p. (single dose)   93 + 23 (1)  65.8 + 8.3 (2)

(1)Unpaired t-test p-value: 0.0022

(2) Unpaired t-test p-value: < 0.0001 (after transformation of data)

Conclusions:
There was no statistically significant increase in the mean number of micronucleated erythrocytes compared to the concurrent negative control animals. Under the conditions of the study, exposure to hexafluorobutadiene at up to 50 ppm by inhalation did not cause chromosomal damage to the bone marrow cells in male rats.
Executive summary:

In male rats exposed by inhalation for 28 days to 5, 15 or 50 ppm hexafluorobutadiene, bone marrow was collected from one of the femurs and processed to prepare 4 bone marrow smears per animal, that were air-dried and fixed in methanol, then stained. The slides were randomly coded and polychromatic erythrocytes (PE) and normochromatic erythrocytes (NE) were recorded in at least 500 erythrocytes per animal. Micronucleated erythrocytes (MPE or MNE) were scored until a total number of 4000 polychromatic erythrocytes was reached.

The group mean number of MPE per 4000 PE of the negative control group (group 1: clean air) and the positive control group (1.5 mg/kg-bw Mitomycin C) showed the expected responses. Therefore, the study was considered valid.

There was no statistically significant change in the mean number of micronucleated erythrocytes in a total of 4000 scored polychromatic erythrocytes and the mean number of polychromatic erythrocytes /500 erythrocytes in any treatment group compared to the negative control group.

Systemic availability of the test substance was demonstrated by slight effects on body weight change and food consumption, as well as by the blue pattern on the incisors indicating plasmatic exposure to the substance or fluoride derivative resulting in enamel change. There was a slight concentration dependent decrease in the ratio of immature polychromatic erythrocytes/total erythrocytes although not statistically significant.

It can be concluded that under the conditions of the study, the exposure to hexafluorobutadiene at up to 50 ppm for 28 days in male rats did not show evidence of chromosomal damage and/or damage to the mitotic spindle apparatus of the bone marrow cells.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

The registered substance was not found mutagenic in a reverse mutation assay in bacterial systems, with and without metabolic activation. It was found to produce statistically significant clastogenic effects only following continuous treatment in the absence of metabolic activation in human lymphocytes cells in vitro.

However, these effects were no confirmed in an in vivo micronucleus assay conducted on bone marrow cells sampled from male rats exposed by inhalation 6h/day, 5 days/week for 28 days to up to 50 ppm.

Based on the available information, hexafluorobutadiene is not considered to be genotoxic and does not need to be classified as a germ cell mutagen according to EU CLP criteria in Regulation (EC) No. 1272/2008.