Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

HFO-1234yf was only mutagenic in S. typhimurium strain TA100 and E. coli WP-2 (in the presence, but not in the absence of S9) and not in other S. typhimurium strains (TA 1535, TA 1537, TA 98). It was found not to be clastogenic in cultured human lymphocytes, but did induce gene mutations in an in vitro mammalian cell gene mutation test.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium (with HEPES and Glutamax-I) supplemented with heat-inactivated horse serum (10% v/v for growing in flasks, and 20% for growing in microtiter plates), sodium pyruvate and penicillin/streptomycin.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: Yes
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: S9 liver homogenate
Test concentrations with justification for top dose:
76, 60, 40, 20 and 10% (v/v) (all ± 10%)
Vehicle / solvent:
Since the test material is a gas, cells in culture flasks were exposed in modular incubator chambers (Billups-Rothenburg, USA) to various concentrations. The atmosphere in the chamber consisted of 19% O2; 5% CO2; and the test material supplemented with N2. Air without the test material was used as negative control.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Sham exposed
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: The test substance is a gas therefore a test atmosphere was generated by mixing a mass flow controlled amount of gaseous test material with a mass flow controlled stream of nitrogen, together accounting for 76% of the air. A mass flow controlled stream of oxygen (19%) and a mass flow controlled stream of carbon dioxide (5%) were added. The resulting test atmosphere was directed to the inlet of the exposure boxes. The boxes were flushed with test atmosphere for a sufficient amount of time to achieve at least 99% of the steady state target concentration

DURATION
- Exposure duration: 4 hours and 24 hours in the absence of S9-mix, and for 4 hours in the presence of S9-mix
- Selection time: 10-14 days

SELECTION AGENT: Trifluorothymidine

NUMBER OF REPLICATIONS: Duplicate

DETERMINATION OF CYTOTOXICITY
- Method: The cytotoxicity of the test material was determined by measuring the relative initial cell yield, the relative suspension growth (RSG) and the relative total growth (RTG).
Evaluation criteria:
See: "Any other information on materials and methods incl. tables"
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
In the presence of S9-mix. See: "additional information on results"
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see: "additional information on results"
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Positive and negative controls:
The negative controls were within the acceptance criteria and treatment with the positive controls yielded the expected significant increase in mutant frequency compared to the negative controls for all treatment conditions tested. Therefore, the test was considered valid.

Concentration levels and visual observations before and after treatment:
The actual concentrations were measured at start and end of exposure. Based on the results of these measurements it can be concluded that at the start of the exposure, all target concentrations were met taking into account the margin of 10% mentioned in the study plan. In one case (lowest concentration of the 24 hour exposure) the concentration seemed just below target, but as the concentration at the end of exposure was higher, this was considered to be an error in the measurement. At the end of the exposure the target concentration was met in all but one case (taking into account the margin of 10%), which was the lowest concentration of the 4 hour exposure. In this case the deviation was -25%. It should be noted that the second sample is taken to demonstrate that the container/incubator was not leaking. In general, the lower values obtained from the second sample may also indicate that the test material was taken up in the culture medium or metabolized during exposure. As the four higher concentrations were all within the 10% margin of the target concentrations, and the guideline requires four concentrations tested in duplicate for a valid assay, the test was considered valid. At the start and end of both the 4 hours and 24 hours treatment in the absence and presence of S9-mix, no visual abnormalities were observed. At the end of the treatment period, the viability of the cells at 76% of the test substance was at or above 90% for all treatment conditions tested.

Cytotoxicity:
In the presence of S9-mix, the test material was toxic to the cells resulting in a reduction in the RTG. The mean RTG at the highest three concentrations evaluated in the absence of S9-mix (76%, 60% and 40%) was 61%. In the absence of S9-mix after 4h treatment no toxicity was observed, but after 24h treatment the test material was slightly toxic to the cells resulting in a mean RTG at the highest concentration evaluated (76%) of 83%.

Mutagenicity:
In the presence of S9-mix (4 hours exposure), a dose related increase in the mean MF by more than 88 or 126 mutants per 1,000,000 clonable cells compared to the negative control was observed from a concentration of 20% onwards. In the absence of S9-mix, both after 4 and 24 hours, no increase in the mean MF by more than 88 or 126 mutants per 1,000,000 clonable cells, i.e. no equivocal or positive response, compared to the negative control was observed at any dose level.

Colony sizing:
At concentrations causing an increase in MF the mutant colonies were scored using the criteria of small and large colonies. In the presence of S9-mix an induction in both the number of small and large colonies was observed. The mean percentage of small colonies at 20, 40, 60 and 76 % (positive responses in MF) was 57%, 57%, 57% and 54% compared to 44%, 44%, 43% and 46% of large colonies. Although in general slightly more small than large colonies were formed, the amount of both small and large colonies were increased, and therefore, none of the mechanisms (clastogenicity and mutagenicity) can be excluded.

Table 1. Summary of the results in the absence of S9-mix

Conc. (%)

absence of S9-mix (24h)

Conc. (%)

absence of S9-mix (4h)

MF*

¿MF*

RTG*

MF*

¿MF*

RTG*

76

89

13

83

76

68

15

118

60

101

25

90

60

76

24

130

40

73

-3

92

40

46

-7

127

20

90

14

91

20

65

12

101

10

55

-20

112

10

52

0

106

0

76

0

100

0

53

0

100

*Mean of duplicate cultures

Table 2. Summary of the results in the presence of S9-mix

Conc. (%)

presence of S9-mix (4h)

MF*

¿MF*

RTG*

76

447

376

61

60

427

356

61

40

306

235

61

20

228

157

94

10

134

63

96

0

72

0

100

* Mean of duplicate cultures

Values were increased by more than 126 mutants per 1,000,000 clonable cells compared to the negative control.

MF = mutant frequency; ¿MF = increase in mutant frequency; RTG = relative total; growth; conc. = concentration of the test material

Conclusions:
Under the conditions of this study, the test material is mutagenic at the TK-locus of mouse lymphoma L5178Y cells in the presence of metabolic activation (S9-mix).
Executive summary:

According to OECD guideline 476 and GLP the test substance was examined for its potential to induce gene mutations at the TK-locus of cultured mouse lymphoma L5178Y cells, in both the absence and the presence of a metabolic activation system (S9-mix). A single test was conducted. In the test, five duplicate cultures were treated for 4 hours in the presence and absence of S9-mix, and 24 hours in the absence of S9-mix.

The highest nominal concentration of the test substance evaluated for mutagenicity was 76%. As the atmosphere in the chamber consisted of 19% O2, 5% CO2 and the test material (supplemented with N2 to achieve lower concentrations), the highest achievable concentration was 76%. Additional concentrations of 60, 40, 20 and 10% (v/v) (all ± 10%) were used in the chambers to expose the cells. Air without the test material was used as negative control. Methyl methanesulphonate (MMS) and 3-methylcholanthrene (MCA) were used as positive control substances in the absence and in the presence of S9-mix, respectively and clear air served as negative control in both tests. As all acceptance criteria were met, the test was considered valid. In the presence of S9-mix the test material was toxic to the cells resulting in a reduction in the relative total growth (RTG). The mean RTG at the highest three concentrations evaluated in the absence of S9-mix (76%, 60% and 40%) was 61%. In the absence of S9-mix after 4h treatment no toxicity was observed, but after 24h treatment the test material was slightly toxic to the cells resulting in a mean RTG at the highest concentration evaluated (76%) of 83%. In the presence of S9-mix, a dose related increase in the mean mutant frequency (MF) by more than 88 or 126 mutants per 1,000,000 clonable cells compared to the negative control was observed as from a concentration of 20% onwards. In the absence of S9-mix, both after 4 and 24 hours, no increase in the mean MF by more than 88 or 126 mutants per 1,000,000 clonable cells, i.e. no equivocal or positive response, compared to the negative control was observed at any dose level.

It is concluded that under the conditions used in this study, the test substance is mutagenic at the TK-locus of mouse lymphoma L5178Y cells, in the presence of metabolic activation (S9-mix).

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
0, 10, 20, 40, 60, and 76% in the atmosphere
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
sham exposed
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: Lymphocytes were exposed in a modular incubator chamber in atmospheric concentrations of 19% O2; 5% CO2; and up to 76% test substance.

DURATION
- Preincubation period: 48 hours
- Exposure duration: 4 hours
- Expression time: 20 or 44 hours
- Fixation time: 26 to 50 hours

SPINDLE INHIBITOR: Colcemid

STAIN: Geimsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index
Evaluation criteria:
Biological relevance and statistical significance are both considered in the evaluation of the results.
Statistics:
Fischer's exact probability test (two sided)
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Slight cytotoxicity at highest concentration with metabolic activation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Test substance did not induce a statistically significant increase in the number of aberrant cells at any concentation or time point analysed with compared to the number of aberrant cells observed in the negative (control air) control cultures.

The positive controls mitomycin C (without S9) and cyclophosphamide (with S9) induced the expected statistically significant increase in the incidence of structural chromosomes.
Conclusions:
Under the conditions of this study, the test substance was not clastogenic in cultured human lymphocytes.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: Aroclor 1254-induced rat liver homogenate (S9-mix).
Test concentrations with justification for top dose:
10, 20, 40, 60, 76% in the atmosphere
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
sham exposed
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: Aminoacridine - S. thypimurium 80.0 µg/plate; N-ethyl-N-nitrosourea - E. coli 100 µg/plate; 2-aminoanthracene - E. coli 80 µg/plate
Details on test system and experimental conditions:
- Exposure duration: 4 hours and 15 minutes to test substance in gas exposure chamber followed by 48-72 hour exposure at 37°C
Evaluation criteria:
- Study is considered valid if the mean colony counts of the control values of the strains are within acceptable ranges, if the results of the positive controls meet the criteria for a positive response, and if no more than 5 % of the plates are lost through contamination or other unforeseen events.
- A test substance is considered to be positive in the bacterial gene mutation test if the mean number of revertant colonies on the test plates is concentration-related increased or if a two-fold or more increase is observed compared to that on the negative control plates.
- A test substance is considered to be negative in the bacterial gene mutation test if it produces neither a dose-related increase in the mean number of revertant colonies nor a positive response at any of the test points.
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA1535, 1537, and 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
only in TA100 at 10% and greater
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
STUDY RESULTS
- Concurrent vehicle negative and positive control data : The positive control for WP2 in the absence of S9 -mix induced a 2.5 fold increase not the 3 fold increase as required. However, the test substance induced a greater than 3 fold increase in revertants indicating the validity of this test system. The remaining controls all behaved as expected.
- Ames test results: The substance caused a dose related increase at and above the 10% dose, and a more than two-fold increase at and above the 20% dose to strains TA100 and WP2uvrA, only in the presence of S9 mix.

Table 1. Mean Number of Revertants Counted (triplicate samples)

 

TA1535

TA 1537

TA 98

TA 100

WP2uvrA

 

-S9

+ S9

-S9

+S9

-S9

+ S9

-S9

+ S9

-S9

+ S9

0 %

46

33

23

22

39

61

224

146

44

43

10 %

32

32

18

28

45

59

134

197

46

83

20 %

53

33

14

30

43

57

122

318

43

106

40 %

26

40

19

34

52

65

153

317

54

153

60 %

37

47

23

30

35

58

174

383

51

174

76 %

28

50

20

26

53

60

157

372

46

175

Pos Control

552

394

3651

173

807

556

688

1160

133

1218

 

Conclusions:
The test substance was mutagenic under the conditions employed in this study (TA100 and WP-2 in the presence of S9) but was not mutagenic in these strains in the absence of S9 or in any of the other strains with or without metabolic activation.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

HFO-1234yf was negative in the following three in vivo genotoxicity studies: micronucleus – mice (acute inhalation exposure) and rats (4-week inhalation exposure) and UDS test (unscheduled DNA synthesis) in rats following 4-week inhalation exposure. Furthermore, the substance was also found to be negative in a combined micronucleus and comet assay in rats following inhalation exposure.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to guideline
Guideline:
other: OECD Guideline for the Testing of Chemicals 489. In Vivo Mammalian Alkaline Comet Assay, adopted 26st September 2014.
Principles of method if other than guideline:
An combined in vivo micronucleus test and comet assay was performed.
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: Micronucleus and comet assay
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan, Horst, the Netherlands
- Age at study initiation: Approximately 7 weeks
- Weight at study initiation: Body weight variation did not exceed ± 20 % of the mean weight.
- Assigned to test groups randomly: Animals were allocated to the dose groups by computer randomization and proportionally to body weight.
- Home cages: The animals were housed with a maximum of 5 animals per macrolon cage (type IV) with wood shavings (Lignocel) as bedding material and a wooden block and strips of paper as environmental enrichment (Enviro-dri).
- Caging during exposure: During the exposure periods, the rats were individually housed in the exposure unit and did not have access to food or water. Immediately after each exposure, the animals were returned to their home cages.
- Diet: Cereal-based rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3) from a commercial supplier (SDS Special Diets Services, Whitham, England), ad libitum.
- Water: Tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC), ad libitum.
- Acclimation period: At least nineteen days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 45 - 65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: gas
Details on exposure:
EXPOSURE EQUIPMENT
The animals were exposed to the test atmosphere in nose-only exposure units consisting of a cylindrical (PVC) column with a volume of about 75 liters, surrounded by a transparent hood. The test atmosphere was introduced at the bottom of the central column, and was exhausted at the top. A cooling coil, located in the top of the column, was used to control the temperature of the atmosphere. Each column included three rodent tube sections containing 34 ports each. Several empty ports were used for test atmosphere sampling and measurement of temperature, relative humidity, oxygen and carbon dioxide. The animals were secured in plastic animal holders (Battelle), positioned radially around the central column. The remaining ports were closed. Only the nose of the rats protruded into the interior of the column. Habituation to the restraint in the animal holders was not performed because in our experience habituation did not help to reduce possible stress (Staal et al., 2012). In our experience, the animal’s body does not exactly fit in the animal holder which always results in some leak from high to low pressure side. By securing a positive pressure in the central column and a slightly negative pressure in the outer hood, which encloses the entire animal holder, dilution of test atmosphere by air leaking from the animals’ thorax to the nose was avoided. The units were illuminated externally by normal laboratory fluorescent tube lighting. The average total air flow through the unit was at least 1 L/min for each rat. The atmosphere in the unit was maintained at a temperature of 22 ± 3ºC and a relative humidity between 30 and 70%.

GENERATION OF THE TEST ATMOSPHERE
The inhalation equipment was designed to expose rats to a continuous supply of fresh test atmosphere. A mass flow controlled (Bronkhorst, Hi Tec, Ruurlo, The Netherlands) stream of test material was mixed with a mass flow controlled stream of oxygen and a mass stream meter controlled flow of humidified compressed air. The resulting test atmosphere was directed to the bottom inlet of an exposure unit, led to the noses of the animals and exhausted at the top of the unit. The exposure unit for the control animals was supplied with a mass stream meter controlled flow of humidified compressed air only. The animals were placed in the exposure unit after stabilization of the test atmosphere.

MONITORING OF EXPOSURE CONDITIONS
Actual concentration: The actual concentration of the test material in the test atmospheres was measured, after dilution, by total carbon analysis (Sick GMS 810, Maihak EuroFID total carbon analyser; Sick Instruments Benelux, Hedel, the Netherlands). The response of the analyser was recorded on a PC every minute using a CAN transmitter (G. Lufft Mess- und Regeltechnik GmbH, 70719 Felbach, Germany). The responses of the analysers were converted to concentrations by means of calibration graphs. For each exposure day, the mean concentration was calculated from the values determined every minute. Representative test atmosphere samples were taken continuously from the exposure unit at the animals’ breathing zone and were diluted (for groups 15,000 and 50,000 ppm only) and passed to the total carbon analyser (TCA) through a sample line. Prior to the first exposure, the output of the flame ionization detector of the TCA was dynamically calibrated. For this purpose test atmospheres were generated as described in "generation of the test atmosphere" and led directly to the TCA (instead of to the inlet of the exposure unit); the excess air was exhausted. At different settings of the mass flow controllers (which were calibrated at the flow settings used for calibrating the TCA prior to use for generation of the calibration concentrations), the response of the TCA was recorded (in duplicate). The calibration settings were selected to generate about 80%, 100% and 120% of each target concentration. After exposure, the calibration was checked. Therefore, a test atmosphere was generated at the target concentration using mass flow controllers, the flow of which was measured using a Dry Cal DC-2 flow calibrator. Based on the measured flow, the concentration was calculated. This calculated concentration was compared to the measured concentration. The deviations for all groups were below 5%, indicating that the calibration graphs accurately represented the actual concentration.

Group 2 (target 5,000 ppm)
- On 17 December 2014, concentrations of 6098, 5004, 4036, 6098, 5004 and 4036 ppm were analysed to calibrate the TCA used for group 2. The response (Y) of the analyser (in % of full scale) was linearly related to the concentration (X) of the test material with a correlation coefficient of 1.000: Y (%) = 1.252E-2 * X (ppm) + 1.723. This concentration-response relation was used to calculate the test atmosphere concentrations for group 2.
Group 3 (target 15,000 ppm)
- On 18 December 2014, concentrations of 17881, 14783, 11962, 17881, 14783 and 11962 ppm were analysed to calibrate the TCA used for group 3. The response (Y) of the analyser (in % of full scale) was linearly related to the concentration (X) of the test material with a correlation coefficient of 1.000: Y (%) = 4.580E-3 * X (ppm) + 1.463. This concentration-response relation was used to calculate the test atmosphere concentrations for group 3.
Group 4 (target 50,000 ppm)
- On 17 December 2014, concentrations of 59181, 49563, 40036, 59181, 49563 and 40036 ppm were analysed to calibrate the TCA used for group 4. The response (Y) of the analyser (in % of full scale) was linearly related to the concentration (X) of the test material with a correlation coefficient of 0.9998: Y (%) = 1.233E-3 * X (ppm) + 4.668. This concentration-response relation was used to calculate the test atmosphere concentrations for group 4.

-Time to attain chamber equilibration (T95): The time to reach 95% of the steady state concentration (T95) was calculated as: 3V/F. This calculation followed from the formula C = C8 (1 – e^-(FT/V)), describing the increase in concentration C in a perfectly stirred chamber with volume V [L] and flow F [L/min], where T [min] was the time and C8 was the steady state concentration.

- Nominal concentration and generation efficiency: Test material usage was determined by the total amount of test material used (determined by weighing the cylinder at the start and end of the generation period). This was compared to the calculated amount used, based on the actual concentration and total flow through the inhalation chamber. The generation efficiency was calculated from the calculated daily use and the actual (weighed) daily use of test material, as follows: Efficiency = 100 x total calculated use of test material for groups 5,000 and 15,000 ppm / actual use.

- Total air flow, temperature, relative humidity and oxygen and carbon dioxide concentration: The total flow through the inhalation chamber consisted of the air flow, oxygen flow and test material flow. The oxygen and test material flow were recorded about hourly by means of the settings of the flow controllers. The air flow was recorded every minute. The temperature and the relative humidity of the test atmospheres were measured continuously in the breathing zone of the animals and recorded every minute using a CAN transmitter with temperature and relative humidity probes (G.Lufft Mess- und Regeltechnik GmbH, 70719 Fellbach, Germany). The concentrations of oxygen (oxygen analyser type PMA-10, M&C Products Analysentechnik GmbH, Ratingen-Lintorf, Germany) and carbon dioxide (GM70, Vaisala, Helsinki, Finland) in the test atmosphere were measured several times during exposure.
Duration of treatment / exposure:
3 days
Frequency of treatment:
The animals were exposed for two days during 6 hours/day and for 2 hours on the third day (i.e. the day of sacrifice).
Dose / conc.:
5 000 ppm
Remarks:
Group 2: Low dose. Actual concentration 5039 ± 20 ppm (mean + SD)
Dose / conc.:
15 000 ppm
Remarks:
Group 3: Mid dose. Actual concentration 15128 ± 27 (mean + SD).
Dose / conc.:
50 000 ppm
Remarks:
Group 4: High dose. Actual concentration 50211 ± 339 ppm (mean + SD)
No. of animals per sex per dose:
5 and 1 reserve animal
Control animals:
yes, concurrent vehicle
Positive control(s):
The animals of the positive control group of the micronucleus test were treated once intraperitoneally with the mutagen Mitomycin C (stock concentration 0.15 mg/mL; dosing volume 10 mL/kg bw; dose level 1.5 mg/kg bw), ca. 24 h before sacrifice. The animals of the positive control group of the comet assay for lung cells were treated intraperitoneally with the mutagen Methyl methane sulfonate (stock concentration 4 mg/mL; dosing volume 10 mL/kg-bw; dose level 40 mg/kg-bw), ca. 2-6 h before sacrifice. The animals of the positive control group of the comet assay for liver cells (hepatocytes) were treated once orally (by gavage) with the mutagen 2-acetaminofluorene (2-AAF) (stock concentration 2.3 mg/mL; dosing volume 20 mL/kg-bw; dose level 46 mg/kg-bw), ca. 12-15 h before sacrifice. Just before each dosing the animals were weighed and the dosing volume was adjusted to the body weight. The concentrations of the dose formulations of the positive controls were not determined analytically.
Tissues and cell types examined:
Liver, lung cells (comet assay) and bone marrow (micronucleus test)
Details of tissue and slide preparation:
COMET ASSAY LIVER AND LUNG CELLS
- Isolation of hepatocytes: At the end of the last exposure, the animals were immediately transported from the animal facilities to another room and sacrificed for isolation of hepatocytes in a random order. The animals of the positive control group (group 7) were sacrificed 12-15 h after oral dosing. Hepatocytes were isolated from the liver using the perfusion technique described by Williams et al. (1977) with minor modifications. Briefly, the liver of each rat was perfused in situ with a HEPES buffer (0.01 M) whilst under sodium pentobarbital anesthesia and exsanguination from the abdominal aorta, followed by an in vitro perfusion with a HEPES-buffered (0.1 M) collagenase solution. Directly after the start of the perfusion to remove the blood from the tissue, a small part of the caudate lobe was tied off using a ligature. Subsequently, part of the lobe was removed and preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde (10% solution of formalin) for possible histopathological examination. In case of a positive response, this sample would be used for histological evaluation to determine whether cytotoxicity was involved. After isolation, the dissociated cells were incubated for 5-10 minutes in a shaking water bath at 37 ºC. Thereafter, the cells were filtered over a nylon filter (70 µm mesh), centrifuged and resuspended in Williams medium E. Cell counts were made and the viability of the hepatocytes was determined by trypan blue exclusion.

- Isolation of lung cells: Immediately after liver perfusion (groups 1-4) or sacrifice (group 6), lung tissue was dissected and kept in ice-cold Krebs-Ringer bicarbonate buffer (Sigma-Aldrich Chemie B.V., pH set at 7.3) until further processing. The tissue was finely minced and homogenized in 2 mL pre-warmed (37ºC) collagenase solution (1 mg/mL in Krebs-Ringer buffer (pH ca. 7.3), supplemented with 1.26 g/L NaHCO3, 6.3 mM CaCl2, and 1% bovine serum albumin). After a short incubation (ca. 5 min) at ambient temperature, the cell suspension was filtered twice (500 µm mesh, followed by 70 µm) and centrifuged (100 g, 5 min, ca. 4 ºC). The cells were resuspended in a sufficient volume for slide preparation. The viability of the cells was determined by trypan blue exclusion. In addition, a small piece of lung was preserved in neutral aqueous phosphate buffered 4% solution of formaldehyde (10% solution of formalin) for possible histopathological examination. In case of a positive response, this sample would be used for histological evaluation to determine whether cytotoxicity was involved.

- Preparation of slides: Microscopic slides were prepared by mixing an aliquot of the cell suspension with a low-melting agarose solution (0.5 % (w/v) in PBS). Subsequently, this mixture was loaded on a glass slide, pre-coated with normal-melting agarose (1.5 % (w/v) in PBS), and mounted with a coverslip. Four slides per animal were prepared (of which one slide was kept in reserve). The slides were stored on a cold plate until the agarose has solidified. Subsequently, the coverslip will be removed and the slide were incubated in lysis buffer (2.5 M NaCl, 0.1 M Na2EDTA, 0.175 M NaOH, 0.01 M Tris in Milli-Q water, supplemented with 1 % Triton X-100 (w/v), pH 10) overnight at 2-10ºC. Subsequently, slides were incubated in ice-cold electrophoresis buffer (0.3 M NaOH, 0.001 M Na2EDTA in Milli-Q water, pH >13) for 30 min, following electrophoresis (ca. 25V and 300 mA) for 30 min in ice-cold electrophoresis buffer, while cooled on ice. After incubation in neutralization buffer (0.4 M Tris in Milli-Q water, pH 7.5) for at least 5 min, slides were dehydrated by incubating in ethanol at ambient temperature and air-dried.

-Slide analysis and counting: Slides were coded by a person not involved in analysing the slides to enable ‘blind’ scoring. Slides were stained with ethidium bromide solution (20 µg/mL in Milli-Q water) which was directly pipetted on the slide and covered with a coverslip just before analysis. A fluorescent microscope connected to a camera and Comet Assay IV software (Perceptive Instruments) was used for the analysis of the slides. Fifty cells (randomly selected starting from the centre of the slide) per slide and three slides per animal were analysed to yield a total number of 150 cells per animal. Ghost cells, with a small head and a diffuse and large tail, were excluded from analysis, but their presence was recorded as an indication of cytotoxicity.

MICRONUCLEUS TEST
- Bone marrow collection and processing: Immediately following liver perfusion (groups 1-4) or sacrifice (group 5), the bone marrow cells of one of the femurs were collected into foetal calf serum and processed into glass-drawn smears according to the method described by Schmid (1976). Four bone marrow smears per animal were made, air-dried and fixed in methanol. Two fixed smears were stained with a May-Grünwald Giemsa solution. The other fixed smears were kept in reserve.

- Microscopic examination of the bone marrow smears: The slides will be randomly coded by a person not involved in the scoring of the slides. 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 evaluation 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.

The numbers of PE and NE were recorded in a total of at least 500 erythrocytes (E) per animal. 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 was scored for the presence of micronuclei until a total of 4000 PE has been scored.
Evaluation criteria:
COMET ASSAY: The test material is considered to be positive in the in vivo comet assay if a statistically significant increase would be observed at one or more dose levels compared to the group mean of the negative control group and/or if a dose related increase in the group mean tail intensity is observed. Positive results from the in vivo comet assay indicate that the test material has the potential to induce primary DNA damage in vivo in the tissue evaluated, under the conditions used in this study. The test material would be considered to be negative in the in vivo comet assay if no statistically significant increase is observed at any of the dose levels compared to the group mean of the negative control group. Negative results indicate that the test material does not have the potential to induce DNA damage in vivo in the tissue evaluated, under the test conditions used in this study. Biological relevance was taken into account for interpretation of the results. If a positive response in the comet assay would be obtained, the possibility that the increase in DNA migration is not associated with genotoxicity, but with severe toxicity, would be assessed.

MICRONUCLEUS TEST: The test material would be considered to cause chromosomal damage and/or damage to the mitotic apparatus if it shows a dose related positive response or a statistically significant increase of micronucleated cells in one or more dose groups when compared to the negative control group. The test material would be considered to be negative in the micronucleus test if it produces no positive response at any of the dose levels analysed. Biological relevance of the results was considered. Statistical methods were used as an aid in evaluating the test results.
Statistics:
See: "Any other information on materials and methods incl. tables"
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
MONITORING OF EXPOSURE CONDITIONS
- Time to attain chamber equilibration: The time to reach 95% of the steady state concentration (T95) was calculated to be about 23 minutes (based on a chamber volume of 75 L and airflow of about 10 L/min).
- Nominal concentration and generation efficiency: The calculated amount of test material was close to the actual amount of test material used, indicating a generation efficiency of 91.0%. This is slightly low for this kind of test material.
- Airflow, temperature and relative humidity: The overall mean (± standard deviation) chamber airflows were 9.91 (± 0.06), 9.83 (± 0.06), 9.60 (± 0.06) and 9.65 (± 0.06) L/min for exposure chambers 1 (control), 2 (low), 3 (mid) and 4 (high) respectively. The air temperature in the exposure chambers during exposure was within the target range of 20 – 24°C for all groups (Table A1.4). The overall mean temperature was 20.4, 20.4, 20.5 and 20.3°C for chambers 1 (control), 2 (low), 3 (mid) and 4 (high) respectively. The relative humidity during exposure was within the target range of 30-70% for all groups. The overall mean relative humidity was 47.6, 46.5, 44.4 and 45.3% in exposure chambers 1 (control), 2 (low), 3 (mid) and 4 (high) respectively. Average oxygen concentrations were 20.6, 20.5, 20.4 and 20.3% (v/v) in chambers 1 (control), 2 (low), 3 (mid) and 4 (high) respectively. Average carbon dioxide concentrations were 0.149, 0.179, 0.158 and 0.274% (v/v) in exposure chambers 1 (control), 2 (low), 3 (mid) and 4 (high) respectively.

CLINICAL SIGNS
All animals survived until scheduled sacrifice. No abnormalities were observed in animals exposed to the test material or to clean air or in animals of the positive control groups for the micronucleus test and comet assay. One male of the positive control group for the comet assay in lung tissue (group 6) showed an ear wound during the acclimatization period and a skin wound on day 0 of exposure. A second male of the positive control group for the comet assay in the liver (group 7) demonstrated testes cryptorchidism during the study. No abnormalities were seen at the group-wise observations made about halfway each 6-hour exposure period (no table presented).

BODY WEIGHTS
Group mean body weights in all groups were considered within the normal range as expected for healthy rats of this age and strain. A slight reduction in body weight gain was observed in groups 2-4. As this reduction was not dose related it is considered that this was not due to treatment with the test substance. In group 2 a slight reduction in the mean body weight was observed on day 1. On day 2 the mean body weight had increased again, but was still slightly lower than on day 0. However, as this was the low dose group and this phenomenon was not observed for the mid and high dose group, this observation is considered not treatment related.

COMET ASSAY
Viability of the cells of the animals of the negative control group was at least 76% for the liver and 80% for the lung. The group mean tail intensity and percentage of ghost cells are presented in "Any other information on results incl. tables". For the positive control rats of both the liver (treated with 2-acetamidofluorene) and the lung (treated with methyl methanesulfonate) a statistically significant increase (p value: 0.0079) in the mean tail intensity was observed, when compared to the negative control animals. For the liver, the mean tail intensity measured in the positive control was comparable to the mean of historical control data. For the lung, no historical data was available for the positive control, but based on statistical analysis the results obtained in this study reflected a clear positive response. The mean tail intensity in the negative control group were within the historical range for both the liver and lung. These results demonstrate the validity of the test system.

There was neither a statistically significant increase in mean tail intensity observed for the liver nor for the lung. For the rats exposed to the test material, when compared to the negative control. The mean percentage of ghost cells of the rats exposed to the test material was comparable to the negative control, indicating that the cell preparations were of good quality and that there was no evidence for cytotoxicity. These results indicate that that treatment with the test material up to 50,000 ppm did not induce primary DNA damage in liver and lung cells of male rats under the conditions used in this study.

MICRONUCLEUS TEST
The group mean PE per 500 E and MPE per 4000 PE are presented in "any other information on results incl. tables". For the positive control rats treated with Mitomycin C, a statistically significant increase (p value: 0.0040) in the mean number of MPE was observed, when compared to the negative control animals. The mean number of MPE found in the positive control group was within historical range when related to 2000 PE. In addition, a statistically significant decrease (p-value: 0.0088) was observed in the mean number of PE per 500 E in the positive control Mitomycin C, when compared to the negative control. This indicated that the positive control substance Mitomycin C reached the bone marrow and induced damage to the chromosomes and/or to the spindle apparatus of the bone marrow cells of male rats. The mean numbers of MPE per 2000 PE in the negative control group were within the historical range. These results demonstrate the validity of the test system.

There was neither a statistically significant increase in the mean number of MPE per 4000 PE nor a statistically significant decrease in the mean number of PE per 500 E for the rats exposed to the test material, when compared to the negative control. This indicated that treatment with the test material up to 50,000 ppm did not result in damage to the chromosomes and/or to the spindle apparatus of the bone marrow cells of male rats nor the erythropoiesis in the bone marrow, under the conditions used in this study.

COMET ASSAY

Table 1. Group mean tail intensity and percentage of ghost cells±SD in isolated male rat hepatocytes after exposure.

Group

No.

Group

description

Target

concentration

in air (ppm)

Tail intensity

(%tail DNA)

(mean±SD)

Percentage

ghost cells

(mean±SD)

1

Negative

0

1.32±0.79

6±4

2

Low

5,000

1.03±0.34

4±2

3

Mid

15,000

1.71±0.81

5±2

4

High

50,000

2.40±0.96

7±3

7

Positive control

2-AAF

17.06±2.68 *

4±1

* Mann-Whitney p-value: 0.0079

 

Table 2. Group mean tail intensity and percentage of ghost cells±SD in isolated male rat lung cells after exposure.

Group

No.

Group

description

Target

concentration

in air (ppm)

Tail intensity

(%tail DNA)

(mean±SD)

Percentage

ghost cells

(mean±SD)

1

Negative

0

1.08±1.09

8±3

2

Low

5,000

0.59±0.36

7±2

3

Mid

15,000

0.56±0.33

7±4

4

High

50,000

0.85±0.71

7±1

6

Positive control

MMS

45.39±7.15 *

10±3

* Mann-Whitney p-value: 0.0079

 

MICRONUCLEUS TEST

Table 3. The group mean number of PE per 500 E and group mean number of MPE per 4000 PE observed in the micronucleus test in male rats after exposure.

Group

No.

Group

description

Target

concentration

in air (ppm)

PE per 500 E

(mean±SD)

MPE per 4000 PE

(mean±SD)

1

Negative

0

322±68

2.6±2.2

2

Low

5,000

305±52

4.0±2.0

3

Mid

15,000

318±59

4.4±1.9

4

High

50,000

272±32

3.0±2.3

5

Positive control

MMC

203±58 *

48.4±12.2 **

* Mann-Whitney p-value: 0.0088

** Mann-Whitney p-value: 0.0040

Conclusions:
Under the conditions used in this study, it is concluded that test material did neither induce chromosomal damage and/or damage to the mitotic spindle apparatus in bone marrow cells, nor induced primary DNA damage in liver and lung cells, of rats exposed to the test material by inhalation on three successive days up to the maximum concentration tested of 50000 ppm.
Executive summary:

According to OECD guidelines 474 and 489 and in compliance with GLP, the test material was examined for its potential to cause damage to the chromosomes and/or the mitotic apparatus of erythroblasts by analysis of erythrocytes as sampled in bone marrow (micronucleus test), and to cause primary DNA damage (such as single and double strand DNA breaks, alkali labile sites and incomplete repair sites) in lung and liver cells (comet assay) of rats, after administration of the test material by inhalation. For both assays, rats were exposed to clean air (negative control; group 1) or three concentrations of the test material (i.e. 5000, 15000 or 50000 ppm; groups 2, 3 and 4, respectively) by inhalation on three successive days (i.e. 6 hours exposure on the first two days and 2 hours exposure on the third day). The interval between start of the first and second exposure was about 24 hours, and the interval between the start of the second and third exposure ranged from 19 to 25 hours. Rats were sacrificed within 3 to 6 hours after the start of the last exposure. The inhalation route was selected because humans may be exposed to the test material by inhalation. The rats of the positive control groups were treated once intraperitoneally (i.p., 10 mL/kg-bw) with Mitomycin C (MMC; 1.5 mg/kg-bw; group 5) ca. 24 hours prior to sacrifice (micronucleus test), once i.p. (10 mL/kg-bw) with Methyl methane sulfonate (MMS; 40 mg/kg-bw; group 6) 2 to 6 hours prior to sacrifice, or once orally (20 mL/kg-bw; group 7) by gavage with 2-acetylaminofluorene (2-AAF, 50 mg/kg-bw) 12 to 15 hours before sacrifice. Prior to each dosing the rats were weighed and the dosing volume was adjusted to the body weight. For the comet assay, the liver of rats of groups 1, 2, 3, 4 and 7 were perfused to obtain single cells, and immediately following liver perfusion (groups 1, 2, 3 and 4) or sacrifice (group 6), lung tissue was dissected and processed to obtain single cells for preparation of slides for comet analysis. For the micronucleus test, immediately following liver perfusion (groups 1, 2, 3 and 4) or sacrifice (group 5), the bone marrow cells of one of the femurs of each rat were collected and processed into smears for microscopic examination. The negative and positive controls of both the micronucleus test and comet assay showed the expected responses and therefore the study was considered valid. Under the conditions used in this study, it is concluded that test material did neither induce chromosomal damage and/or damage to the mitotic spindle apparatus in bone marrow cells, nor induced primary DNA damage in liver and lung cells, of rats exposed to the test material by inhalation on three successive days up to the maximum concentration tested of 50000 ppm. The TK study performed with the test substance (study no. 20567) demonstrated the presence of the test material in blood, indicating that the negative responses observed in this study are not due to lack of systemic availability of the test material or its metabolites.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland
- Assigned to test groups randomly: Yes
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: gas
Vehicle:
- Vehicle used: None, test material is a gas and exposure was by inhalation.
Details on exposure:
TYPE OF INHALATION EXPOSURE: Nose only


GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:nose-only inhalation chamber - cylindrical column, surrounded by a transparent hood. Test atmosphere from the bottom and the exhaust at the top. The column volume was ~ 70 liters.
- Method of holding animals in test chamber: plastic animal holders
- Source and rate of air:
- Temperature, humidity, pressure in air chamber: 20-24C, 30-70% humidity,
- Air flow rate: 10.7 - 20.0 l/min (20 l/min group had 10 animals)
- Air change rate:
- Treatment of exhaust air:


TEST ATMOSPHERE
- Brief description of analytical method used: total carbon analyzer
- Samples taken from breathing zone: yes
Duration of treatment / exposure:
4 hour
Frequency of treatment:
Once
Post exposure period:
24 to 48 hours
Dose / conc.:
12 500 ppm
Remarks:
Group 2: Low dose.
Dose / conc.:
50 000 ppm
Remarks:
Group 3: Mid dose.
Dose / conc.:
200 000 ppm
Remarks:
Group 4: High dose.
No. of animals per sex per dose:
5 at control, low and mid dose and 10 at high dose.
Control animals:
yes, sham-exposed
Positive control(s):
Mitomycin C
- Justification for choice of positive control: Known to cause micronuclei
- Route of administration: Intraperitoneal
- Doses: 0.75 mg/kg
Tissues and cell types examined:
Bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: 200000 ppm is 20% of the atmosphere which can reduce the amount of oxygen in the air.

DETAILS OF SLIDE PREPARATION: Method according to Schmidd (1976). Two smears per animals, air dried and fixed with methanol, stained with May-Grunwald Geimsa solution.

METHOD OF ANALYSIS: Microscopic evaluation
Evaluation criteria:
A test substance is considered to cause chromosomal damage and/or damage to the mitotic apparatus, if the mean number of MPF2000 PE is statistically significantly higher, when compared to the mean number of the vehicle controls.
A test substance is considered to be negative in the micronucleus test if it produces no positive response at the used dose level.
Both statistical significance and biological relevance are considered together in the evaluation.
Statistics:
ANOVA: MPE and PE. If significant, followed by pooled error variance t-test (pooled or separate depending if the variances were homogenous or not).
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test substance did not cause cytotoxicity to the bone marrow cells.
In the positive control group, the mean number of the micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) was statistically significantly higher from the negative control group A (***p <0.001). This demonstrates the validity of the test system.
Conclusions:
From the results obtained it is concluded that this micronucleus test did not show any indication of chromosomal damage and/or damage to the mitotic apparatus of the bone marrow target cells in male mice following acute Inhalation exposure to the test substance.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

HFO-1234yf was shown to be nongenotoxic in all in vivo studies. Based on these results of this battery of in vitro and in vivo studies it is concluded that HFO-1234yf is not genotoxic in mammalian systems.

Therefore the substance does not need to be classified for genetic toxicity in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008 and its amendments.