1-Propene, 1,3,3,3-tetrafluoro-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

HFO-1234ze was not mutagenic in S. typhimurium strains TA100, TA1535, 1537, TA98 and E. coli WP-2 (with or without S9). It was found not to be clastogenic in cultured human lymphocytes.

Link to relevant study records

Reference

Reference 1

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

Cytokinesis block (if used):

Colcemid

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:

yes

Remarks:

sham exposed

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: substance is a liquified gas. Lymphocytes were exposed in a modular incubator chamber in atmospheric concnetrations of 19% O2, 5% CO2, and up to 76% test substance


DURATION
- Preincubation period: 48hrs
- Exposure duration: 4hrs
- Expression time (cells in growth medium):20 or 44 hrs
- Fixation time (start of exposure up to fixation or harvest of cells): 26-50 hrs


SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): 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)

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:

not applicable

Untreated negative controls validity:

valid

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 concentration 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.

Reference 2

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:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Guideline of Reverse Mutation Assay in Bacteria

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:

Phenobarbital and 5,6-benzoflavone-induced rat liver homogenate (S9-mix).

Test concentrations with justification for top dose:

0.05, 0.1, 0.5, 1, 5 ,10 and 50 % in the atmosphere

Untreated negative controls:

yes

Remarks:

sham exposed bacteria

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

9-aminoacridine

sodium azide

Details on test system and experimental conditions:

- Exposure duration to test substance in gas exposure chamber followed by 24 - 48 hour exposure to test substance 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.

Statistics:

None

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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 and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

HFO-1234ze was not mutagenic under the conditions employed in this study with or without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

HFO-1234ze was negative in the following three in vivo genotoxicity studies: micronucleus – mouse (acute inhalation exposure) and rats (4-week inhalation exposure) and UDS test (unscheduled DNA synthesis) following 4-week inhalation exposure.

Link to relevant study records

Reference

Reference 1

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

Reason / purpose for cross-reference:

reference to same 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:

rat

Strain:

Sprague-Dawley

Sex:

male

Route of administration:

inhalation: gas

Duration of treatment / exposure:

28 days

Frequency of treatment:

6 hours/d, 5 days/wk for 4 weeks

Post exposure period:

None

Dose / conc.:

1 000 ppm (analytical)

Remarks:

Group 2: Low dose

Dose / conc.:

5 000 ppm (analytical)

Remarks:

Group 3: Low-mid dose

Dose / conc.:

10 000 ppm (analytical)

Remarks:

Group 4: High-mid dose.

Dose / conc.:

15 000 ppm (analytical)

Remarks:

Group 5: High dose

No. of animals per sex per dose:

5

Control animals:

yes, sham-exposed

Positive control(s):

mitomycin C
- Justification for choice of positive control(s): substance known to cause micronuclei
- Route of administration: intra-peritoneal injection
- Doses / concentrations: 0.15 mg/kg

Tissues and cell types examined:

bone marrow - polychromatic and normocromatic erythrocytes

Evaluation criteria:

A response is considered to be positive and cause chromosomal damage and/or damage to the mitotic apparatus if the mean number of MPE/200 PE is statistically significantly higher compared to controls. Statistical and biological relevance are considered together in the evaluation.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay) - postive control animals exhibited a statistically signifant increase compared to negative control animals
- Appropriateness of dose levels and route: Highest dose tested by relevant route of exposure for 28 days

Conclusions:

No damage to the chromosomes and/or mitotic spindle apparatus (micronuclei) in the bone marrow target cells of male rats was induced following 28 day inhalation exposure to up to 15000 ppm HFO-1234ze.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

GLP compliance:

yes (incl. QA statement)

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Route of administration:

inhalation: gas

Duration of treatment / exposure:

28 days

Frequency of treatment:

6 hours/d, 5 days/wk for 4 weeks

Post exposure period:

none

Dose / conc.:

5 000 ppm (analytical)

Remarks:

Group 2: Low dose.

Dose / conc.:

15 000 ppm (nominal)

Remarks:

Group 3: High dose.

No. of animals per sex per dose:

5

Control animals:

yes, sham-exposed

Positive control(s):

2-acetylaminofluorene
- Justification for choice of positive control(s): sustance known to produce unscheduled DNA synthesis
- Route of administration: oral gavage
- Doses / concentrations: 50 mg/kg
- Dose volume 10 ml/kg of a 5 ml/kg solution

Tissues and cell types examined:

cultured hepatocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Isolated hepatocytes were prepared within 24 hours of the last expsosure for negative control and test article treated animals. Hepatocytes from AAF treated animals were prepared 12-16 hours after exposure

Evaluation criteria:

The study is considered valid if the positive control gives a positive response and if the negative control gives a clear negative response.
A response at a data point is considered positive if the population average NNG > 5, and if at least 20 % of the cells are "in repair" and weakly positive if the population average NNG is between 0 and 5.
A test material is considered to cause DNA damage and induce DNA repair in liver cells if at least one dose level at one time point results in an increase in the mean NNG compared to the vehicle control.

A test material is considered non-genotoxic under the conditions of the test if all dose levels and time points produce NNG < O.
Both numerical significance and biological relevance are considered together in the evaluation.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

other: did not meet criteria but were different from controls and HFO-1234ze treated

Additional information on results:

Both the test material HFO-1234yf and the negative control (clean air) yielded net nuclear grains (NNG)< O. Since exposure to the test material did not induce NNG> 5, it is demonstrated that HFO-1234ze did not induce unscheduled DNA synthesis in rat hepatocytes. The positive control substance 2-AAF induced NNG3.25 with less 20 % of the cells in repair. While the positive control did not exhibit as robust examination as expected, the results did show that these animals had a higher NNG and % cells in repair compared to controls or HFO-1234ze tested cells.

The mean NNG determined for 2 -AAF (-6.310 was clearly higher than the NNG of the negative control (-9.78) or test substance (-13.04 and -11.82). The cells in repair was 3.25% for 2 -AAF and <0.5 for control and 0.2% for the test substance. No technical problems were noted and the negative controls and background counts were within historical controls. The response of the groups treated with HFO-1234ze is considered to be correct.

Conclusions:

It is concluded that the test material HFO-1234ze did not induce unscheduled DNA synthesis (UDS) in liver cells of male rats, exposed to the test material at concentrations up to 15000 ppm by inhalation, under the conditions used in this study.

Reference 3

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

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Remarks:

US laboratory, no certificate available

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Kingston NY
- Assigned to test groups randomly: yes
- Diet : ad libitum
- Water :ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.0 to 22. 1
- Humidity (%): 20.0 to 22. 1
- Photoperiod (hrs dark / hrs light): 12/12 hours

Route of administration:

inhalation: gas

Vehicle:

- Vehicle(s)/solvent(s) 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 litres.
- Method of holding animals in test chamber: plastic animal holders
- Source and rate of air:
- Temperature, humidity, pressure in air chamber: 20 - 24 °C, 30 - 70 % humidity,
- Air flow rate: 10.7 - 20.0 L/min (20 L/min group had 10 animals)

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 - 48 hours

Dose / conc.:

10 000 ppm (analytical)

No. of animals per sex per dose:

10

Control animals:

yes, sham-exposed

Positive control(s):

Cyclophosphamide
- Justification for choice of positive control(s): known to cause micronuclei
- Route of administration: intraperitoneal
- Doses / concentrations: 40 mg/kg

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: 100000 ppm is 10 % 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 positive response is normally indicated by a statistically significant increase in the incidence of
micronucleated polychromatic erythrocytes for the treatment group compared with the concurrent
negative control group (P<0.01); individual and/or group mean values should exceed the laboratory
historical negative control range (Morrison and Ashby 1995). A negative result is indicated where
individual and group mean incidences of micronucleated polychromatic erythrocytes for the group treated
with the test substance are not significantly greater than incidences for the concurrent negative control
group (P>0.01) and where these values fall within the historical negative control range. An equivocal
response is obtained when the results do not meet the criteria specified for a positive or negative
response.

Statistics:

The results for each treatment group were compared with the results for the concurrent negative control group using non-parametric statistics.As there was considered to be no difference between the sexes, the induced micronuclei frequency for both sexes were combined to facilitate interpretation and maximise the power of statistical analysis.
For incidences of micronucleated polychromatic erythrocytes, exact one-sided P-values are calculated by permutation (StatXact, CYTEL Software Corporation, Cambridge, Massachussetts). For assessment of effects on the proportion of polychromatic erythrocytes, equivalent permutation tests based on rank scores are used, ie exact versions of Wilcoxon's sum of ranks test.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not applicable

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:

Interpretation of results: negative
HFO-1234ze did not show any evidence of causing an increase in micronuclei or bone marrow cell toxicity when administered to CD-1 mice for a single 4-hour nose-only inhalation exposure in this in vivo test procedure.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Based on these results of this battery of in vitro and in vivo studies it is concluded that HFO-1234ze is not genotoxic.

Justification for classification or non-classification

HFO-1234ze was not shown to be genotoxic in in vitro or in vivo genotoxicity studies. No classification is deemed necessary.