Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial Reverse Mutation Assay/Ames test): The substance 1,1,1,2,2,4,5,5,5-nonafluoro-4-(trifluoromethyl )-3-pentanone did not induce mutagenicity in S. typhimurium TA1535, TA1537, TA100, TA98 and Escherichia coli WP2 uvrA in the presence or absence of Aroclor 1254-induced rat liver S9 metabolic activation (OECD 471, GLP);

Gene mutation (mammalian cell gene mutation assay): there was no evidence of induced mutant colonies over background in Chinese hamster lung fibroblast (V79) cells exposed to 1,1,1,2,2,4,5,5,5-nonafluoro-4-(trifluoromethyl)-3-pentanone in the presence or absence of mammalian metabolic activation (phenobarbital and ß-naphthoflavone-induced rat liver S9) (OECD 476/GLP).  

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: other guideline: EC Directive 2000/32, B13/14 OECD 471, 1997
GLP compliance:
yes
Type of assay:
other: not available
Species / strain / cell type:
bacteria, other: Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) Escherichia coli WP2uvrA
Metabolic activation system:
S9 liver homogenate of rat induced with Aroclor 1254.
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 10 ... 5000 μg/plate
Concentration range in the main test (without metabolic activation): 10 ... 5000 μg/plate
Vehicle / solvent:
Solvent: ethanol
Species / strain:
other: as specified above
Metabolic activation:
with
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(> 5000 μg/plate)
Species / strain:
other: as specified above
Metabolic activation:
without
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(> 5000 μg/plate)
Species / strain:
other: as specified above
Metabolic activation:
with
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(> 5000 μg/plate)
Species / strain:
other: as specified above
Metabolic activation:
without
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(> 5000 μg/plate)
Additional information on results:
Observations:
The bacterial background lawn was not reduced at all concentrations tested and no biologically relevant decrease in the number of revertants was observed. The presence of 5 and 10% (v/v) liver microsomal activation did not influence these findings. T-7479 did not induce a dose-related, two-fold increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.
Remarks on result:
other: Test system: preliminary test
Conclusions:
Interpretation of results
negative with metabolic activation
negative without metabolic activation
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8th May 2018 - 24th August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Shanghai Waysmos Fine Chemical Co., LTD.; 8180402
- Purity: 99.83%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: store in cool, protected from light
- Stability under test conditions: instable after repeated contact to air and to light


Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: ATCC, CCL-93
- Suitability of cells: V79 cells in vitro have been widely used to examine the ability of chemicals to induce cytogenetic changes and thus identify potential carcinogens or mutagens.
- Cell cycle length, doubling time or proliferation index: 12 - 14 h doubling time

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Minimal Essential Medium (MEM) and cultured at a humidified atmosphere of 5% CO2 and at 37 °C incubation temperature.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically 'cleansed' against high spontaneous background: Yes; For purifying the cell population of pre-existing HPRT- mutants cells were exposed to HAT medium containing 10 µM hypoxanthine, 3.2 µM aminopterin, 5 µM thymidine and 10 µM glycine for several cell doublings (2 - 3 days) with a subsequent recovery period in medium supplemented with 10 µM hypoxanthine and 5 µM thymidine
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and Beta-naphthoflavone-induced rat liver S9
Test concentrations with justification for top dose:
Preliminary test: 25, 50, 100, 250, 500, 1000, 1500 and 2000 µg/mL
Main tests (4 hrs; + and – S9): 25, 50, 100, 250, 500, 1000, 1500 and 2000 µg/mL

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: A solubility test was performed with different solvents and vehicles. Based on the results of the solubility test ethanol was be used as solvent.
The test item was dissolved in ethanol and different stock solutions were prepared. The stock solutions were diluted with cell culture medium and added to the cells. The solvent was compatible with the survival of the cells and the S9 activity.
Untreated negative controls:
yes
Remarks:
Medium
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium;

DURATION
- Exposure duration: 4 hrs + and – S9
- Expression time (cells in growth medium): 7-9 days
- Selection time (if incubation with a selection agent): 9-11 days (mutant frequency); 6-8 days (cloning efficiency)

SELECTION AGENT (mutation assays): selective medium containing 11 µg/mL 6-thioguanine

NUMBER OF REPLICATIONS: Single cell culture (test item; positive controls); duplicates (solvent and medium negative controls)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: After the selection period, colonies were fixed with methanol, stained with Giemsa and counted.

NUMBER OF CELLS EVALUATED: 4 x 10~5 cells (mutant selection); 200 cells in 2 flasks (cloning efficiency)

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was evaluated by relative survival (RS). The cloning efficiency (CE) of cells plated immediately after treatment was adjusted by any loss of cells during treatment as compared with adjusted cloning efficiency in negative / solvent controls (assigned a survival of 100%).



Evaluation criteria:
A test chemical is considered to be clearly negative if, in all experimental conditions examined
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend-test
- all results are inside the distribution of the historical negative control data
A test chemical is considered to be clearly positive if, in any of the experimental conditions examined
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control, and
- the increase is concentration-related when evaluated with an appropriate trend test, and
- any of the results are outside the distribution of the historical negative control data.
- if there is by chance a low spontaneous mutant frequency in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results.
Statistics:
The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative/solvent controls. Mutant frequencies of the negative/solvent controls were used as reference.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Remarks:
4 hrs
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
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH-value detected with the test item was within the physiological range (pH 7.0 ± 0.4).
- Effects of osmolality: Osmolality of the highest test item concentration was 472 mOsm/kg.
- Precipitation: No precipitation of the test item was noted in the experiments.

RANGE-FINDING/SCREENING STUDIES:
The toxicity of the test item was determined in a pre-experiment. Eight concentrations [25, 50, 100, 250, 500, 1000, 1500 and 2000 µg/mL] were tested with and without metabolic activation for the 4 h exposure assay (Table 1, 2). Toxicity of the test item was evaluated using the relative survival (RS). A cytotoxic effect is observed as soon as the relative survival decreased below 70%. The test item was tested at the following concentrations in the main experiments: without and with metabolic activation - 25, 50, 100, 250, 500, 1000, 1500 and 2000 µg/mL

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Yes; January 2015 – April 2018
- Negative (solvent/vehicle) historical control data: Yes; January 2015 – April 2018
Conclusions:
In an in vitro mammalian cell gene mutation test, the test item 1,1,1,2,2,4,5,5,5-nonafluoro-4-(trifluoromethyl)-3-pentanone is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.
Executive summary:

In an in vitro gene mutation study in mammalian cells (HPRT; 183288), Chinese hamster lung fibroblasts (V79) were exposed to 1,1,1,2,2,4,5,5,5-nonafluoro-4-(trifluoromethyl)-3-pentanone (99.83%) in ethanol at concentrations of 25, 50, 100, 250, 500, 1000, 1500 and 2000 µg/mL for 4 hrs with and without phenobarbital and ß-naphthoflavone-induced rat liver S9 metabolic activation.

1,1,1,2,2,4,5,5,5-nonafluoro-4-(trifluoromethyl)-3-pentanone was tested up to the limits of cytotoxicity. The positive controls induced the appropriate response. There was no evidence of induced mutant colonies over background in the presence or absence of metabolic activiation.

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

Genetic toxicity in vivo

Description of key information

Mammalian erythrocyte micronucleus test: 1,1,1,2,2,4,5,5,5-nonafluoro-4-(trifluoromethyl )-3-pentanon did not increase the mean frequency of micronucleated polychromatic erythrocytes in the bone marrow of male NMLI treated mice compared to controls. 1,1,1,2,2,4,5,5,5-nonafluoro-4-(trifluoromethyl )-3-pentanone is not clastogenic in the micronucleus test.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
other: Data sharing dispute
Adequacy of study:
key study
Study period:
October 2005 to February 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other:
Remarks:
other: Study performed by OECD guidelines and under GLP with no deviations. All finding clearly sup port the "not clastogenic" conclusions
Qualifier:
according to
Guideline:
other: OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test) in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test
Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles river, Sulzfeld, Germany
- Age at study initiation: 6 weeks old
- Housing: polycarbonate cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.7-22.1 deg C.
- Humidity (%): 30 to 70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: October to Dec 2005
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: 1% CMC
- Justification for choice of solvent/vehicle: Suitably stable emulsion
- Concentration of test material in vehicle:200 mg/mL
- Amount of vehicle (if gavage or dermal): 10mL/kg
Duration of treatment / exposure:
Single exposure
Frequency of treatment:
Once
Post exposure period:
24, 48 hours
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide;
- Justification for choice of positive control(s): Common usage
- Doses / concentrations: 50 mg/kg
Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: According to OECD Guidelines

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Single injections with collection after 24 and 48 hours.

DETAILS OF SLIDE PREPARATION: Slides were automatically stained using the "Wright-stain-procedure"

METHOD OF ANALYSIS: The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. Averages and standard deviations were calculated
Evaluation criteria:
All validation critieria were met.
a. Positive control substance performed as desired.
b. Incidence of micronucleated polychromatic erhthrocytes were within historical control ranges. Me an +/- 3 standard deviations
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg
- Solubility: suspension
- Clinical signs of toxicity in test animals: negative
- Evidence of cytotoxicity in tissue analyzed:none
- Rationale for exposure: well tolerated high dose
- Harvest times: Study duration of 3 days

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): None
- Ratio of PCE/NCE (for Micronucleus assay): None
- Appropriateness of dose levels and route: Appropriate
- Statistical evaluation: Yes
Conclusions:
Negative - not clastogenic under the conditions of this assay.
Executive summary:

MTDID 5789 (Lot #2) was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect on erythrocytes in bone marrow. The study procedures described in this report were based on the most recent OECD and EEC guidelines. Batch Lot #2 of MTDID 5789 was a clear colourless liquid with a purity of 99.91%. The test substance was emulsified in 1% (w/v) carboxymethylcellulose. Five male animals were used in each of the four treatment groups, including negative and positive controls. All groups received a single intraperitoneal injection. The negative and positive control groups were treated with vehicle and 50 mg/kg body weight of cyclophosphamide (CP), respectively. Animals were dosed with MTDID 5789 at 2000 (two groups) mg/kg body weight. All animals showed no abnormalities after dosing. Bone marrow of the groups treated with MTDID 5789 was sampled 24 or 48 hours after dosing. Bone marrow of the negative and positive control groups was harvested 24 and 48 hours after dosing, respectively.

No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with MTDID 5789. The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, both criteria for an acceptable assay were met. The groups that were treated with MTDID 5789 showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The groups that were treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, demonstrating toxic effects on erythropoiesis.

It is concluded that MTDID 5789 is not clastogenic in the micronucleus test under the experimental conditions described in this report.

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

Additional information

There is one in vitro reverse gene mutation assay in bacteria and one in vitro gene mutation study in mammalian cells available for

1,1,1,2,2,4,5,5,5-nonafluoro-4-(trifluoromethyl )-3-pentanone. The in vitro cytogenicity study is waived as an in vivo mammalian erythrocyte micronucleus test in mice is available.

In vitro

In a reverse gene mutation assay in bacteria (OECD 471/GLP), strains of S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvr A were exposed to 1,1,1,2,2,4,5,5,5-nonafluoro-4-(trifluoromethyl )-3-pentanone in ethanol at concentrations of 10 -5000 µg/plate in the presence and absence of Aroclor 1254-induced rat liver S9 metabolic activation. 1,1,1,2,2,4,5,5,5-nonafluoro-4-(trifluoromethyl )-3-pentanone was tested up to the limit concentration (5000 µg/plate). The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

In an in vitro gene mutation study in mammalian cells (HPRT; OECD 476/GLP), Chinese hamster lung fibroblasts (V79) were exposed to 1,1,1,2,2,4,5,5,5-nonafluoro-4-(trifluoromethyl)-3-pentanone (99.83%) in ethanol at concentrations of 25, 50, 100, 250, 500, 1000, 1500 and 2000 µg/mL for 4 hrs with and without phenobarbital and ß-naphthoflavone-induced rat liver S9 metabolic activation. 1,1,1,2,2,4,5,5,5-nonafluoro-4-(trifluoromethyl)-3-pentanone was tested up to the limits of cytotoxicity. The positive controls induced the appropriate response. There was no evidence of induced mutant colonies over background in the presence or absence of metabolic activiation.

In vivo

In a mammalian erythrocyte micronucleus test (OECD 474/GLP), groups of male NMRI mice (5/dose) were treated intraperitoneally with 1,1,1,2,2,4,5,5,5-nonafluoro-4-(trifluoromethyl )-3-pentanone (99.91%) in 1% (w/v) carboxymethylcellulose at a single dose of 2000 mg/kg bw. Bone marrow cells were harvested at 24h and 48 hrs post-treatment. 1,1,1,2,2,4,5,5,5-nonafluoro-4-(trifluoromethyl )-3-pentanone was tested at an adequate dose. The positive control (cyclophosphamide) induced the appropriate response. No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of treated animals compared to controls. 1,1,1,2,2,4,5,5,5-nonafluoro-4-(trifluoromethyl )-3-pentanone is not clastogenic in the micronucleus test.

The results from these studies are suitable for use in the human health risk assessment.

Justification for classification or non-classification

Based on the available information in the dossier, the substance 1,1,1,2,2,4,5,5,5-nonafluoro-4-(trifluoromethyl)-3-pentanone does not need to be classified for germ cell mutagenicity when the criteria outlined in Annex I of 1272/2008/EC are applied.