Registration Dossier

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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test: not mutagenic up to 5000 µg/plate with and without metabolic activation (OECD 471, GLP, K, rel. 1)

- HL/CAT: not clastogenic up to 400 µg/mL with and without metabolic activation (OECD 473, GLP, K, rel. 1)

- MLC/MLA: not mutagenic up to 400 µg/mL with and without metabolic activation (OECD 490, GLP, K, rel. 1)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02-Dec-15 to 07-Mar-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Adopted July 21, 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Remarks:
Except for the quality environment in which the characterisation of the test item was performed was not known.
Type of assay:
bacterial reverse mutation assay
Target gene:
- S. typhimurium: Histidine gene
Species / strain / cell type:
other: S. Typhimurium (TA98, TA100, TA102, TA1535 and TA1537)
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by Aroclor 1254
Test concentrations with justification for top dose:
Preliminary test (without and with S9) TA100: 200, 500, 1000, 2000 and 5000 µg/plate

Main study:
- Experiment 1 (pre incubation): TA1535, TA1537, TA102 and TA98: Without and with S9-mix: 200, 500, 1000, 2000 and 5000 µg/plate
- Experiment 2 (pre incubation): TA1535, TA1537, TA98, TA100 and TA102: Without and with S9-mix: 200, 500, 1000, 2000 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: tetrahydrofuran
- Justification for choice of solvent/vehicle:
Test compound was soluble in tetrahydrofuran and tetrahydrofuran has been accepted and approved by authorities and international guidelines.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9: 650 µg/plate in DMSO for TA100
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without S9: 10 µg/plate in DMSO for TA98 and 15 µg/plate for TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Tert-butyl hydroperoxide (TBH)
Remarks:
without S9: 250 µg/plate in DMSO for TA102
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9: 5 µg/plate in saline for TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene in DMSO for all tester strains
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: pre incubation

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.


ACCEPTABILITY OF THE ASSAY:
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at WIL Research Europe.
b) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or TA102 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) The total number of revertants in the tester strains TA100 and TA102 is greater than two (2) times the concurrent control, or the total number of revertants in the tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate
Remarks on result:
other: not mutagenic
Conclusions:
Under the test conditions, test substance is not considered as mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98, TA100 and TA102.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471, EU Method B.13/14 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 TA100 and TA 102) were exposed to the test substance diluted in tetrahydrofuran at the following concentrations both in the presence and absence of metabolic activation system (S9 -mix).

Preliminary test (without and with S9) TA100: 200, 500, 1000, 2000 and 5000 µg/plate

Main study:

- Experiment 1 (pre incubation): TA1535, TA1537, TA102 and TA98: Without and with S9-mix: 200, 500, 1000, 2000 and 5000 µg/plate

- Experiment 2 (pre incubation): TA1535, TA1537, TA98, TA100 and TA102: Without and with S9-mix: 200, 500, 1000, 2000 and 5000 µg/plate

Negative and positive control groups were also included in mutagenicity tests.

The negative control values were within the laboratory historical control data ranges, except the responses for TA102 (absence of S9-mix, first experiment) and TA1535 (absence of S9-mix, second experiment). However since the mean number of revertant colonies showed a characteristic number of revertant colonies (237 and 3 revertant colonies, respectively) when compared against relevant historical control data (248 and 5 relevant colonies, respectively), the validity of the test was considered to be not affected.

The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except the response for TA1535, TA1537 and TA98 in the second experiment. The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was more than 3 times greater than the concurrent solvent control values, this deviation in the mean plate count of the positive control had no effect on the results of the study.

No cytotoxic effect was observed. All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.

Under the test conditions, test substance is not considered as mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98, TA100 and TA102.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30-Nov-2015 to 03-Jun-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: Cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
- Type and identity of media:
Blood samples
Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.
- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not applicable, immediately after blood collection lymphocyte cultures were started.
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: not applicable, immediately after blood collection lymphocyte cultures were started.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone.
Test concentrations with justification for top dose:
Dose range finding test:
Without S9-mix, 3hr exposure; 24 hr fixation: 105, 200, 300, 400 and 500 µg/mL
Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 105, 200, 300, 400 and 500 µg/mL
With S9-mix, 3hr exposure; 24 hr fixation: 105, 200, 300, 400 and 500 µg/mL
First cytogenetic test:
Without and with S9-mix, 3 h exposure time, 24 h fixation time: 100, 300 and 400 µg/mL
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 100, 200, 300 and 400 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 100, 200, 300, 325, 350, 375 and 400 µg mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: tetrahydrofuran
- Justification for choice of solvent/vehicle:
Test compound was soluble in tetrahydrofuran and tetrahydrofuran has been accepted and approved by authorities and international guidelines
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9: in Hank's Balanced Salt Solution: 0.5 and 0.75 µg/mLfor a 3 h exposure period, 0.2 and 0.3 µg/mL for a 24 h exposure period and 0.1 and 0.15 µg/mL for a 48 h exposure period
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9: in Hank's Balanced Salt Solution: 10 µg/mL for a 3h exposure period
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration: 3 hr (with and without S9-mix), 24 and 48 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hr

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

A chromosome aberration test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item induces a statistically significant increase in the number of cells with chromosome aberrations. The positive control data will be analysed by the Fisher’s exact test (one-sided, p < 0.05).
Evaluation criteria:
A test item is considered positive (clastogenic) in the chromosome aberration test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with an Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided,
p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with an Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

Statistics:
Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) ) was used for statistical analysis of the data.
Key result
Species / strain:
lymphocytes: human pheripheral blood
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 400 µg/ml and above.

RANGE-FINDING/SCREENING STUDIES:
Toxicity was observed at the highest dose levels tested.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide induced appropriate responses.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxicity was observed up to and including the highest precipitating tested dose
Remarks on result:
other: not clastogenic
Conclusions:
Under the experimental conditions, the test substance is not considered as clastogenic in human lymphocytes with and without metabolic activation.
Executive summary:

A chromosome aberration study with Perfluoro methoxy dioxole was performed according to OECD 473 guideline, EU Method B.10 and GLP principles, in cultured peripheral human lymphocytes in presence and absence of a metabolic activation system in order to assess its clastogenic potential.

Two independent experiments were performed. In the first cytogenetic assay, the test item was tested up to 400 μg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. Perfluoro methoxy dioxole precipitated in the culture medium at this dose level. In the second cytogenetic assay, the test item was also tested up to 400 μg/ml for a 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time in the absence of S9-mix. Perfluoro methoxy dioxole precipitated in the culture medium at this dose level.

Negative and positive control groups were also included in clastogenic tests.

The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Perfluoro methoxy dioxole did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments.

No biologically relevant effects of Perfluoro methoxy dioxole on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.

Finally, it is concluded that this test is valid and that Perfluoro methoxy dioxole is not clastogenic in human lymphocytes under the experimental conditions described in this report.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09-Nov-15 to 29-Dec-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
- Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin.
- Growth medium: basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Exposure medium: For 3 hour exposure, cells were exposed to the test item in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium) // For 24 hour exposure, cells were exposed to the test item in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).
- Selective medium: Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 μg/ml trifluorothymidine (TFT) (Sigma).
- Non-selective medium: Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test:
Without and with S9-mix, 3 hours treatment: 105, 300, 500 and 1000 µg/mL
Without S9-mix, 24 hours treatment: 105, 300, 500 and 1000 µg/ml

Experiment 1:
With and Without S9-mix, 3 hours treatment: 105, 150, 200, 250, 300, 350, 400, 450 and 500 μg/ml
Experiment 2:
Without S9-mix, 24 hours treatment: 105, 150, 200, 250, 300, 350 and 400 µg/ml

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: tetrahydrofuran
- Justification for choice of solvent/vehicle: Test compound was soluble in tetrahydrofuran and tetrahydrofuran has been accepted and approved by authorities and international guidelines.

Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9: 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: cyclophosphamide
Remarks:
with S9: 7.5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)
Evaluation criteria:
ACCEPTABILITY OF THE ASSAY
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120%. An acceptable number of surviving cells (10^6) could be analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The positive control should demonstrate an absolute increase in the total mutation frequency above the spontaneous background MF (an induced MF (IMF) of at least 300 x 10-6). At least 40% of the IMF should be reflected in the small colony MF. Furthermore, the positive control should have an increase in the small colony MF of at least 150 x 10^6 above that seen in the concurrent solvent/control (a small colony IMF of at least 150 x 10-6).

DATA EVALUATION
Any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
- Precipitation: The test item precipitated in the exposure medium at concentrations of 350 μg/ml and above.

RANGE-FINDING/SCREENING STUDIES:
Dose range finding:
In the absence of S9-mix, the relative suspension growth was 4% at the test item concentration of 1000 μg/ml compared to the relative suspension growth of the solvent control (3 hour of treatment).
In the presence of S9-mix, the relative suspension growth was 3% at the test item concentration of 1000 μg/ml compared to the relative suspension growth of the solvent control (3 hour of treatment).
The relative suspension growth was 84% at the test item concentration of 300 μg/ml compared to the relative suspension growth of the solvent control. No cell survival was observed at the test item concentrations of 500 μg/ml and upwards after 24 hours treatment.
Muation assays:
No severe toxicity was observed in de absence and presence of S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No obvious toxicity was observed up to and including the highest tested precipitating dose level in both experiments in the absence and presence of S9-mix.
Remarks on result:
other: not mutagenic
Conclusions:
Under the experimental conditions, the test substance is not considered as mutagenic in the mouse lymphoma L5178Y with and without metabolic activation.
Executive summary:

In a mouse lymphoma assay conducted according to OECD 490 guideline and GLP principles, L5178Y mouse lymphoma cells were exposed to the test substance, in a first experiment to 105, 150, 200, 250, 300 and 350 μg/ml in the absence of S9 -mix and 105, 150, 250, 300, 350 and 400 μg/ml in the presence of S9 -mix for a 3 hours exposure period and in a second experiment to 105, 150, 250, 300, 350 and 400 μg/ml in the absence of S9 -mix for a 3 hours exposure period.

No cytotoxicity was observed at these dose levels in the absence and presence of S9-mix in both experiments. The test item precipitated in the culture medium at dose levels of 350 μg/ml and above.

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range.

The growth rate over the two-day expression period for cultures treated with THF was between 16 and 20 (3 hours treatment) and 136 and 141 (24 hours treatment).

 

Mutation frequencies in cultures treated with positive control chemicals were increased 16- and 22-fold in the absence of S9-mix, and 8-fold for CP in the presence of S9-mix. In addition the observed mutation frequencies of the positive control items were within the acceptability criteria of this assay. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate for the detection of a mutagenic response and that the metabolic activation system (S9-mix) functioned properly.

 

In the absence of S9-mix, Perfluoro methoxy dioxole did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modification in the duration of treatment.

In the presence of S9-mix, Perfluoro methoxy dioxole did not induce a significant increase in the mutation frequency.

It is concluded that Perfluoro methoxy dioxole is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Three key studies were identified for the genetic toxicity assessment.

Mutagenicity:

A key study (CRL, 2016a, Rel.1) was performed to evaluate the mutagenic potential of the test substance in bacteria. This reverse gene mutation assay was performed according to the OECD test guideline No. 471, EU Method B.13/14 and in compliance with GLP, using the strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 TA100 and TA 102). No cytotoxic effect was observed up to 5000 µg/plate with and without metabolic activation. All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiment at any dose level.

Therefore, test substance is not considered as mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98, TA100 and TA102.

A second key study (CRL, 2016c, Rel.1) was performed to evaluate the mutagenic potentiel of the test substance in mammalian cells. This in vitro genetic mutation study was performed according to the OECD 490 guideline and in compliance with GLP. L5178Y mouse lymphoma cells were exposed to test substance up to 400 µg/mL with and without metabolic activation. The test substance did not induce a significant increase in the mutation frequency in absence and in presence of metabolic activation at any dose level.

Therefore, Perfluoro methoxy dioxole is not considered as mutagenic in the mouse lymphoma L5178Y test system.

Clastogenicity:

A key study (CRL, 2016b, Rel.1) was performed to evaluate the clastogenicity potential of the test substance. This chromosome aberration study was performed according to OECD 473 guideline, EU Method B.10 and in compliance with GLP, in cultured peripheral human lymphocytes exposed to test substance up to 400 µg/mL with and without metabolic activation. The test substance did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments at any dose level. Therefore, test substance is not considered as clastogenic in human lymphocytes with and without metabolic activation.

CONCLUSION: test substance is not considered as mutagenic or clastogenic.

Justification for classification or non-classification

Harmonized classification:

The substance does not have an harmonized classification according to the Regulation (EC) No. 1272/2008 (CLP).

Self-classification:

Based on the available data, no self-classification is proposed for the substance regarding genetic toxicity according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the UN GHS.