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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro Genetic toxicity:

The available set of in vitro studies includes a mutagenicity in bacteria (according to OECD TG 471, EC method B.14, or equivalent methodology in the Japanese standards), an in vitro mammalian chromosome aberration test (according to OECD TG 473, EC method B.10) using peripheral human lymphocytes and a mouse lymphoma assay (according to OECD TG 490) conducted in compliance with the good laboratory practice standards.


- Gene mutation (Bacterial reverse mutation assay): S. typhimurium TA98, TA 100, TA102, TA 1535 and TA 1537: negative with and without metabolic activation. The results were consistent with another study conducted according to Japanase standard guidelines in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, with methodology equivalent to the OECD guideline.
- in vitro mammalian chromosome aberration test: peripheral human lymphocytes: negative with and without metabolic activation.

- in vitro gene mutation in L5178Y mouse lmphoma cells, negative with and without metabolic activation in two independent experiments.

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:
1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
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:
S9 Mix
Test concentrations with justification for top dose:
Preliminary test; 5, 15, 50, 150, 1500, 5000 micro gram/plate
Test; 156.3, 312.5, 625, 1250, 2500, 5000 micro gram/plate
Vehicle / solvent:
DMSO: Dimethylsulfoxide
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Details on test system and experimental conditions:
Two plates were used for each of seven different concentrations of the sample in the range-finding study and six different concentrations of the sample in the main test. The liver microsome fraction (S9) was prepared from the liver of Slc:Sprague-Dawley rats prepared with phenobarbital and 5,6-benzoflavone.
Evaluation criteria:
The result was considered positive if the number of colonies found was twice the number of colonies of the control, which was exposured to dimethylsulfoxide, the solvent for test sample, and concentration-related increase over the range tested and reproducible increase at range finding trial and main test were observed.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
negative with and without metabolic activation
The number of revertant colonies in six concentrations, 156.3, 312.5, 625, 1250, 2500 and 5000 microgram/plate did not increase more than twice compare with the solvent control in five bacteria strains with and without activation.
The growth inhibition of the bacteria was not observed.
Executive summary:

The number of revertant colonies in six concentrations, 156.3, 312.5, 625, 1250, 2500 and 5000 microgram/plate did not increase more than twice compare with the solvent control in five bacteria strains with and without activation.

The growth inhibition of the bacteria was not observed either.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From October, 1995 To December, 1995.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983
Deviations:
yes
Remarks:
there was no positive control for the assay with S. typhimurium TA1535 and TA1537 tested with metabolic activation
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
there was no positive control for the assay with S. typhimurium TA1535 and TA1537 tested with metabolic activation
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
no data
Test concentrations with justification for top dose:
50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: common solvent.
Untreated negative controls:
other: sterility check with test substance
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
9-aminoacridine
mitomycin C
other: hydrazine sulphate; Doxorubicine HCl
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) for trial 1 with and without S9, and for trial 2 without S9; preincubation for trial 2 with S9

DETERMINATION OF CYTOTOXICITY
- Method: observation of the background plate

OTHER: triplicates
2 independent experiments were performed:
1st trial: plate test with and without S9
2nd trial: plate test without S9 and pre-incubation test with S9

3 plates/dose/strain, both for the test with and without metabolic activation.

- positive controls:
9-AA: TA1537
mitomycin C: TA102
2-AF: TA98, TA100, TA102 with S9
Doxorubicin HCl: TA98, TA100 without S9
Hydrazine sulfate: TA1535
Evaluation criteria:
Comparison of the spontaneous reversions (in the negative control) with the ones in the test article plates and in the positive control plates were done.
Criteria used in the study: The positive controls should induce a number of revertant colonies statistically greater than and at least double the mean number of spontaneous reverted colonies.
Statistics:
Comparison of the spontaneous reversions (in the negative control) with the ones in the test article plates and in the positive control plates were done by Student's "t" test.
Key result
Species / strain:
S. typhimurium TA 1535
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
Key result
Species / strain:
S. typhimurium TA 1537
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
Key result
Species / strain:
S. typhimurium TA 98
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
Key result
Species / strain:
S. typhimurium TA 100
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
Key result
Species / strain:
S. typhimurium 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:
CYTOTOXICITY: At the doses tested no cytotoxic effect was observed.

Table 1: Test without metabolic activation (1 trial)

1 trial (without S9) Reversions/plate Mean S.D. (P)
TA1535 Solvent control 19 28 27 24.67 4.933 /
50 µg/plate 27 18 26 23.67 4.933 0.816
150 µg/plate 20 23 25 22.67 2.517 0.566
500 µg/plate 29 23 20 24.00 4.583 0.872
1500 µg/plate 26 23 21 23;33 2.517 0.698
5000 µg/plate 24 21 27 24.00 3.00 0.851
Positive control 133 115 131 126.33 9.866 < 0.001***
TA1537 Solvent control 9 13 14 12.00 2.646 /
50 µg/plate 9 12 11 10.67 1.528 0.492
150 µg/plate 12 10 11 11.00 1 0.573
500 µg/plate 13 8 14 11.67 3.215 0.896
1500 µg/plate 7 11 12 10.00 2.646 0.407
5000 µg/plate 11 10 13 11.33 1.528 0.725
Positive control 61 58 59 59.33 1.528 < 0.001***
TA98 Solvent control 46 39 47 44.00 4.359 /
50 µg/plate 37 43 44 41.33 3.786 0.469
150 µg/plate 46 40 38 41.33 4.163 0.486
500 µg/plate 45 43 41 43.00 2 0.736
1500 µg/plate 43 42 38 41.00 2.646 0.366
5000 µg/plate 37 48 41 42.00 5.568 0.650
Positive control 931 1040 878 949.67 82.597 < 0.001***
TA100 Solvent control 181 173 193 182.33 10.066 /
50 µg/plate 153 184 171 169.33 15.567 0.291
150 µg/plate 185 171 138 164.67 24.132 0.307
500 µg/plate 174 170 152 165.33 11.719 0.129
1500 µg/plate 146 182 191 173.00 23.812 0.566
5000 µg/plate 172 160 158 163.33 7.572 0.059
Positive control 600 490 575 555.00 57.663 < 0.001***
TA102 Solvent control 215 271 249 245.00 28.213 /
50 µg/plate 230 280 248 252.67 25.325 0.744
150 µg/plate 260 230 241 243.67 15.177 0.946
500 µg/plate 213 246 250 236.33 20.306 0.688
1500 µg/plate 265 217 240 240.67 24.007 0.849
5000 µg/plate 295 210 260 255.00 42.72 0.752
Positive control 1320 1074 1240 1211.33 125.48 < 0.001***

* P < 0.05

** P < 0.01

*** P < 0.001

Table 2: Test with metabolic activation (1 trial)

1 trial (with S9) Reversions/plate Mean S.D. (P)
TA1535 Solvent control 26 32 29 29.00 3.000 /
50 µg/plate 24 31 27 27.33 3.512 0.566
150 µg/plate 30 24 24 26.00 3.464 0.320
500 µg/plate 29 27 24 26.67 2.517 0.360
1500 µg/plate 21 22 30 24.33 4.933 0.234
5000 µg/plate 26 21 31 26.00 5.000 0.423
Positive control / / / / / /
TA1537 Solvent control 13 8 14 11.67 3.215 /
50 µg/plate 6 15 13 11.33 4.726 0.924
150 µg/plate 9 8 9 8.67 0.577 0.187
500 µg/plate 7 11 13 10.33 3.055 0.630
1500 µg/plate 10 7 12 9.67 2.517 0.444
5000 µg/plate 5 12 8 8.33 3.512 0.292
Positive control / / / / / /
TA98 Solvent control 41 50 47 46.00 4.583 /
50 µg/plate 48 46 38 44.00 5.292 0.647
150 µg/plate 49 48 44 47.00 2.646 0.760
500 µg/plate 40 47 38 41.67 4.726 0.318
1500 µg/plate 50 44 45 46.33 3.215 0.923
5000 µg/plate 50 42 45 45.67 4.041 0.929
Positive control 960 1070 966 998.67 61.849 < 0.001***
TA100 Solvent control 190 179 183 184.00 5.568 /
50 µg/plate 180 157 177 171.33 12.503 0.184
150 µg/plate 190 152 184 175.33 20.429 0.517
500 µg/plate 179 194 176 183.00 9.644 0.884
1500 µg/plate 180 160 171 170.33 10.017 0.108
5000 µg/plate 164 181 194 179.67 15.044 0.664
Positive control 830 947 950 909.00 68.432 < 0.001***
TA102 Solvent control 279 315 281 291.67 20.232 /
50 µg/plate 290 284 261 278.33 15.308 0.414
150 µg/plate 276 300 240 272.00 30.199 0.402
500 µg/plate 246 280 264 263.33 17.010 0.137
1500 µg/plate 277 284 290 283.61 6.506 0.550
5000 µg/plate 303 290 267 286.67 18.230 0.766
Positive control 784 990 870 881.33 103.467 < 0.001***

* P < 0.05

** P < 0.01

*** P < 0.001

Conclusions:
Negative with and without metabolic activation.
Under the conditions of this test, the test article did not induce any significant increase in the number of reversions, either in the absence or in the presence of metabolic activation, up to the concentration of 5000 µg/plate on TA1535, TA1537, TA98, TA100 and TA102 Salmonella thyphimurium strains, in two independent experiments.
Executive summary:

The ability of the test substance 1,6 -divinylperfluorohexane to induce gene mutation in strain TA1535, TA1537, TA98, TA100 and TA102 of Salmonella typhimurium was assessed. The test substance, dissolved in ethanol, was tested at five concentrations ranging from 50 to 5 000 µg/plate, both with and without metabolic activation. Two independent experiments were performed. No appreciable increase in the number of reversions in comparison with the negative control was evident in either experiment at any tested doses for any strains, wether in the presence or in the absence of metabolic activation. The reference mutagens induced a number of reverted colonies statistically greater than and at least double the mean number of spontaneous reverted colonies. Therefore, the test substance did not induce any significant increase in the number of reversions, either in the absence or in the presence of metabolic activation, up to the concentration of 5 000 µg/plate on TA1535, TA1537, TA98, TA100 and TA102 Salmonella thyphimurium strains.

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:
23 June 2014 to 21 August 2014
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)
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
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: peripheral human lymphocytes from 3 healthy adult, non-smoking, male volunteers 25-, 31-, 35-years old
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat S9 fraction liver homogenate was obtained from Trinova Biochem GmbH, Giessen, Germany and is prepared from male rats (SD) that have been treated orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).
Test concentrations with justification for top dose:
First cytogenetic assay:
Without and with S9-mix : 512, 1600 and 3540 μg/ml culture medium
(3 h exposure time, 24 h fixation time).

Second cytogenetic assay:
512, 1600 and 3540 μg/ml culture medium
(24 h exposure time, 24h fix
48 h exposure time, 24 and 48 h fixation time).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Common solvent
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
Mitomycin C: without S9 ; Cyclophosphamide: with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 +/- 2h
- Exposure duration: 3h, 24h or 48h
- Expression time (cells in growth medium): 20 - 22h
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48h

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

NUMBER OF CELLS EVALUATED:
1,6-divinylfluorohexane was tested in the absence and presence of 1.8% (v/v) S9-fraction in duplicate in two independent experiments.
At least two slides were prepared per culture. At least three analysable concentrations were used for scoring of the cytogenetic assay. Chromosomes of metaphase spreads were analysed from those cultures with an inhibition of the mitotic index of about 50% or above whereas the mitotic index of the lowest concentration level was approximately the same as the mitotic index of the solvent control.
To prevent bias, all slides were randomly encoded before examination of chromosome aberrations and scored. One hundred metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
The mitotic index of each culture was determined by counting the number of metaphases from at least 1000 cells (with a maximum deviation of 5%)

STRUCTURAL CHROMOSOME ABERRATIONS
Breaks, gaps, minutes, dicentrics and exchange figures.
Evaluation criteria:
A chromosome aberration test is considered acceptable if it meets the following criteria:
a) The number of chromosome aberrations found in the solvent control cultures should reasonably be within the laboratory historical control data range.
b) The positive control substances should produce a statistically significant (Chi-square test, onesided, p < 0.05) increase in the number of cells with chromosome aberrations.
c) A homogeneous response between the replicate cultures is observed.
d) A possible precipitate present on the slides should not interfere with the scoring of chromosome aberrations.
Statistics:
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a concentration-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear concentration-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics:

X2 =(N-1) (ad-bc)2/(a+b) (c+d) (a+c) (b+d)
where b = the total number of aberrant cells in the control cultures.
d = the total number of non aberrant cells in the control cultures.
n0 = the total number of cells scored in the control cultures.
a = the total number of aberrant cells in treated cultures to be compared with the control.
c = the total number of non aberrant cells in treated cultures to be compared with the control.
n1 = the total number of cells scored in the treated cultures.
N = sum of n0 and n1

If P [ X2 > (N-1) (ad-bc)2/(a+b) (c+d) (a+c) (b+d)] (one-tailed) is small (p< 0.05) the hypothesis that the incidence of cells with chromosome aberrations is the same for both the treated and the solvent control group is rejected and the number of aberrant cells in the test group is considered to be significantly different from the control group at the 95% confidence level.
Key result
Species / strain:
lymphocytes: peripheral human lymphocytes
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
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Under the experimental conditions of this test, 1,6-divinylfluorohexane is not clastogenic in human lymphocytes, both in the absence and presence of metabolic activation.
Executive summary:

The effect of 1,6-divinylfluorohexane on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix) has been assessed. The possible clastogenicity of 1,6-divinylfluorohexane was tested in two independent experiments.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

The test substance was soluble in dimethyl sulfoxide, at concentrations of 160 mg/ml and below but formed a suspension at a concentration of 354 mg/ml. In the first cytogenetic assay, 1,6-divinylfluorohexane was tested up to the recommended concentration level of 0.01 M (3540 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. 1,6-divinylfluorohexane precipitated in the culture medium at this concentration level. In the second cytogenetic assay, 1,6-divinylfluorohexane was tested up to 3540 μg/ml for a 24 and 48 h continuous exposure time with a 24 and 48 h fixation time in the absence of S9-mix. 1,6-divinylfluorohexane precipitated in the culture medium at this concentration level. The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C (without S9) and cyclophosphamide (with S9), both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. 1,6-divinylfluorohexane did not induce a 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 effects of 1,6-divinylfluorohexane on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that 1,6-divinylfluorohexane does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Finally, it is concluded that this test is valid and that 1,6-divinylfluorohexane is not clastogenic in human lymphocytes under the experimental conditions of this study.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-09-09 to 2019-12-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
Thymidine kinase (TK) locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: L5178Y/TK+/- -3.7.2C mouse lymphoma cells from the American Type Culture Collection, (ATCC, Manassas, USA) (2001)
- Suitability of cells: Recommended test system in international guidelines
- Normal cell cycle time (negative control): not specified

For cell lines:
- Absence of Mycoplasma contamination: verified
- Cell cycle length, doubling time or proliferation index : no data
- Modal number of chromosomes: not specified
- Periodically checked for karyotype stability: no data
- Periodically ‘cleansed’ of spontaneous mutants: yes, cleansing was performed prior to the DRF and mutagenicity tests

MEDIA USED
- Type and composition 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 2mM L-glutamin.
* Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).
* Exposure medium for 3-hour exposure: basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium).
* Exposure medium for 24-hour exposure: basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).
* Selective medium: basic medium, supplemented with 20% (v/v) heat-inactivated horse serum (R20-medium) and 5 μg/mL trifluorothymidine (TFT).
* Non-selective medium: basic medium, supplemented with 20% (v/v) heat-inactivated horse serum (R20-medium).
- CO2 concentration: 5.0 ± 0.5% CO2
- humidity level: target 80 - 100%, actual range: 52 - 95%
- temperature: target 37.0 ± 1.0°C, actual range: 34.6 - 37.6°C
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: Rat S9 homogenate purchased from Trinova Biochem GmbH, Giessen, Germany, prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).

- method of preparation of S9 mix : prepared immediately before use and kept refrigerated. Components per mL physiological saline: 1.63 mg MgCl2.6H2O; 2.46 mg KCl; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 μmol HEPES. The solution is filtered (0.22 μm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction is added (50% (v/v) S9-fraction) to complete the S9-mix.

- concentration or volume of S9 mix and S9 in the final culture medium : 4% (v/v) in the exposure medium

- quality controls of S9 : metabolic capability was confirmed in the experiment with the positive control cyclophosphamide
Test concentrations with justification for top dose:
The test item was assessed in a solubility assay, and in a dose-range finding assay to assess cytotoxicity.
Cytotoxicity data were obtained by treating 8 x 106 cells (106 cells/mL for 3 hour treatment) or 6 x 106 cells (1.25 x 105 cells/mL for 24 hour treatment) with a
number of test item concentrations increasing by approximately half log steps.
Assays were done with a 3-hour treatment in a sterile 30 ml centrifuge tube incubated in a shaking incubator at 37°C.
For the 24 hours treatment, 2 sets of cells were treated, one in closed flasks and one in venting flasks. The test item was tested in the absence and presence of S9-mix.
The highest tested concentration was 2000 µg/mL exposure medium, as recommended in the test guideline.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: based on solubility test (visual homogeneity) and previous studies. Test item concentrations were used within 2 hours after preparation to limit potential volatilisation.
- Justification for percentage of solvent in the final culture medium: maximum recommended concentration in the test guideline (1% v/v)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: a single culture per concentration
- Number of independent experiments : 2 independent experiments under test different conditions (a short and a long treatment periods)

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium: 8x10E6 cells per culture were used (10E6 cells/ml) for the 3-hr treatment with and without S9, or 6x10E6 cells (1.25x10E5 cells/ml) for the 24 hour treatment, in closed flasks.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 hrs with and without S9; 24 hours without S9.

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): cells were subcultured for 2 days after the treatment period. During this culture period at least 4 x 106 cells (where possible) were subcultured every day in order to maintain log phase growth
- Selection time (incubation with a selective agent): 11-12 days
- Method used: 96-microwell plates.
- selective agent: 5 µg/mL trifluorothymidine (TFT), incubation for 11 or 12 days
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: for cloning efficiency, one cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium ; for mutation frequency, 4x10E6 cells were subcultured every day.
- Criteria for small (slow growing) and large (fast growing) colonies: The small colonies are morphologically dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphologically less dense colonies with a hazy contour and with a diameter larger than a quarter of a well. A well containing more than one small colony is classified as one small colony. A well containing more than one large colony is classified as one large colony. A well containing one small and one large colony is classified as one large colony.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: For the mutation assays: cloning efficiency and relative total growth (RTG). For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium.
- Any supplementary information relevant to cytotoxicity: the plates for the TFT-selection were stained for 1.5-2 hours, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Mutation frequency (MF):
For determination of the mutation frequency (MF) a total number of 9.6 x 10E5 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 105 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection).
The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 1.5-2 hours, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.
The colonies were divided into small and large colonies.

Evaluation criteria:
ACCEPTABILITY CRITERIA
a) Absolute cloning efficiency of the solvent controls (CEday2) between 65 and 120% to have an acceptable number of surviving cells analysed for expression of the TK mutation.
b) spontaneous MF in the solvent control ≥ 50 per 10E6 survivors and ≤ 170 per 10E6 survivors.
c) Suspension growth (SG) over the 2-day expression period for the solvent controls between 8 and 32 for the 3-hr treatment, and between 32 and 180 for the 24-hr treatment.
d) positive control should demonstrate an absolute increase in the total MF, i.e, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10E-6. At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10E-6 above the concurrent solvent control (a small colony IMF of 150 x 10-6).
If (one of) the acceptability criteria are not met, the test will be rejected and repeated. In case no clear conclusion for positive or negative result can be made an additional confirmation study will be performed to confirm the study results.

ANALYSIS
In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data (HCD) range.
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the microwell version, the GEF is 126 x 10E-6.

* positive/mutagenic: if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. Any observed increase should be biologically relevant and will be compared with the HCD range.
* equivocal/questionable: if no clear conclusion for positive or negative result can be made after an additional confirmation study.
* negative/not mutagenic): if none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
Statistics:
Usually not used in MLA assay.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
3-hour treatment
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Remarks:
one solvent control was used for the determination of MF
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
24-hour treatment
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
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-SPECIFIC CONFOUNDING FACTORS
- Data on pH: 7.4 at the concentration 2000g/mL vs 7.4 in the solvent control.
- Data on osmolality: 428 mOsm/kg at a concentration of 2000 µg/mL vs 454 mOsm/kg in the solvent control.
- Possibility of evaporation from medium: the 3-treatment was performed in a closed tube, under agitation. The 24-hour treatment was performed in closed flasks since no severe toxicity was observed in the dose-range findings under these conditions.
- Water solubility: low, therefore the test item was solubilised in DMSO prior to treatment of the culture medium.
- Precipitation and time of the determination: none noted.
- Definition of acceptable cells for analysis: survival
- Other confounding effects: none known

RANGE-FINDING/SCREENING STUDIES: results are reported in Tables 1 (3-hour treatment) and 2 (24-hour treatment)

STUDY RESULTS
- Concurrent vehicle negative and positive control data are reported in Tables 3 and 4
The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.
Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. The results showed that the test conditions were adequate and the metabolic activation system (S9-mix) functioned properly.

Criteria for data analysis and interpretation:
- the interpretation of the MLA results was based on a GEF value of 126; MF results of treated were compared to the MF of the corresponding controls + 126.

Gene mutation tests in mammalian cells are reported in Tables 3 (3-hour treatment) and 4 (24-hour treatment):
- Results from cytotoxicity measurements:
o Relative total growth (RTG) and cloning efficiency (CEday2)

- Genotoxicity results:
In the experiment with 3-hour treatment with and without metabolic activation, the cloning efficiency of one of the duplicate solvent controls was below the acceptability criteria (CEday2 within the range 65 - 120 %). Since the other solvent control was within the acceptability criteria, and duplicate solvent control groups are no longer requested the revised test guideline OECD 490, the determination of the mutation frequency of the test item was done with only one solvent control (deviation to the study plan).
The determination of the mutagenicity of the test item with only one solvent control had no effect on the results of the study as no increase in the mutation frequency was observed in the treatment groups compared to the solvent control.

HISTORICAL CONTROL DATA
- Negative (solvent/vehicle) historical control data: in Table 5
- Positive historical control data: in Table 6

Dose-range finding tests

Table 1 - Dose range-finding test: cytotoxicity following 3-hour treatment

 Dose  cell count after 24-h of subculture   cell count after 48-h of subculture  SG(1)  RGS(2)
 (µg/ml)  (cells/ml x 10E5)   (cells/ml x 10E5)    %
  without metabolic activation
 Solvent control (DMSO)  4.2  9.8  21  100
 63  3.5  7.0  12  59

 125

 2.7

 8.8

 12

 58

 250

 3.3

 9.8

 16

 79

 500

 1.3

 12.6

 8

 40

 1000

 1.4

 12.7

 9

 43

 2000

 1.6

 13.1

 10

 51

 

with metabolic activation

 Solvent control (DMSO)

3.1

 8.4

 13

 100

 63

 3.5

8.1

 14

 108

 125

 2.7

 7.6

 10

 78

 250

 3.5

 8.1

 14

 108

 500

 3.5

 8.2

 14

 109

 1000

 3.7

 8.3

 15

 118

 2000

 3.6

 8.5

 15

 117

(1) Suspension growth

(2) Relative Suspension Growth

Table 2 - Dose range-finding test: cytotoxicity following 24-hour treatment

 Dose  cell count after 24-h of subculture   cell count after 48-h of subculture  SG(1)  RGS(2)
 (µg/ml)  (cells/ml x 10E5)   (cells/ml x 10E5)    %
  without metabolic activation (closed flasks)
 Solvent control (DMSO)  9.2  7.6  45  100
 63  8.2  7.5  39  174

 125

 8.2

6.6

 35

 154

 250

 8.5

 5.6

 30

 135

 500

 8.0

 4.0

 20

 91

 1000

 9.6

 3.2

 20

 88

 2000

 9.5

 4.9

 30

 132

 

without metabolic activation (open flasks)

 Solvent control (DMSO)

9.7

 8.4

 52

 100

 63

 8.8

7.9

 44

 85

 125

 7.6

 6.7

 33

 62

 250

 8.0

 5.6

 28

 54

 500

 7.4

 4.6

 22

 41

 1000

 8.7

 3.5

 20

 38

 2000

 8.3

 4.6

 25

 47

(1) Suspension growth

(2) Relative Suspension Growth

MUTATIONS EXPERIMENTS

Table 3 - Mutation Experiment 1 (3-hour treatment)

 dose

 RSG

 CE day2

 RCE

 RTG

 mutation frequency per 10E6 survivors     

 (µg/ml)

 (%)

 (%)

 (%)

 (%)

 total

 (small

 large)

 

without metabolic activation

 

3-hour treatment

 SC

 100

 95

 100

 100

 78

 (23

 52)

 15.6

 98

 88

 92

 90

 70

 (16

 52)

 125

 86

 84

 88

 75

 71

 (21

 48)

 250

 102

 78

 82

 84

 68

 (20

 46)

 500

 90

 88

 92

 82

 89

 (28

 58)

 1000

 97

 66

 69

 67

 101

 (37

 61)

 2000

 85

 98

 103

 87

 54

 (16

 36)

 MMS

 72

 50

 53

 38

 661

 (301

 306)

 

with metabolic activation

 

 3-hour treatment 

 SC

 100

 81

 100

 100

 106

 (23

 79)

 15.6

 96

 88

 108

 103

 89

 (21

 65)

 31.3

 98

 85

 105

 103

 90

 (25

 62)

 62.5

 90

 91

 113

 101

 80

 (32

 45)

 125

 79

 86

 107

 84

 89

 (30

 56)

 250

 71

 62

 77

 54

 98

 (10

 86)

 500

 62

 83

 102

 63

 94

 (32

 58)

 1000

 72

 71

 88

 64

 93

 (30

 61)

 2000

 65

 69

 86

 56

 93

 (39

 51)

 CP

 73

 68

 84  62  520  (203  273)

Table 4 - Mutation Experiment 2 (24-hour treatment)

 dose

 RSG

 CE day2

 RCE

 RTG

 mutation frequency per 10E6 survivors     

 (µg/ml)

 (%)

 (%)

 (%)

 (%)

 total

 (small

 large)

 

without metabolic activation

 

24-hour treatment

 SC1

 100

 91

 100

 100

 159

 (24

 128)

 SC2

 

 76

 

 

 144

 (20

 120)

 15.6

 89

 127

 152

 134

 164

 (22

 132)

 31.3

 106

 105

 126

 133

 162

 (24

 130)

 62.5

 94

 107

 128

 120

 88

 (28

 56)

 125

 76

 111

 133

 101

 95

 (29

 62)

 250

 74

 135

 161

 119

 90

 (26

 59)

 500

 68

 141

 168

 114

 88

 (23

 60)

 1000

 63

 123

 147

 93

 116

 (35

 73)

 2000

 59

 116

 139

 81

 125

 (49

 67)

 CP

 103

 91

 109  113  667  (282  285)

HISTORICAL CONTROL DATA

Table 5 - Historical Negative Control Data of the spontaneous Mutation Frequencies of the solvent control for the Mouse Lymphoma Assay

 

           Mutation frequency per 10E6 survivors

 

 without S9-mix  

 with S9-mix

 

 3-hr exposure

 24-hr exposure

 3-hr exposure

 mean

101

98

100

 SD

30

31

30

 n

279

262

293

 Upper control limit

(95% control limits)

170

162

165

 Lower control limit

(95% control limits)

31

34

36

SD: Standard Deviation

n = Number of observations

Distribution of historical negative control data from experiments performed between September 2015 and September 2018

Table 6 - Historical Control Data of the Mutation Frequencies of the positive controls for the Mouse Lymphoma Assay

             Mutation frequency per 10E6 survivors
   without S9-mix    with S9-mix
   3-hr exposure  24-hr exposure  3-hr exposure

 mean

803 695 1545
 SD 253 223 887
 n 142 132 151

 Upper control limit

(95% control limits)

1533 1270 3954

 Lower control limit

(95% control limits)

72 119 -864

SD: Standard Deviation

n = Number of observations

Distribution of historical negative control data from experiments performed between September 2015 and September 2018

Conclusions:
The test substance 1,6-divinylperfluorohexane is not mutagenic in the L5178Y Mouse Lymphoma Cells with and without metabolic activation.
Executive summary:

The mutagenic potential of 1,6-divinylperfluorohexane was assessed for its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix). The TK mutational system detects base pair mutations, frame shift mutations and small deletions.

The test was performed in the absence of S9-mix with 3 and 24 hour treatment periods and in the presence of S9-mix with a 3 hour treatment period. 

The study procedures described in this report were based on the most recent OECD test guideline 490. The vehicle of the test item was dimethyl sulfoxide.

The vehicle of the test item was dimethyl sulfoxide.

The test item was tested up to the maximum recommended conncentration of 2000 µg/mL in both experiments, without causing severe cytotoxicity. In the first experiment the incubation time was 3 hours, with or without metabolic activation. In the second experiment, the incubation time was 24-hours, in closed flasks, in the absence of metabolic activation.

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency 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.

In the absence of S9-mix, the test item did not induce a biologically relevant increase in the mutation frequency in the first experiment with a 3-hour treatment. This result was confirmed in an independent experiment with modification in the duration of treatment (24 hours).

In the presence of S9-mix, the test item did not induce a biologically relevant increase in the mutation frequency.

In conclusion, 1,6-divinylperfluorohexane is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions of this study.

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

Additional information

Genetic toxicity assessment:

In vitro genetic toxicity:

There are two in vitro studies available addressing mutagenicity in bacteria (according to OECD TG 471, EC method B.14, or equivalent methodology in the Japanese standards), and an in vitro mammalian chromosome aberration test (according to OECD TG 473, EC method B.10) using peripheral human lymphocyte conducted in compliance with the good laboratory practice standards.

The ability of the test substance 1,6-divinylperfluorohexane to induce gene mutation in strain TA1535, TA1537, TA98, TA100 and TA102 of Salmonella typhimurium was assessed. The test substance, dissolved in ethanol, was tested at five concentrations ranging from 50 to 5 000 µg/plate, both with and without metabolic activation. Two independent experiments were performed. No appreciable increase in the number of reversions in comparison with the negative control was evident in either experiment at any tested doses for any strains, wether in the presence or in the abence of metabolic activation. The reference mutagens induced a number of reverted colonies statistically greater than and at least double the mean number of spontaneous reverted colonies. Therefore, the test substance did not induce any significant increase in the number of reversions, either in the absence or in the presence of metabolic activation, up to the concentration of 5 000 µg/plate on TA1535, TA1537, TA98, TA100 and TA102 Salmonella typhimurium strains.

Negative results were also reported in another good quality study in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, at concentrations up to 5000 µg/plate, conducted according to the Japanese standards.

The effect of 1,6-divinylfluorohexane on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix) has been assessed. The possible clastogenicity of 1,6-divinylfluorohexane was tested in two independent experiments.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

1,6-divinylfluorohexane was soluble in dimethyl sulfoxide, at concentrations of 160 mg/ml and below but formed a suspension at a concentration of 354 mg/ml. In the first cytogenetic assay, 1,6-divinylfluorohexane was tested up to the recommended concentration level of 0.01 M (3540 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. 1,6-divinylfluorohexane precipitated in the culture medium at this concentration level. In the second cytogenetic assay, 1,6-divinylfluorohexane was tested up to 3540 μg/ml for a 24 and 48 h continuous exposure time with a 24 and 48 h fixation time in the absence of S9-mix. 1,6-divinylfluorohexane precipitated in the culture medium at this concentration level. The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C (without S9) and cyclophosphamide (with S9), both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. 1,6-divinylfluorohexane did not induce a 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 effects of 1,6-divinylfluorohexane on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that 1,6-divinylfluorohexane does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Finally, it is concluded that this test is valid and that 1,6-divinylfluorohexane is not clastogenic in human lymphocytes under the experimental conditions described in this report.

The ability of the test substance 1,6-divinylperfluorohexane to induce gene mutation in mammalian cells was nvestigated in mouse lymphoma assay, in the absence of metabolic activation with 3- and 24-hour treatment period, and in the presence of metabolic activation with a 3-hr treatment period. The test substance was tested at the highest recommended dose, and did not produce severe toxicity. The test conditions met the acceptability criteria and no increase in the mutation frequency was observed.

In conclusion, based on available in vitro data, 1,6-divinylperfluorohexane has no mutagenic potential in three types of studies, so no in vivo follow up study is necessary.


Justification for selection of genetic toxicity endpoint
Studies were performed according to OECD test guidelines and corresponding EC methods in compliance with GLP.

Justification for classification or non-classification

The test material appears to have no mutagenic potential in vitro. Therefore, 1,6-divinylperfluorohexane does not have to be classified for genetic toxicity according to the criteria of the CLP regulation No. 1272/2008.