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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reliable Ames test performed according to OECD 471 guideline and in accordance with GLP principles with and without metabolic activation.

Reliable mouse lymphoma assay performed according to OECD 490 guideline and in accordance with GLP principles with and without metabolic activation.

Reliable chromosome aberration study performed according to OECD 473 guideline and in accordance with GLP principles with and without metabolic activation.

All these 3 in vitro studies were negative.

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:
September 2015 - October 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
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:
Rat liver S9-mix induced by Aroclor 1254
Test concentrations with justification for top dose:
Experiment 1: direct plate assay
Preliminary test (without and with S9) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 52, 164, 512, 1600 and 5000 µg/plate

Experiment 2: pre-incubation assay
Without and with S9-mix: 52, 164, 512, 1600 and 5000 µg/plate
Vehicle / solvent:
- Vehicle used: Water
Test item is stable in water and completely miscible
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 5 µg/plate in saline for TA1535 (direct plate + pre-incubation)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: ICR-191
Remarks:
without S9 2.5 µg/plate in DMSO for TA1537 (direct plate)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene
Remarks:
without S9 10 µg/plate in DMSO for TA98 (direct plate + pre-incubation) and 15 µg/plate in DMSO for TA1537 (pre-incubation)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 650 µg/plate in DMSO for TA100 (direct plate + pre-incubation)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 10 µg/plate in DMSO for WP2uvrA (direct plate + pre-incubation)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene in DMSO for all tester strains
Remarks:
with S9 2.5 µg/plate for TA1535,TA1537 (direct plate + pre-incubation); 1 µg/plate for TA98,TA100 (direct plate); 5 µg/plate for TA100 (pre-incubation); 15 µg/plate for WP2uvrA (direct plate + pre-incubation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

Experiment 2 included a pre-incubation step
Fresh bacterial culture (0.1 mL 10E9 cells/mL) of one of the tester strains and a dilution of the test item in Milli-Q water (0.1 mL) were pre-incubated for 30 minutes by 70 rpm at 37°C, either with 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays).

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.
Rationale for test conditions:
To obtain more information about the possible mutagenicity of the test substance, a pre-incubation experiment was performed in the absence and presence of S9-mix.
Evaluation criteria:
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.

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA 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 follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat 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.
Statistics:
No formal hypothesis testing was done.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1600 µg/plate and above after pre-incubation
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1600 µg/plate and above after pre-incubation
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1600 µg/plate and above after pre-incubation
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1600 µg/plate and above after pre-incubation
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1600 µg/plate and above after pre-incubation
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- No precipitation was observed up to and including the top dose of 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
In strain TA100, fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no dose-relationship was observed, the reductions are not considered to be caused by toxicity of the test substance. In all other strains, no biologically significant decrease in the number of revertants was observed.

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 in the direct plate assay
- pre-incubation assay
TA1535: without S9: 1600 µg/plate and above and with S9: no toxicity
TA1537: without S9: 1600 µg/plate and above and with S9: no toxicity
TA98: without S9: 1600 µg/plate and above and with S9: no toxicity
TA100: without S9: 1600 µg/plate and above and with S9: 5000 μg/plate
WP2uvrA: without S9: 1600 µg/plate and above and with S9: no toxicity
Conclusions:
In an AMES test, performed according to OECD guideline and GLP principles, Tetrabutylphosphonium Bromide was found not to be mutagenic with or without metabolic activation.
Executive summary:

An AMES test was performed according to the OECD guideline 471 and GLP principles. All bacterial strains showed negative responses up to and including 5000 ug/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. No cytotoxicity and/or precipitation of the test substance was observed in the direct-plate experiment. In the pre-incubation assay, toxicity was observed in all tester strains in the absence of S9-mix and in tester strain TA100 in the presence of S9-mix at the two highest concentrations tested. The negative and 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.

Based on the results of this study it is concluded that Tetrabutylphosphonium Bromide; CYPHOS® 442 Phosphonium Salt is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 2015 - October 2015
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)
Version / remarks:
2014
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes
Type of assay:
other: in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: Cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
- Source of cells:
Blood samples were collected by venapuncture. 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.

- AGT: 12.6-12.8 h
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone. The concentration of the S9-fraction in the exposure medium was 1.8% (v/v).
Test concentrations with justification for top dose:
Dose range finding test:
With and without S9-mix, 3h exposure; 24 h fixation: 52, 164, 512, 1600 and 2000 μg/mL culture medium
Without S9-mix, 24/48 h exposure; 24/48 h fixation: 52, 164, 512, 1600 and 2000 µg/mL


First cytogenetic test:
With and without S9-mix, 3 h exposure time, 24 h fixation time: 50, 750, 1500, 1600, 1700 and 1800 µg/mL
Based on the results of the first cytogenetic assay the following dose levels were selected for cytogenetic assay 1A:
Without S9-mix, 3 h exposure, 24 h fixation time: 50, 500, 750, 1000, 1250, 1500 and 1600 µg/ mL

The following dose levels were selected for scoring of chromosome aberrations:
Without S9-mix, 3 h exposure, 24 h fixation time: 50, 1500 and 1600 μg/mL culture medium
With S9-mix, 3 h exposure, 24 h fixation time: 50, 750 and 1600 μg/mL culture medium


Second cytogenetic test:
Without S9-mix, 24 h and 48 h exposure; 24 h and 48 h fixation: 25, 50, 75, 100, 150 and 200 µg/mL

the following doses were selected for scoring of chromosome aberrations:
Without S9-mix, 24 h exposure; 24 h fixation: 25, 50 and 100 μg/mL culture medium
Without S9-mix, 48 h exposure; 48 h fixation: 25, 50 and 75 μg/mL culture medium
Vehicle / solvent:
- Vehicle used: culture medium
The test item was melted at 120°C (3 hour). The melted test item was flushed with nitrogen. A solubility test was performed. In the dose range finding study Tetrabutylphosphonium Bromide; CYPHOS® 442 Phosphonium Salt was dissolved in RPMI 1640 medium. The stock solution was treated with ultrasonic waves until the test item was completely dissolved. In the cytogenetic assays the test item was suspended in culture medium at concentrations of 50 mg/mL and above. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. The test item was dissolved in culture medium at concentrations of 20 mg/mL and below. Tetrabutylphosphonium Bromide; CYPHOS® 442 Phosphonium Salt concentrations were used within 1.5 hours after preparation.
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/mL for 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 3 h exposure period
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

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

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

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 150 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
Rationale for test conditions:
Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level had an inhibition of the mitotic index of 50% or greater whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control.

Based on the mitotic index of the dose range finding test and the first cytogenetic assay appropriate dose levels were selected for the second cytogenetic assay. As clear negative results were obtained in the presence of metabolic activation, the repetition of the experiment was not considered necessary.
Evaluation criteria:
Acceptability of the assay
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).

A test item is considered positive (clastogenic) in the chromosome aberration test if:
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) 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) All results are inside the 95% control limits of the negative historical control data range.
Statistics:
GraphPad PRISM version 4.03 and ToxRat Professional version 3.0.0 were used for statistical analysis of the data.
Since the Fisher’s exact test showed that there was a statistically significant difference between one of the test item groups and the vehicle control group a Cochran Armitage trend test (p < 0.05) was performed to test whether there is a significant trend in the induction.
Species / strain:
lymphocytes: cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: No precipitation was observed up to and including the top dose of 2000 µg/mL

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 2000 µg/mL and above in the absence of S9, 3 h treatment/24 h fixation; at dose levels of 164 µg/mL and above in the absence of S9 for the continuous treatment of 48 h and at dose levels of 1600 µg/mL and above in the presence of S9, 3 h treatment, 24 h fixation

FIRST CYTOGENIC ASSAY
- Toxicity was observed at the higest dose level (1600 µg/mL) in the presence and absence of S9, 3 h treatment/24 h fixation
- In the absence of S9-mix, test item induced a statistically significant increase in the number of cells with chromosome aberrations at the highest tested concentration (1600 µg/mL) only. However since no significant trend was observed this increase is not biologically relevant.
- In the presence of S9-mix: No statistically significant increase in the number of cells with chromosome aberrations.

- In the absence of S9-mix, at the concentration of 1600 μg/mL, in one of the duplicate cultures, 3 polyploid cells were observed. This number of polyploidy cells is above the 95% control limits for polyploid cells. However, both in the absence and presence of S9-mix, Tetrabutylphosphonium Bromide; CYPHOS® 442 Phosphonium Salt did not significantly increase the number of polyploid cells and cells with endoreduplicated chromosomes. Therefore, this increase in the number of polyploid cells is an accidental finding and not biologically relevant.

SECOND CYTOGENETIC ASSAY
- Toxicity was observed at dose levels of 100 µg/mL and above in the absence of S9, 24 h treatment/24 h fixation and at dose levels of 75 µg/mL and above in the absence of S9, 48 h treatment/48 h fixation.
- In the absence of S9-mix: No statistically significant increase in the number of cells with chromosome aberrations.
- No increase the number of polyploid cells and cells with endoreduplicated chromosomes.


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. Although at the 24 h exposure time the response of MMC-C was just above the upper control limits, these limits are 95% control limits and a slightly higher response is within the expected response ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Appropriate toxicity was reached at the dose levels selected for scoring.
Conclusions:
A chromosome aberration study with Tetrabutylphosphonium Bromide was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. It is concluded that Tetrabutylphosphonium Bromide is not clastogenic in human lymphocytes.
Executive summary:

In a chromosome aberration study, to test the ability to induce structural chromosome aberrations in cultured human lymphocytes, cultured peripheral human lymphocytes were exposed to different concentrations of Tetrabutylphosphonium Bromide (dissolved in culture medium), in the presence and absence of S9-mix according to OECD/ EC guidelines and GLP principles. In the first cytogenetic assay, Tetrabutylphosphonium Bromide was tested up to and including 1600 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. Appropriate toxicity was reached at this dose level. In the second cytogenetic assay, Tetrabutylphosphonium Bromide was tested up to and including 100 μg/mL for a 24 h continuous exposure time with a 24 h fixation time and up to and including 75 μg/mL for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels. The mean number of cells with chromosome aberrations found in the solvent control and positivce control cultures was within the expected response ranges of the historical control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Tetrabutylphosphonium Bromide 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 repeated experiments. No effects of Tetrabutylphosphonium Bromide 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 Tetrabutylphosphonium Bromide does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations in vitro.

 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 2015
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)
Version / remarks:
July 2015
Deviations:
no
GLP compliance:
yes
Type of assay:
other: 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):
CELLS USED
- L5178Y/TK+/--3.7.2C mouse lymphoma cells
- Source: American Type Culture Collection, (ATCC, Manassas, USA) (2001).

Type and identity of media:
- 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 supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- 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 h treatment: 52, 164, 512, 1600 and 2000 µg/mL
Without S9-mix, 24 h treatment: 52, 164, 512, 1600 and 2000 µg/mL

Experiment 1:
with and Without S9-mix, 3 h treatment: 50, 100, 250, 500, 750, 1000, 1250, 1500, 1750 and 2000 µg/mL

Experiment 2
Without S9-mix, 24 h treatment: 5, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400 and 500 µg/mL
Vehicle / solvent:
- Vehicle used: exposure medium

The test item was melted at 120°C (3 h). The melted test item was flushed with nitrogen. In the dose range finding test and first mutation experiment, at concentrations of 25 mg/mL and higher the test item was suspended in dimethyl sulfoxide. At concentrations of 16 mg/mL and lower the test item was dissolved in dimethyl sulfoxide. In the second mutation experiment, the stock solution of 50 mg/ml was fully soluble. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension or had completely dissolved.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 15 µg/mL for the 3 h treatment period and 5 µg/mL for the 24 h treatment period
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 7.5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Dose range finding study: 3 h in the presence of S9-mix and for 3 h and 24 h in the absence of S9-mix
Mutagenicity test 1: 3 h in the presence and absence of S9-mix
Mutagenicity test 2: 24 h in the absence of S9-mix

- 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)
Rationale for test conditions:
The test item did not precipitate in the exposure medium up to and including the concentration of 2000 μg/mL. Therefore, this concentration was used as the highest test item concentration.
Dose range used in experiment 1 was based on the results of the dose range finding test. Dose range used in experiment 2 was based on the dose range finding test and experiment 1.
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% in order to have an acceptable number of surviving cells analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10E6 survivors and ≤ 170 per 10E6 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 h treatment) and 32-180 (24 h 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 10E-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 10E-6 above that seen in the concurrent solvent/control (a small colony IMF of at least 150 x 10E-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 item 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 item 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 item is considered negative (not mutagenic) in the mutation assay if none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
Statistics:
The global evaluation factor (GEF) has been defined by the IWTGP as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
No significant increase in the mutation frequency at the TK locus was observed after treatment with the test item either in the absence or in the presence of S9-mix.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: The test item did not precipitate in the exposure medium up to and including the concentration of 2000 μg/mL.

RANGE-FINDING/SCREENING STUDIES:
- In the absence of S9-mix, the relative suspension growth: 9% at 2000 μg/mL
- In the presence of S9-mix, the relative suspension growth: 8% at 2000 μg/mL
- The relative suspension growth was 3% at 512 μg/mL. No cell survival was observed at 1600 μg/mL and upwards.

MUTATION EXPERIMENT 1:
Evaluation of toxicity
In the absence of S9-mix, the dose levels of 50 to 250 μg/mL showed no cytotoxicity. Therefore, the dose levels of 50 and 250 μg/mL were not regarded relevant for mutation frequency measurement.
In the presence of S9-mix, the dose level of 2000 μg/mL was not used for mutation frequency measurement, since this dose level was too toxic for further testing. The dose levels of 50 and 100 μg/mL showed similar no cytotoxicity. Therefore, the dose level of 50 μg/ml was not regarded relevant for mutation frequency measurement.
The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 100, 500, 750, 1000, 1250, 1500, 1750 and 2000 μg/mL exposure medium.
With S9-mix: 100, 250, 100, 500, 750, 1000, 1250, 1500 and 1750 μg/mL exposure medium.

- In the absence of S9-mix, the relative total growth: 10% at 2000 μg/mL
- In the presence of S9-mix, the relative total growth: 12% at 1750 μg/mL

Evaluation of the mutagenicity
No significant increase in the mutation frequency at the TK locus was observed after treatment with the test item either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

MUTATION EXPERIMENT 2:
Evaluation of toxicity
The dose levels of 250 to 500 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.
The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 5, 10, 25, 50, 100, 150 and 200 μg/mL exposure medium.

- The relative total growth: 19% at 200 μg/mL

Evaluation of mutagenicity
No significant increase in the mutation frequency at the TK locus was observed after treatment with the test item. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

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.
Conclusions:
The mouse lymphoma assay was conducted according to OECD 490 guideline and GLP principles. It was demonstrated that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.Tetrabutylphosphonium Bromide did not induce a significant increase in the mutation frequency, either in the absence or presence of S9. Based on these results, it was concluded that Tetrabutylphosphonium Bromide is not mutagenic under the experimental conditions described in this report.

Executive summary:

The mouse lymphoma assay was conducted with Tetrabutylphosphonium Bromide according to OECD 490 guideline and in accordance with GLP principles. The test was performed in the absence of S9-mix with 3 h and 24 h treatment periods and in the presence of S9-mix with a 3 h treatment period.

In the first experiment, the test item was tested up to and including concentrations of 2000 and 1750 μg/mL in the absence and presence of S9-mix, respectively. The incubation time was 3 h. Relative total growth (RTG) was 10 and 12% in the absence and presence of S9-mix, respectively.

In the second experiment, the test item was tested up to and including concentrations of 200 μg/mL in the absence of S9-mix. The incubation time was 24 h. The RTG was 19%.

The mutation frequency found in the solvent control and positive control cultures was within the acceptability criteria of this assay and it was concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, Tetrabutylphosphonium Bromide did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, Tetrabutylphosphonium Bromide did not induce a significant increase in the mutation frequency.

It is concluded that Tetrabutylphosphonium Bromide 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

Ames test:

An Ames test was performed according to the OECD guideline 471 and GLP principles. All bacterial strains showed negative responses up to and including 5000 ug/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. No cytotoxicity and/or precipitation of the test substance was observed in the direct-plate experiment. In the pre-incubation assay, toxicity was observed in all tester strains in the absence of S9-mix and in tester strain TA100 in the presence of S9-mix at the two highest concentrations tested. The negative and 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.

Chromosome aberration test:

In a chromosome aberration study, to test the ability to induce structural chromosome aberrations in cultured human lymphocytes, cultured peripheral human lymphocytes were exposed to different concentrations of Tetrabutylphosphonium Bromide (dissolved in culture medium), in the presence and absence of S9-mix according to OECD/ EC guidelines and GLP principles. In the first cytogenetic assay, Tetrabutylphosphonium Bromide was tested up to and including 1600 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. Appropriate toxicity was reached at this dose level. In the second cytogenetic assay, Tetrabutylphosphonium Bromide was tested up to and including 100 μg/mL for a 24 h continuous exposure time with a 24 h fixation time and up to and including 75 μg/mL for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels. The mean number of cells with chromosome aberrations found in the solvent control and positivce control cultures was within the expected response ranges of the historical control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. Tetrabutylphosphonium Bromide 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 repeated experiments. No effects of Tetrabutylphosphonium Bromide 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 Tetrabutylphosphonium Bromide does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations in vitro.

Mouse lymphoma assay:

The mouse lymphoma assay was conducted with Tetrabutylphosphonium Bromide according to OECD 490 guideline and in accordance with GLP principles. The test was performed in the absence of S9-mix with 3 h and 24 h treatment periods and in the presence of S9-mix with a 3 h treatment period. In the first experiment, the test item was tested up to and including concentrations of 2000 and 1750 μg/mL in the absence and presence of S9-mix, respectively. The incubation time was 3 h. Relative total growth (RTG) was 10 and 12% in the absence and presence of S9-mix, respectively. In the second experiment, the test item was tested up to and including concentrations of 200 μg/mL in the absence of S9-mix. The incubation time was 24 h. The RTG was 19%. The mutation frequency found in the solvent control and positive control cultures was within the acceptability criteria of this assay and it was concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. In the absence of S9-mix, Tetrabutylphosphonium Bromide did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, Tetrabutylphosphonium Bromide did not induce a significant increase in the mutation frequency.

Justification for classification or non-classification

Based on the available data, Tetrabutylphosphonium Bromide is not classified for genotoxicity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) (including all amendments) and CLP Regulation EC (No.) 1272/2008.