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Genetic toxicity in vitro

Description of key information

No data for MTBAC is available that address mutagenicity. The results of the following studies are read across to MTBAC. The rationale to read across these data to MTBAC is attached in IUCLID section 13.

 

Two AMES tests with tributylmethylammoniummethylsulfate and TMAC, a chromosome aberration test with TMAH and a mouse lymphoma assay with TMAH. The outcome of all studies is negative.

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
For Read Across Justification please refer to Section 13.
Reason / purpose:
read-across source
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see "additional information on results"
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see "additional information on results"
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see "additional information on results"
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see "additional information on results"
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see "additional information on results"
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TOXICITY
A slight decrease in the number of revertants was occasionally observed in the standard plate test.In the preincubation assay bacteriotoxicity (reduced background growth, decrease in the number of revertants) was observed using the Salmonella strains depending on the test conditions from about 2,500 μg/plate onward.
Conclusions:
A bacterial mutagenicity test was performed with tributylmethylammoniummethylsulfate according to OECD TG 471 and 472 and in comliance with GLP. There was no increase in the number of revertants in any of the S. typhimurium or E. coli tester strains at any dose, neither in the standard plate test nor in the preincubation test, with or without metabolic activation. These negative results were read-across to MTBAC.
Executive summary:

In a GLP compliant bacterial mutagenicity test, performed according to OECD TG 471 and 472, 4Salmonella typhimuriumstrains (TA 1535, TA 1537, TA 98, and TA 100) andE. colistrain WP2 uvr A were used to test the mutagenic potential of tributylmethylammoniummethylsulfate both with and without metabolic activation. One standard plate test and one preincubation test were performed at test concentrations of 0; 20; 100; 500; 2500 and 5000 µg/plate. Three plates per experiment per concentration or control were used. No precipitation of the test substance was found. A slight decrease in the number of revertants was occasionally observed in the standard plate test. In the preincubation assay bacteriotoxicity (reduced background growth, decrease in the number of revertants) was observed using the Salmonella strains depending on the test conditions from about 2500 µg/plate onward. There was no increase in the number of revertants in any of the S. typhimurium or E. coli tester strains at any dose, neither in the standard plate test nor in the preincubation test, with or without metabolic activation. It was therefore concluded that the test substance is not mutagenic under these experimental conditions. These results were read-across to MTBAC.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
The rationale to read across the data is attached in section 13.
Reason / purpose:
read-across source
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
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.
Conclusions:
In an AMES test, performed according to OECD guideline and GLP principles, TMAC was found not to be mutagenic with or without metabolic activation. This result is read across to MTBAC.
Executive summary:

An AMES test was performed with TMAC according to OECD guideline and GLP principles. All bacterial strains showed negative responses up to 5000 ug/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. 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 TMAC is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with and without metabolic activation. This result is read across to MTBAC.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: AMTBC14001
- Expiration date of the lot/batch: August 22, 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: The stability of the test item under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.
Target gene:
N/A
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (minimal essential medium with Earle's salts) containing a L-glutamine source
supplemented with
10% (v/v) fetal calf serum (FCS)
1% (v/v) penicillin/streptomycin (10 000 IU / 10 000 μg/mL)
1% (v/v) amphotericine B (250 μg/mL)

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (phenobarbital and beta-naphthoflavone induced rat liver)
Test concentrations with justification for top dose:
1st Experiment
4 hours exposure, 24 hours harvest time, without S9 mix
0; 75.0; 150.0; 300.0; 600.0; 1200.0; 2400.0 μg/mL
4 hours exposure, 24 hours harvest time, with S9 mix
0; 75.0; 150.0; 300.0; 600.0; 1200.0; 2400.0 μg/mL
2nd Experiment
24 hours exposure, 24 hours harvest time, without S9 mix
0; 150.0; 300.0; 600.0; 1200.0; 2400.0 μg/mL
4 hours exposure, 44 hours harvest time, with S9 mix
0; 150.0; 300.0; 600.0; 1200.0; 2400.0 μg/mL
Vehicle / solvent:
- Vehicle used: culture medium (MEM)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 20 - 24 h
- Exposure duration: 1st experiment: 4 h (±S9); 2nd experiment: 4 h (+S9) and 24 h (-S9)
- Recovery time (cells in growth medium): 1st experiment: 20 h (±S9); 2nd experiment: 40 h (+S9) and 0 h (-S9)
- Fixation time (start of exposure up to harvest of cells): 1st experiment: 24 h (±S9); 2nd experiment: 24 h (-S9) and 44 h (+S9)

STAIN (for cytogenetic assays): a mixture of 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI; stock: 5 mg/mL; Sigma-Aldrich, Cat.No. D9542) and propidium iodide (stock: 5 mg/mL; Sigma-Aldrich, Cat.No. P4170) in Fluoroshield™ (Sigma-Aldrich, Cat.No. F6182) at a concentration of 0.25 μg/mL each

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group.

DETERMINATION OF CYTOTOXICITY
- Method: other: Proliferation index cytostasis (CBPI)

OTHER:
- pH: Changes in the pH were recorded by a change in the color of the indicator in the culture medium (phenol red: no color change from pH 6.7 - 8.3). The pH was measured for the top concentration and for the negative control with and without S9 mix.
- Osmolarity: Osmolarity was measured for the top concentration and for the negative control with and without S9 mix.
- Solubility: Test substance precipitation was checked immediately after start of treatment of the test cultures (macroscopically) and at the end of treatment (macroscopically / microscopically).
Evaluation criteria:
Acceptance criteria
The in vitro micronucleus assay is considered valid if the following criteria are met:
• The quality of the slides allowed the evaluation of a sufficient number of analyzable cells both in the control groups (vehicle/positive) and in at least three exposed test groups.
• Sufficient cell proliferation was demonstrated in the vehicle control.
• The number of cells containing micronuclei in the vehicle control was within the range of our laboratory’s historical negative control data (95% control limit). Weak outliers can be judged acceptable if there is no evidence that the test system is not “under control”.
• The positive control substances both with and without S9 mix induced a distinct, statistically significant increase in the number of micronucleated cells in the expected range).

Assessment criteria
A test substance is considered to be clearly positive if the following criteria are met:
• A statistically significant increase in the number of micronucleated cells was obtained.
• A dose-related increase in the number of cells containing micronuclei was observed.
• The number of micronucleated cells exceeded both the value of the concurrent vehicle control and the range of our laboratory’s historical negative control data (95% control limit. A test substance is considered to be clearly negative if the following criterion is met:
• Neither a statistically significant nor dose-related increase in the number of cells containing micronuclei was observed under any experimental condition.
• The number of micronucleated cells in all treated test groups was close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit).
Statistics:
The statistical evaluation of the data was carried out using an appropriate statistical analysis. The proportion of cells containing micronuclei was calculated for each test group. A comparison of the micronucleus rates of each test group with the concurrent vehicle control group was carried out for the hypothesis of equal proportions (i.e. one-sided Fisher's exact test, BASF SE).
If the results of this test were statistically significant compared with the respective vehicle control, labels (* p ≤ 0.05, ** p ≤ 0.01 or S for both categories) have been printed in the tables.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
MICRONUCLEUS ANALYSIS

In this study, no biologically relevant increase in the number of micronucleated cells was observed either without S9 mix or after the addition of a metabolizing system.
In both experiments in the absence and presence of metabolic activation after 4 and 24 hours treatment with the test substance the values (0.2 – 0.6% micronucleated cells) were within the range of the concurrent negative control values (0.2 – 1.0% micronucleated cells) and clearly within the range of the 95% control limit of our historical negative control data (0.0 - 1.0% micronucleated cells). Besides, in the 1st Experiment in the absence of S9 mix the positive control group EMS 400 μg/mL did not lead to the expected statistically significant increased micronucleus frequency (0.1% micronucleated cells). Therefore. the second positive control group, EMS
500 μg/mL, was evaluated (2.6% micronucleated cells). The positive control substances EMS (without S9 mix; 400 or 500 μg/mL) and CPP (with S9 mix; 0.5 μg/mL) induced statistically significant increased micronucleus frequencies in both independently performed experiments in at least one positive control group each. In this study,
in the absence and presence of metabolic activation the frequency of micronucleated cells (2.0 – 6.3% micronucleated cells) was clearly above the range of our historical negative control data (0.1 - 1.5% micronucleated cells) and close to our historical positive control data range (2.3 – 13.8% micronucleated cells).

CYTOTOXICITY - RELATIVE POPULATION DOUBLING
In addition, cytotoxicity indicated by reduced RPD of below 50% of control was observed only after 24 hours continuous test substance treatment in the absence of S9 mix at the highest applied test substance concentrations (1200 μg/mL and 2400 μg/mL). These values were calculated based on cell numbers determined at the end of each experiment. However, in the main experiments due to the use of the cytokinesis block method it is a measure of cell proliferation only until addition of CytB to the cultures. But, it also gives an useful information on cell loss due to test substance exposure.

CYTOTOXICITY - PROLIFERATION INDEX
In this study, reduced proliferative activity was observed after 24 hours continuous treatment at the highest test group scored for cytogenetic damage (55.1% cytostasis at 600 μg/mL).

CELL MORPHOLOGY
In this study, cell morphology/attachment was not adversely influenced (grade > 2) at any concentration tested for the occurrence of micronuclei. The slides were not scorable for cytogenetic damage due to strong cytotoxicity in the 2nd Experiment in the absence of S9 mix at 1200 μg/mL and above.

TREATMENT CONDITIONS
Osmolality and pH values were not influenced by test substance treatment. No precipitation of the test substance in culture medium was observed.

According to the results of the present in vitro micronucleus assay, the test substance did not lead to a biologically relevant increase in the number of micronucleated cells either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other. The frequencies of micronuclei after test substance treatment were within the range of the concurrent negative control values at both exposure times and clearly within the range of the 95% control limit of our historical negative control data.The number of micronucleated cells in the negative control groups were within our historical negative control data range (95% control limit) and, thus, fulfilled the acceptance criteria of this study. The proficiency of the laboratory to perform the micronucleus test in V79 cells was demonstrated either by the laboratory’s historical control database on vehicle and positive controls or by X-bar chart to identify the variability of the vehicle control data. The increase in the frequencies of micronuclei induced by the positive control substances EMS and CPP clearly demonstrated the sensitivity of the test system and/or the metabolic activity of the S9 mix employed. The values were close to the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: AMTBC14001
- Expiration date of the lot/batch: August 22, 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: The stability of the test item under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.



Target gene:
HPRT
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media:
All media were supplemented with:
- 1% (v/v) penicillin/streptomycin (stock solution: 10000 IU / 10000 μg/mL)
- 1% (v/v) amphotericine B (stock solution: 250 μg/mL)
Culture medium
Ham's F12 medium containing stable glutamine and hypoxanthine (Biochrom; Cat. No. FG 0815) supplemented with 10% (v/v) fetal calf serum (FCS).
Treatment medium (without S9 mix)
Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10% (v/v) FCS.
Treatment medium (with S9 mix)
Ham's F12 medium containing stable glutamine and hypoxanthine.
Pretreatment medium ("HAT" medium)
Ham's F12 medium supplemented with:
- hypoxanthine (13.6 x 10E-3 mg/mL)
- aminopterin (0.18 x 10E-3 mg/mL)
- thymidine (3.88 x 10E-3 mg/mL)
- 10% (v/v) FCS
Selection medium ("TG" medium)
Hypoxanthine-free Ham's F12 medium supplemented with:
- 6-thioguanine (10 μg/mL)
- 1% (v/v) stable glutamine (200 mM)
- 10% (v/v) FCS
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Test concentrations with justification for top dose:
1st Experiment
without S9 mix
0; 156.3; 312.5; 625.0; 1250.0; 2500.0 μg/mL
with S9 mix
0; 156.3; 312.5; 625.0; 1250.0; 2500.0 μg/mL
2nd Experiment
without S9 mix
0; 250.0; 500.0.; 1000.0; 2000.0; 2500.0 μg/mL
with S9 mix
0; 250.0; 500.0.; 1000.0; 2000.0; 2500.0 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: culture medium (Ham's F12)
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, culture medium (Ham's F12) was selected as vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with S9-Mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9-Mix
Details on test system and experimental conditions:
TEST SYSTEM
Cell line and storage
The CHO (Chinese hamster ovary) cell line (1, 2) is a permanent cell line derived from the Chinese hamster and has a
- high proliferation rate (doubling time of about 12 - 16 hours)
- high plating efficiency (about 90%)
- karyotype with a modal number of 20 chromosomes.
Stocks of the CHO cell line (1-mL portions) are maintained at -196°C in liquid nitrogen using 7% (v/v) DMSO in culture medium as a cryoprotectant. Each batch used for mutagenicity testing was checked for mycoplasma contamination.

Culture media
All media were supplemented with:
- 1% (v/v) penicillin/streptomycin (stock solution: 10000 IU / 10000 μg/mL)
- 1% (v/v) amphotericine B (stock solution: 250 μg/mL)
Culture medium
Ham's F12 medium containing stable glutamine and hypoxanthine (Biochrom; Cat. No.FG 0815) supplemented with 10% (v/v) fetal calf serum (FCS).
Treatment medium (without S9 mix)
Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10% (v/v) FCS.
Treatment medium (with S9 mix)
Ham's F12 medium containing stable glutamine and hypoxanthine.
Pretreatment medium ("HAT" medium)
Ham's F12 medium supplemented with:
- hypoxanthine (13.6 x 10E-3 mg/mL)
- aminopterin (0.18 x 10E-3 mg/mL)
- thymidine (3.88 x 10E-3 mg/mL)
- 10% (v/v) FCS
Selection medium ("TG" medium)
Hypoxanthine-free Ham's F12 medium supplemented with:
- 6-thioguanine (10 μg/mL)
- 1% (v/v) stable glutamine (200 mM)
- 10% (v/v) FCS

Cell culture
For cell cultivation, deep-frozen cell suspensions were thawed at 37°C in a water bath, and volumes of 0.5 mL were transferred into 25 cm2 plastic flasks containing about 5 mL Ham's F12 medium including 10% (v/v) FCS. Cells were grown with 5% (v/v) CO2 at 37°C and ≥ 90% relative humidity up to approximate confluence and subcultured twice weekly (routine passage in 75 cm2 plastic flasks). Routine passage (preparation of a single cell suspension) *
- Cell medium was removed and cells were washed with 5 mL PBS or HBSS (both Ca-Mg-free).
- Cells were trypsinized with 2 mL HBSS (Hanks balanced salt solution; Ca-Mg-free) and 2 mL trypsin (0.25% [w/v]) to remove the cells from the bottom of the plastic flasks.
- This reaction was stopped by adding 6 mL culture medium incl. 10% (v/v) FCS.
- Cells were pipetted up and down to separate them and to prepare a homogeneous single cell suspension.
- Cells were counted in a counting chamber or using a cell counter.
- Cell suspensions were diluted with complete culture medium to the desired cell count.

EXOGENOUS METABOLIC ACTIVATION
S9 fraction
The S9 fraction was prepared according to Ames et al. (3) at BASF SE in an AAALAC-approved laboratory in accordance with the German Animal Welfare Act and the effective European Council Directive.
At least 5 male Wistar rats [Crl:WI(Han)] (200 - 300 g; Charles River Laboratories Germany GmbH) received 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally (both supplied by Sigma-Aldrich, 82024 Taufkirchen, Germany) each on three consecutive days. During this time, the animals were housed in polycarbonate cages: central air conditioning with a fixed range of temperature of 20 - 24°C and a fixed relative humidity of 30 - 70%. The day/night rhythm was 12 hours: light from 6 am – 6 pm and darkness from 6 pm – 6 am. Standardized pelleted feed and drinking water from bottles were available ad libitum.

24 hours after the last administration, the rats were sacrificed and the livers were prepared using sterile solvents and glassware at a temperature of +4°C. The livers were weighed and washed in a weight-equivalent volume of a 150 mM KCl solution and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4°C, 5 mL portions of the supernatant (S9 fraction) were stored at -70°C to -80°C.
S9 mix
The S9 mix was prepared freshly prior to each experiment (3). For this purpose, a sufficient amount of S9 fraction was thawed at room temperature; 1 part S9 fraction was mixed with 9 parts S9 supplement (cofactors) in the pre-experiment and both main experiments. This preparation, the S9 mix (10% S9 fraction), was kept on ice until used.
The concentrations of the cofactors in the S9 mix were:
− MgCl2 8 mM
− KCl 33 mM
− glucose-6-phosphate 5 mM
− NADP 4 mM
− phosphate buffer (pH 7.4) 15 mM
The phosphate buffer (4) is prepared by mixing a Na2HPO4 solution with a NaH2PO4 solution in a ratio of about 4:1.

SELECTION OF VEHICLE, TEST GROUPS AND DOSES
Choice of the vehicle
Due to the good solubility of the test substance in water, culture medium (Ham's F12) was selected as vehicle.

Pretest for dose selection
Following the requirements of the current international guidelines and the ICPEMC Task Group (5) a test substance should be tested up to a maximum concentration of 5 mg/mL, 5 μL/mL or 10 mM, whichever is the lowest. In case of toxicity, the top dose should result in approximately 10 - 20% relative survival (relative cloning efficiency), but not less than 10%. For relatively insoluble test substances at least one concentration should be scored showing no precipitation in culture medium at the end of the exposure period. In the pretest for toxicity based on the purity and the molecular weight of the test substance 2400 μg/mL (approx. 10 mM) was used as top concentration both with and without S9 mix at 4 hour exposure time. The pretest was performed following the method described for the main experiment. The cloning efficiency 1 (survival) was determined as a toxicity indicator for dose selection and various parameters were checked for all, or at least some, selected doses. In the pretest the pH value was not influenced by the addition of the test substance preparation to the culture medium at the concentrations measured. In addition, precipitation of the test substance in culture medium was not observed up to the highest required concentration of 2400.0 μg/mL in the absence and presence of S9 mix. After 4 hours treatment in the absence and in the presence of S9 mix cytotoxicity was not observed as indicated by a reduced relative cloning efficiency of about or below 20%.

Test groups and doses
Based on the data and the observations from the pretest and taking into account the current guidelines, the following doses were selected in this study.

1st Experiment
without S9 mix
0; 156.3; 312.5; 625.0; 1250.0; 2500.0 μg/mL
with S9 mix
0; 156.3; 312.5; 625.0; 1250.0; 2500.0 μg/mL
2nd Experiment
without S9 mix
0; 250.0; 500.0.; 1000.0; 2000.0; 2500.0 μg/mL
with S9 mix
0; 250.0; 500.0.; 1000.0; 2000.0; 2500.0 μg/mL

Duplicate cultures (referred to as A and B in the results tables) were used for all experimental groups. At least four concentrations were evaluated to describe a possible dose-response relationship. In general, in this study, the concentrations are given as rounded values by using a dilution factor of 2.

TEST SUBSTANCE PREPARATION
The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. To achieve a solution of the test substance in the vehicle, the test substance preparation was treated with ultrasonic waves thoroughly. The further concentrations were diluted from the stock solution according to the planned doses. All test substance solutions were prepared immediately before administration.

ANALYSIS OF TEST SUBSTANCE PREPARATION
This study was performed in an aqueous test system. Due to the use of culture medium (Ham’s F12) as vehicle the verification of the stability of the test substance in the vehicle was not required.

EXPERIMENTAL PROCEDURE
Controls
Negative control
Negative controls, with and without S9 mix, were treated with culture medium without test substance in parallel to the other treatment groups.

Positive controls
The following positive control substances were used to demonstrate the sensitivity of the test method and/or the activity of the S9 mix:
Without metabolic activation:
400 μg/mL ethyl methanesulfonate (EMS; SIGMA, M-0880)
EMS (stock solution: 4 mg/mL) was dissolved in Ham's F12 medium without FCS and added
in a volume of 2 mL to the cultures.
With metabolic activation:
1.25 μg/mL 7,12-dimethylbenz[a]anthracene (DMBA; SIGMA, D3254)
DMBA was dissolved in DMSO (stock solution: 125.0 μg/mL) and added in a volume of
200.0 μL to the cultures.
The stability of EMS and DMBA is well-defined under the selected culture conditions since both
positive control substances are well-established reference mutagens.
The positive control substances are stored at -80°C and thawed shortly before application. The
preparation of the positive controls is documented in a specific record sheet.

Time schedule
Day 1: Seeding of the cells pretreated with "HAT" medium: in 175 cm² flasks (1x10E6 cells in 20 mL) and in 25 cm² flasks (200 cells in 5 mL)
Day 2: Test substance incubation (20 – 24 hours after seeding); exposure period (4 hours); removal of test substance by intense washing; 1st cytotoxicity determination (cloning efficiency 1: survival)
Day 5: 1st passage of the treated cells
Day 7 - 9: Drying, fixation, staining and counting of the cloning efficiency 1; 2nd passage of the treated cells; addition of selection medium; 2nd cytotoxicity determination (cloning efficiency 2: viability)
From day 16: Drying, fixation, staining and counting of the selected colonies and cloning efficiency 2
In this study, all incubations were performed at 37°C with a relative humidity of ≥ 90% in a 5% (v/v) CO2 atmosphere.

Preparation of test cultures
Cell stocks (1.0-mL portions) stored in liquid nitrogen were thawed at 37°C in a water bath. 0.5 mL of stock cultures were pipetted into 25 cm2 plastic flasks containing 5 mL Ham's F12 medium (incl. 10% [v/v] FCS). After 24 hours, the medium was replaced to remove any dead cells. At least 2 passages were performed before cells were taken for the experiment. A further passage was also necessary in order to prepare test cultures.

Pretreatment of cells with "HAT" medium
During the week prior to treatment, any spontaneous HPRT-deficient mutants were eliminated by pretreatment with "HAT" medium. 3 – 5x10E5 cells were seeded per flask (75 cm²) and incubated with "HAT" medium for 3 - 4 days. A subsequent passage in Ham's F12 medium incl. 10% (v/v) FCS was incubated for a further 3 - 4 days.

Attachment period
For each test group, about 1x10E6 logarithmically growing cells per flask (175 cm²) were seeded into about 20 mL Ham's F12 medium supplemented with 10% (v/v) FCS and incubated for about 20 - 24 hours. Two flasks (one flask referred to as A and one flask referred to as B) were used for each test group.

Exposure period
After the attachment period, the medium was removed from the flasks and the treatment medium was added. The cultures were incubated for the respective exposure
period at 37°C, 5% (v/v) CO2 and ≥ 90% relative humidity.

Expression period
The exposure period was completed by rinsing several times with HBSS. Then the flasks were topped up with at least 20 mL Ham's F12 medium incl. 10% (v/v) FCS and left to stand in the incubator for about 3 days (4-hour treatment). This was followed by the 1st passage. After an entire expression period of 7 – 9 days the cells were transfered into selection medium (2nd passage).

Selection period
For selection of the mutants, six 75 cm2 flasks with 3x10E5 cells each from every treatment group, if possible, were seeded in 10 mL selection medium ("TG" medium) at the end of the expression period. The flasks were returned to the incubator for about 6 - 7 days. At the end of the selection period, the medium was removed and the remaining colonies were fixed with methanol, stained with Giemsa and counted.

Cytotoxicity determination
Cloning efficiency (CE) (pre-experiment)
The procedure for the determination of the cloning efficiency in the pre-experiment was similar to that described for the determination of the cloning efficency 1 (CE1) in the main experiments, excepting that every dose group contained only two cultures.

Cloning efficiency 1 (CE1; survival)
For the determination of the influence of the test substance directly after the exposure period, about 200 cells per dose group were seeded in 25 cm² flasks in duplicate using 5 mL Ham's F12 medium incl. 10% (v/v) FCS. Following cell attachment for 20 – 24 hours, cells were treated with the vehicle, test substance or positive control for 4 hours. Following exposure, cells were rinsed several times with HBSS. Finally, cells were cultured in 5 mL Ham's F12 medium incl.10% (v/v) FCS.
Cloning efficiency 2 (CE2; viability)
For the determination of the mutation rate after the expression period, two aliquots of about 200 cells each were reserved from the transfer into selection medium (after 7 – 9 days) and seeded in two flasks (25 cm2) containing 5 mL Ham's F12 medium incl. 10% (v/v) FCS. In all cases, after seeding the flasks were incubated for 6 - 8 days to form colonies. These colonies were fixed, stained and counted. The absolute and relative cloning efficiencies (%) were calculated for each test group.

Evaluation criteria:
Acceptance criteria
The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the negative/vehicle controls should not be less than
50% (with and without S9 mix).
• The background mutant frequency in the negative/vehicle controls should be within historical negative control data range of 0.00 – 16.43 mutants per 10E6 clonable cells.
• The positive controls both with and without S9 mix have to induce distinctly increased mutant frequencies.
• At least 4 dose levels should be tested ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions. Freely soluble and apparently non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM.

Assessment criteria
A finding is assessed as positive if the following criteria are met:
• Increase in the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range.
• Evidence of the reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a dose response relationship.
Isolated increases of mutant frequencies above our historical negative control range (i.e.15 mutants per 10E6 clonable cells) or isolated statistically significant increases without a dose response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
The test substance is considered non-mutagenic according to the following criterion:
• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significantly increased above the concurrent negative control and is within our historical negative control data range.
Statistics:
An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective negative control groups. A trend is judged as statistically significant whenever the one-sided p-value (probability value) is below 0.05 and the slope is greater than 0. However, both, biological and statistical significance will be considered together.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
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:
MUTANT FREQUENCY
In this study, no relevant increase in the number of mutant colonies was observed with or without S9 mix. In both experiments after 4 hours treatment with the test substance the values for the corrected mutation frequencies (MFcorr.: 0.44 – 7.49 per 10E6 cells) were close to the respective negative control values (MFcorr.: 0.77 – 4.97 per 10E6 cells) and clearly within the range of our historical negative control data (without S9 mix: MFcorr.: 0.00 – 16.43 per 10E6 cells;with S9 mix: MFcorr.: 0.00 – 16.12 per 10E6 cells).
In all experiments, no statistically significant dose-related increase in the mutant frequency was found in cells after 4 hours of treatment either in the absence or presence of S9 mix.
The positive control substances EMS (without S9 mix; 400 μg/mL) and DMBA (with S9 mix; 1.25 μg/mL) induced a clear increase in mutation frequencies, as expected. The values of the corrected mutant frequencies (without S9 mix: MFcorr.: 140.80 – 196.88 per 10E6 cells; with S9 mix: MFcorr.: 305.74 – 362.31 per 10E6 cells) were within our historical positive control data range (without S9 mix: MFcorr.: 47.35 – 383.57 per 10E6 cells; with S9 mix: MFcorr.: 41.99 – 812.14 per 10E6 cells).

CYTOTOXICITY
No cytotoxic effects, as indicated by clearly reduced cloning efficiencies of about or below 20% of the respective negative control values were observed up to the required concentration in both experiments in the presence and in the absence of S9 mix.

CELL MORPHOLOGY
After 4 hours treatment either in the absence or presence of metabolic activation, the cell morphology and attachment of the cells was not adversely influenced (grade > 2) in any test
group tested for gene mutations.

TREATMENT CONDITIONS
Osmolarity and pH values were not influenced by test substance treatment.In this study, in the absence and the presence of S9 mix, no precipitation in culture medium
was observed up to the highest required test substance concentration.
Conclusions:
An in vitro mammalian cell gene mutation test (HPRT locus) was performed in CHO cells according to OECD TG 476 and in compliance with GLP. MTBAC did not cause any relevant increase in mutant frequencies with and without metabolic activation and was considered not mutagenic.
Executive summary:

BTMAC was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro according OECD TG 476. Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week.

Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a] anthracene (DMBA), led to the expected increase in the frequencies of forward mutations. The negative controls gave mutant frequencies within the range expected for the CHO cell line.

In this study in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest required concentration evaluated for gene mutations.

Based on the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies both without S9 mix and after the addition of a metabolizing system in two experiments performed independently of each other.

Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In a GLP compliant bacterial mutagenicity test, performed according to OECD TG 471 and 472, 4 Salmonella typhimurium strains (TA 1535, TA 1537, TA 98, and TA 100) and E. coli strain WP2 uvr A were used to test the mutagenic potential of tributylmethylammoniummethylsulfate both with and without metabolic activation. One standard plate test and one preincubation test were performed at test concentrations of 0; 20; 100; 500; 2500 and 5000 µg/plate. Three plates per experiment per concentration or control were used. No precipitation of the test substance was found. A slight decrease in the number of revertants was occasionally observed in the standard plate test. In the preincubation assay bacteriotoxicity (reduced background growth, decrease in the number of revertants) was observed using the Salmonella strains depending on the test conditions from about 2500 µg/plate onward. There was no increase in the number of revertants in any of the S. typhimurium o rE. coli tester strains at any dose, neither in the standard plate test nor in the preincubation test, with or without metabolic activation. It was therefore concluded that the test substance is not mutagenic under these experimental conditions.

An Ames test was performed with TMAC according to OECD guideline and GLP principles. All bacterial strains showed negative responses up to 5000 µg/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. 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 TMAC is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with and without metabolic activation.

These above results are read across to MTBAC. The rationale to read across these data to MTBAC is attached in IUCLID section 13.

In an in vitro micronucleus assay, MTABC did not lead to a biologically relevant increase in the number of micronucleated cells either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other. The frequencies of micronuclei after test substance treatment were within the range of the concurrent negative control values at both exposure times and clearly within the range of the 95% control limit of our historical negative control data. The number of micronucleated cells in the negative control groups were within our historical negative control data range (95% control limit) and, thus, fulfilled the acceptance criteria of this study. The proficiency of the laboratory to perform the micronucleus test in V79 cells was demonstrated either by the laboratory’s historical control database on vehicle and positive controls or by X-bar chart to identify the variability of the vehicle control data. The increase in the frequencies of micronuclei induced by the positive control substances EMS and CPP clearly demonstrated the sensitivity of the test system and/or the metabolic activity of the S9 mix employed. The values were close to the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study.

BTMAC was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro according OECD TG 476. Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- andβ-naphthoflavone induced rats (exogenous metabolic activation). Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week.

Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a] anthracene (DMBA), led to the expected increase in the frequencies of forward mutations. The negative controls gave mutant frequencies within the range expected for the CHO cell line.

In this study in the absence and the presence of metabolic activation no cytotoxicity was observed up to and including the highest required concentration evaluated for gene mutations. Based on the results, MTBAC did not cause any relevant increase in the mutant frequencies both without S9 mix and after the addition of a metabolizing system in two experiments performed independently of each other and is therefore considered not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Justification for classification or non-classification

Based on the available data, MTBAC is not classified for genotoxicity according to CLP Regulation (EC) No. 1272/2008.