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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A negative bacterial reverse mutation test (OECD 471), a negative in vitro mammalian chromosome aberration (OECD 473) and a negative in vitro Mammalian Cell Gene Mutation Test (OECD 490), all performed in accordance with GLP principles, are available.

Link to relevant study records

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 September 2018 - 20 September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The substance can be produced through different process routes, yielding solutions in water or organic solvent (e.g. methanol or ethylene glycol).
It was initially unclear how to register the substance (mono-constituent or multi-constituent substance) and which manufactured substance to test to fulfil the REACH data requirements. After consultation with the ECHA helpdesk, the test program was started with the manufactured substance of the Lead registrant (solvent: methanol) in which the highest amount of solvent could be removed without causing degradation of the substance. This resulted in the selection of a solution of 56-57% BTMAOH in methanol as test substance.
During the course of the test program, and in order to aid meaningful risk assessment, after consultation with ECHA and upon ECHA's recommendation, it was considered to be more appropriate to test the water-based manufactured substance. As a consequence, some testing was performed with a BTMAOH solution in methanol, and some testing was performed with a BTMAOH solution in water.

The current entry reflects a test performed with a water-based test solution.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 May 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:
Type and composition of metabolic activation system:
- source of S9 : rat liver S9-mix induced by Aroclor 1254 (500 mg/kg bw)
- method of preparation of S9 mix: S9-mix per 10 mL: 30 mg NADP, 15.2 mg glucose-6-phosphate in 5.5 mL Milli-Q water; 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution; 1 mL 0.33 M KCl solution. The solution was filter (0.22 μm)-sterilized and 0.5 mL S9-fraction was added
- concentration of S9 in S9 mix: 5%
- quality controls of S9: Each S9 batch was characterized with the mutagens benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively
Test concentrations with justification for top dose:

Justification for top dose: 5 mg is the recommended maximum test concentration according to the guideline.

Direct plate assay
Dose-range finding test (without and with S9; tester strains TA100 and WP2uvrA): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate (reported as part of experiment 1)
First experiment (without and with S9; tester strains TA1535, TA1537 and TA98): 52, 164, 512, 1600, 5000 μg/plate

Pre-incubation assay
Second experiment (without and with S9, tester strains WP2uvrA, TA1535, TA1537, TA 98 and TA100): 52, 164, 512, 1600, 5000 μg/plate
Vehicle / solvent:
- Vehicle used: Milli-Q water

Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene; ICR-191
Remarks:
For details on positive control substances, see Table 1 and Table 2
Details on test system and experimental conditions:
Two individual experiments were performed. The dose range-finding study with tester strains TA100 and WP2uvrA was reported as part of the first experiment. The first experiment was a direct plate assay. The second experiment was a pre-incubation assay and was performed to obtain more information about the possible mutagenicity of the test item.

METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS:
- Doses levels were tested in triplicate in each strain.

METHODS: The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (1E9 cells/mL) of one of the tester strains, 0.1 ml of a dilution of the test item in Milli-Q water
and either 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
- Preincubation period (second experiment): 30 ± 2 minutes at 70 rpm at 37 ± 1°C
- Exposure duration: 48 ± 4 h (in the dark at 37.0 ± 1.0 °C)

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: precipitation of the test item was recorded

COLONY COUNTING
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

ACCEPTABILITY CRITERIA
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 Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
Evaluation criteria:
A test 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 times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three 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 times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three 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.

Any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
First Experiment: Direct Plate Assay
Precipitation:
No precipitation of the test item was observed.

Cytotoxicity:
A moderately or slighty reduced bacterial background lawn was observed in tester strains TA1537 and TA98 in the absence and presence of S9-mix and in tester strain TA1535 in the absence of S9-mix at the top dose level of 5000 μg/plate.

Mutagenicity:
In the direct plate test, no increase in the number of revertants was observed upon treatment with Benzyltrimethylammonium hydroxide under all conditions tested.

Second Experiment: Pre-Incubation Assay
Precipitation:
Precipitation of test item on the plates was observed at the start and at the end of the incubation period at the concentration of 5000 μg/plate.

Cytotoxicity:
A moderately or slighty reduced bacterial background lawn was observed in all tester strains in the absence and presence of S9-mix at the top dose level of 5000 μg/plate.

Mutagenicity:
In the pre-incubation test, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

Acceptability criteria:
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.
Conclusions:
Based on the results of an Ames test, performed according to OECD guideline 471 and GLP principles, Benzyltrimethylammonium hydroxide is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

A bacterial reverse mutation test was performed according to OECD guideline 471 and GLP principles to determine the potential of Benzyltrimethylammonium hydroxide and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537) and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or

absence of S9 -metabolic activation. Bacterial strains were tested in the absence and presence of S9-metabolic activation in two independent experiments, a direct plate assay and a pre-incubation assay, up to and including the highest recommended concentration (5 mg/plate). 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 (S9 -mix) functioned properly. The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. 

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 April 2018 - 08 August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The substance can be produced through different process routes, yielding solutions in water or organic solvent (e.g. methanol or ethylene glycol).
It was initially unclear how to register the substance (mono-constituent or multi-constituent substance) and which manufactured substance to test to fulfil the REACH data requirements. After consultation with the ECHA helpdesk, the test program was started with the manufactured substance of the Lead registrant (solvent: methanol) in which the highest amount of solvent could be removed without causing degradation of the substance. This resulted in the selection of a solution of 56-57% BTMAOH in methanol as test substance.
During the course of the test program, and in order to aid meaningful risk assessment, after consultation with ECHA and upon ECHA's recommendation, it was considered to be more appropriate to test the water-based manufactured substance. As a consequence, some testing was performed with a BTMAOH solution in methanol, and some testing was performed with a BTMAOH solution in water.

The current entry reflects a test performed with a water-based test solution.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: Peripheral human lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED:
- Source of cells: blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
-Suitability of cells: Cells were collected from healthy adult, non-smoking volunteers (approximately 18 to 35 years of age).
- Average Generation Time (AGT) : the cells and the age of the donor at the time the AGT was determined(December 2017)) are presented below:

Dose-range finding study: age 30, AGT = 14.0 h
First cytogenetic assay: age 30, AGT = 14.8 h
Cytogenetic assay 1A: age 32, AGT = 14.8 h
Second cytogenetic assay: age 35, AGT = 13.7 h
Cytogenetic assay 2A: age 26, AGT = 14.2 h

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Culture medium: RPMI 1640 medium supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively) and 30 U/mL heparin
Lymphocyte culture: 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.
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : Rat liver from adult male Wistar rats
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained 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. Solution was filter (0.22 μm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
- concentration or volume of S9 mix and S9 in the final culture medium: The concentration of the S9-fraction in exposure medium was 1.8% (v/v).
Test concentrations with justification for top dose:
Top dose was selected according to guidelines.

Dose-range finding study
3 h exposure, 24 h fixation, with and without S9: 62.5, 125, 250, 500, 1000, and 2000 μg test item/mL
24 h exposure, 24 h fixation, without S9: 62.5, 125, 250, 500, 1000, and 2000 μg test item/mL
48 h exposure, 48 h fixation, without S9: 62.5, 125, 250, 500, 1000, and 2000 μg test item/mL

- First cytogenetic assay:
3 h exposure, 24 h fixation, with and without S9: 500, 1000, and 2000 μg test item/mL
- Cytogenetic assay 1A:
3 h exposure, 24 h fixation, without S9: 500, 1000, and 2000 μg test item/mL

- Second cytogenetic assay:
24 h exposure, 24 h fixation, without S9: 125, 250, 500, 1000, and 2000 μg test item/mL
48 h exposure, 48 h fixation, without S9: 125, 250, 500, 1000, and 2000 μg test item/mL
- Cytogenetic assay 2A:
24 h exposure, 24 h fixation, without S9: 125, 250, 500, 750, 1000, 1250, 1500, 1750, and 2000 μg test item/mL
Vehicle / solvent:
- Vehicle used: Culture medium
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9
Details on test system and experimental conditions:
Two independent cytogenetic assays were performed, preceeded by a dose-range finding assay.

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration experiment 1: 3 h (with and without S9)
- Exposure duration experiment 2: 24, 48 h (without S9)
- Fixation time: 24 h (for 3 or 24 hour exposure period) and 48 h (for 48 hour exposure period)

RINSING AFTER EXPOSURE
3 hour exposure: yes, with Hanks' Balanced Salt Solution
24 hour exposure: no
48 hour exposure: no

ENVIRONMENTAL CONDITIONS:
- Humidity: 55 - 88%
- Temperature: 34.6 - 37.2 °C

SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.5 μg/mL medium)

STAIN (for cytogenetic assays): Giemsa (5% v/v)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated coverslipper.

NUMBER OF REPLICATIONS: duplicates in two independent experiments, several repeat assays were performed for scoring.

NUMBER OF CELLS EVALUATED: 1000

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 150 in each replicate.
- In case the number of aberrant cells, gaps excluded, was ≥ 38 in 75 metaphases, no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analyzed.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

DOSE SELECTION FOR SCORING:
- Chromosomes of metaphase spreads were analyzed from those cultures with an inhibition of the mitotic index of 55 ± 5%, whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control. Also cultures treated with an intermediate dose were examined for chromosome aberrations.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreduplication: yes
- The pH and the osmolarity of the culture medium containing the highest tested concentration were recorded.
Evaluation criteria:
ACCEPTABILITY CRITERIA
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 analyzed by the Fisher’s exact test (one-sided, p < 0.05).

EVALUATION CRITERIA
A test item is considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

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

Since, the Fisher’s exact test showed that there are statistically significant differences between one or more 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.
Key result
Species / strain:
lymphocytes: Peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
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
- Effects of pH: No
- Effects of osmolality: No

DOSE LEVELS SELECTED FOR SCORING:
First cytogenetic assay:
- With S9-mix : 500, 1000 and 2000 μg/mL (3 h exposure time, 24 h fixation time).
Cytogenetic assay 1A:
- Without S9-mix : 500, 1000 and 2000 μg/mL (3 h exposure time, 24 h fixation time).
Second cytogenetic assay:
- 125, 1000 and 2000 μg/mL (24 h exposure time, 24 h fixation time).
- 125, 250 and 1000 μg/mL (48 h exposure time, 48 h fixation time).
Cytogenetic assay 2A:
- 125, 500 and 1000 μg/mL (24 h exposure time, 24 h fixation time).

FIRST CYTOGENETIC ASSAY
- Both in the absence and presence of S9-mix, Benzyltrimethylammonium hydroxide did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.
- The test item did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.

SECOND CYTOGENETIC ASSAY
- For the 24 hours and 48 exposure time the highest dose levels selected for scoring were slightly too toxic (MI of 64% and 61%).
- At the 24 h continuous exposure time Benzyltrimethylammonium hydroxide induced a statistically significant increase in the number of cells with chromosome aberrations at the highest tested concentration only. Since, the toxicity at this dose level was slightly too high (64%) the increase was not biologically relevant and the experiment was repeated in cytogenetic assay 2A.
- At the 24 h continuous exposure time in cytogenetic assay 2A Benzyltrimethylammonium hydroxide induced a statistically significant increase in the number of cells with chromosome aberrations at the highest tested concentration only. Since the Cochran Armitage trend test was negative (p = 0.057) the amount of aberration was within the historical control limits and the type of aberrations observed were mostly only breaks and gaps, the increase was considered not biologically relevant.
- The test item did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.


COMPARISON WITH HISTORICAL CONTROL DATA AND VALIDITY:
The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database (see Appendix 4). The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Conclusions:
A chromosome aberration study with Benzyltrimethylammonium hydroxide was performed according to OECD guideline 473 and GLP principles. Based on the results of two independent experiments in cultured peripheral human lymphocytes, it was concluded that Benzyltrimethylammonium hydroxide is not clastogenic in human lymphocytes under these experimental conditions.
Executive summary:

An in vitro mammalian chromosome aberration test was done according to OECD guideline 473 and GLP principles.The objective of this study was to evaluate Benzyltrimethylammonium hydroxide for its ability to induce structural chromosome aberrations in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix). The possible clastogenicity of the test item was tested in two independent experiments. The vehicle of the test item was culture medium. In the first and second cytogenetic assay the test item was tested up to and including 2000 µg/mL in the absence and presence of 1.8% (v/v) S9-fraction. Based on the results of appropriate positive and vehicle controls, it was concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. Both in the absence and presence of S9-mix Benzyltrimethylammonium hydroxide did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments, except in the absence of S9-mix at the 24 h treatment time with a 24 h fixation time where a statistically significant increase in the number of cells with chromosome aberrations was observed. Since the Cochran Armitage trend test was negative (p = 0.057), the amount of aberrations was within the historical control limits and the type of aberrations observed were mostly only breaks and gaps, the increase was considered not biologically relevant. No biologically relevant effects of the test item 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 Benzyltrimethylammonium hydroxide is not clastogenic in human lymphocytes.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 September 2018 - 03 October 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The substance can be produced through different process routes, yielding solutions in water or organic solvent (e.g. methanol or ethylene glycol).
It was initially unclear how to register the substance (mono-constituent or multi-constituent substance) and which manufactured substance to test to fulfil the REACH data requirements. After consultation with the ECHA helpdesk, the test program was started with the manufactured substance of the Lead registrant (solvent: methanol) in which the highest amount of solvent could be removed without causing degradation of the substance. This resulted in the selection of a solution of 56-57% BTMAOH in methanol as test substance.
During the course of the test program, and in order to aid meaningful risk assessment, after consultation with ECHA and upon ECHA's recommendation, it was considered to be more appropriate to test the water-based manufactured substance. As a consequence, some testing was performed with a BTMAOH solution in methanol, and some testing was performed with a BTMAOH solution in water.

The current entry reflects a test performed with a water-based test solution.
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
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):
- Source of cells: L5178Y/TK+/- -3.7.2C mouse lymphoma cells from American Type Culture Collection, (ATCC, Manassas, USA), (2001)
- Suitability of cells: recommended test system in international guidelines

MEDIA USED
- Type and identity of media
Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin
Growth medium: basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
Exposure medium: 3 hr exposure: basic medium, supplemented with 5% (v/v) heat-inactivated horse serum (=R5 medium); 24 hr 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 (total amount of serum = 20%, R20-medium) and 5 μg/mL trifluorothymidine
Non-selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium)
- Properly maintained: yes, stock cultures of the cells were stored in liquid nitrogen (-196°C).
- Periodically checked for Mycoplasma contamination: yes
- Cleansed against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : Rat liver S9-mix induced by a combination of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight).
- method of preparation of S9 mix: 1.63 mg MgCl2.6H2O; 2.46 mg KCl; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 µmol HEPES. The solution was filter (0.22 µm)-sterilized. To 0.5 mL S9-mix components 0.5 mL
S9-fraction.
- concentration of S9 mix in medium : The concentration of the S9-fraction in the exposure medium was 4% (v/v).
Test concentrations with justification for top dose:
Justification for top dose: 2000 µg/mL was tested due to error instead of the recommend top dose of 0.01 M (1673 µg/mL),

Dose-range finding test (with and without S9-mix, 3 hour treatment; without S9-mix, 24 hour treatment): 63, 125, 250, 500, 1000, and 2000 μg/mL

Experiment 1
3 hour treatment (without S9): 16, 31, 63, 125, 250, 500, 1000, and 2000 μg/mL
3 hour treatment (with S9): 16, 31, 63, 125, 250, 500, 1000, and 2000 μg/mL

Experiment 2
24 hour treatment (without S9): 6, 13, 25, 50, 100, 200, 500, and 750 µg/mL
Vehicle / solvent:
- Vehicle used:
Dose range finding test: RPMI 1640 (exposure medium (R5) Hepes buffered medium)
Experiment 1 and 2: Milli-Q water

Author comment: the vehicle dose range finding test was a accidental deviation of the study protocol.

Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without S9; in DMSO, 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9; 7.5 µg/mL in Hanks’ balanced salt solution without calcium and magnesium.
Details on test system and experimental conditions:
ENVIROMENTAL CONDITIONS
Test item concentrations were used within 3 hours after preparation.
All incubations were carried out in a humid atmosphere (80 - 100%, actual range 35.9 - 37.5%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 68 - 99 °C).

METHOD OF APPLICATION: in medium
- Cell density at seeding: below 1 x 10^6 cells/mL

DURATION
- Cleansing period: Prior to dose-range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10-medium containing 10^-4 M hypoxanthine, 2 x 10^-7 M aminopterine and 1.6 x 10^-5 M thymidine (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10-medium containing hypoxanthine and thymidine only. After this period cells were returned to R10-medium for at least 1 day before starting the experiment.
- Exposure duration: 3 hours (experiment 1; with and without S9-mix); 24 hours (experiment 2; without S9-mix).
- Expression time: 2 days in which at least 4 x 10^6 cells were subcultured every day
- Selection time (if incubation with a selection agent): 11 or 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)
STAIN: 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)

NUMBER OF REPLICATIONS:
- test concentrations: 1
- positive control: 1
- solvent control: 2

DETERMINATION OF THE MUTATION FREQUENCY
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.
For determination of the mutation frequency (MF) a total number of 9.6 x 105 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.

OTHER EXAMINATIONS
The pH and the osmolarity were measured in Charles River Laboratories Study No. 20137532.
Evaluation criteria:
DATA EVALUATION
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 micro well version of the assay the GEF is 126.

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 there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF.

In addition to this criteria, any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No, see CRL20137532
- Effects of osmolarity: No, see CRL20137532

Dose-range Finding Test
After 3 hours of treatment with the test item both in the absence and presence of S9-mix, no toxicity in the relative suspension growth (RSG) was observed up to and including the highest test item concentration of 2000 μg/mL.
After 24 hours of treatment with the test item the RSG was 2% at the test item concentration of 2000 μg/mL compared to the RSG of the solvent control.

Experiment 1
No precipitation was observed.
All dose levels were selected to measure mutation frequencies at the TK-locus.
The relative total growth of the highest test item concentration was 70% and 38% compared to the total growth of the solvent controls in the absence and presence of S9-mix, respectively.

In the absence and presence of S9-mix, Benzyltrimethylammonium hydroxide did not induce a biologically relevant increase in the mutation frequency.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.

Experiment 2
No precipitation was observed.
The dose levels selected to measure mutation frequencies at the TK-locus were: 6, 13, 25, 50, 100, 200, 500 and 750 µg/mL.
The relative total growth of the highest measured test item concentration was 13% compared to the total growth of the solvent controls in the absence of S9-mix.
Dose levels of 1000 and 1500 μg/mL were to cytotoxic to asses for mutation frequency (RSG of 9% and 4% weew calculated, respectively).

In the absence of S9-mix, Benzyltrimethylammonium hydroxide did not induce a biologically relevant increase in the mutation frequency. 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.

Acceptability criteria:
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. The suspension growth over the two-day expression period for cultures treated with Milli-Q water was between 18 and 19 (3 hour treatment) and 108 and 122 (24 hour treatment)

See Illustration for detailed experimental results and historical control data.
Conclusions:
An in vitro mammalian cell gene mutation test was performed according to OECD guideline 490 and GLP principles with Benzyltrimethylammonium hydroxide. Based on the results, Benzyltrimethylammonium hydroxide is not mutagenic with and without metabolic activation in the TK mutation test system under the experimental conditions described in this report.
Executive summary:

An in vitro Mammalian Cell Gene Mutation Test using the Thymidine Kinase Gene was performed according to OECD guideline 490 and GLP principles in order to evaluate the mutagenic potential of Benzyltrimethylammonium hydroxide. The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay. In addition, positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in mutation frequency demonstrating that the test conditions were adequate and that the metabolic activation system (S9 -mix) functioned properly.

Benzyltrimethylammonium hydroxide was tested up to the recommended concentration of 2000 µg/mL in the absence and presence of S9 -mix with a 3 hour treatment period in a first experiment and tested in the absence of S9-mix for a 24 hour treatment period up to 750 µg/mL, as higher concentrations were to toxic to assess, in a second experiment. Benzyltrimethylammonium hydroxide did not induce a biologically relevant increase in the mutation frequency nor a change in numbers of small and large colonies compared to solvent control cultures in both experiments. Therefore it is concluded that Benzyltrimethylammonium hydroxide is not mutagenic in the mouse lymphoma L5178Y test system.

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

Additional information

A bacterial reverse mutation test was performed according to OECD guideline 471 and GLP principlesto determine the potential of Benzyltrimethylammonium hydroxide and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537) and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of S9 -metabolic activation. Bacterial strains were testedin the absence and presence ofS9-metabolic activationintwo independent experiments, a direct plate assay and a pre-incubation assay, up to and including the highest recommended concentration (5 mg/plate).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 (S9 -mix) functioned properly. The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation.Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

An in vitro mammalian chromosome aberration test was done according to OECD guideline 473 and GLP principles.The objective of this study was to evaluate Benzyltrimethylammonium hydroxide for its ability to induce structural chromosome aberrations in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix). The possible clastogenicity of the test item was tested in two independent experiments. The vehicle of the test item was culture medium. In the first and second cytogenetic assay the test item was tested up to and including 2000 µg/mL in the absence and presence of 1.8% (v/v) S9-fraction. Based on the results of appropriate positive and vehicle controls, it was concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. Both in the absence and presence of S9-mix Benzyltrimethylammonium hydroxide did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments, except in the absence of S9-mix at the 24 h treatment time with a 24 h fixation time where a statistically significant increase in the number of cells with chromosome aberrations was observed. Since the Cochran Armitage trend test was negative (p = 0.057), the amount of aberrations was within the historical control limits and the type of aberrations observed were mostly only breaks and gaps, the increase was considered not biologically relevant. No biologically relevant effects of the test item 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 Benzyltrimethylammonium hydroxide is not clastogenic in human lymphocytes.

An in vitro Mammalian Cell Gene Mutation Test using the Thymidine Kinase Gene was performed according to OECD guideline 490 and GLP principles in order to evaluate the mutagenic potential of Benzyltrimethylammonium hydroxide. Benzyltrimethylammonium hydroxide was tested up to the recommended concentration of 2000 µg/mL in the absence and presence of S9 -mix with a 3 hour treatment period in a first experiment and tested in the absence of S9-mixfor a 24 hour treatment periodupto 750 µg/mL, as higher concentrations were to toxic to assess,in a second experiment. Benzyltrimethylammonium hydroxide did not induce a biologically relevant increase in the mutation frequency nor a change in numbers of small and large colonies compared to solvent control cultures in both experiments.

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay. In addition, positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in mutation frequency demonstrating that the test conditions were adequate and that the metabolic activation system (S9 -mix) functioned properly.

Therefore it is concluded that Benzyltrimethylammonium hydroxide is not mutagenic in the mouse lymphoma L5178Y test system.

Justification for classification or non-classification

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