Cetrimonium bromide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The potential of the test item 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 an exogenous mammalian metabolic activation system (S9) was determined in a test performed according to OECD Guideline 471.

 

The possible clastogenicity of the test item was tested in two independent experiments according to OECD Guideline 473. In Vitro Mammalian Chromosome Aberration Test.

 

The mutagenic potential of the test item was determined by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix) in a test performed according to OECD Guideline 490.

Based on the results from the studies, it is concluded that FeF Cetyl Trimethyl Ammonium Bromide (CTAB) USP/NF:

is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay,

is not clastogenic in human lymphocytes under the experimental conditions described in the study,

is not mutagenic in the TK mutation test system under the experimental conditions described in the study.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September-December 2019

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:

adopted 29 July 2016

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

August 1998

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

White powder
Batch (Lot) Number: GX0B433
Expiry date: 31 December 2022
Purity: 100% according to certificate of analysis

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Blood was collected from healthy adult, non-smoking volunteers (approximately 18 to 35 years of age). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2018, December 2019 for donor Cytogenetic assay 2A) are presented below:
Dose-range finding study: age 27, AGT = 12.7 h
First cytogenetic assay: age 32, AGT = 14.0 h
Second cytogenetic assay:age 24, AGT = 12.9 h
Cytogenetic assay 2A: age 29, AGT = 14.6 h

Metabolic activation:

with and without

Metabolic activation system:

Rat S9 homogenate was obtained from Trinova Biochem GmbH, Giessen, Germany and is prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).
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. The 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.
Metabolic activation was achieved by adding 0.2 mL S9-mix to 5.3 mL of a lymphocyte culture (containing 4.8 mL culture medium, 0.4 mL blood and 0.1 mL (9 mg/mL) phytohaemagglutinin). The concentration of the S9-fraction in the exposure medium was 1.8% (v/v).

Test concentrations with justification for top dose:

In the dose-range finding study, blood cultures were treated with 0.49, 0.98, 1.95, 3.9, 7.8 and 15.6 µg test item /mL culture medium with and without S9-mix.

Based on the results of the dose-range finding test the following dose levels were selected for the cytogenetic assay:
With and without S9-mix: 1, 8, 10, 12, 14 and 16 µg/mL culture medium (3 h exposure time, 24 h fixation time).

The following dose levels were selected for the second cytogenetic assay:
Without S9-mix: 0.1, 1, 2, 3, 4, 5 and 6 µg/mL culture medium (24 h exposure time, 24 h fixation time).
0.1, 0.5, 2, 3, 4, 5 and 6 µg/mL culture medium (48 h exposure time, 48 h fixation time).

The experiment was repeated in cytogenetic assay 2A with the following dose levels:
Without S9-mix: 0.1, 2, 4, 5, 6, 7 and 8 µg/mL culture medium (48 h exposure time, 48 h fixation time).

Vehicle / solvent:

The vehicle for the test item was Milli-Q water.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Cytotoxicity of the test item in the lymphocyte cultures was determined using the mitotic index.

Evaluation 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).

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, one-sided, 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.

Statistics:

Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.

Key result

Species / strain:

lymphocytes: Human

Remarks:

Human lymphocytes culture

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity of the test item in the lymphocyte cultures was determined using the mitotic index.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Both in the absence and presence of S9-mix the test item did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments. It was noted that the test item increased the number of polyploid cells both in the absence and presence of S9-mix in a dose dependent manner. This may indicate that the test item has the potential to disturb mitotic processes, which was used as a measure for the cytotoxicity of the test item in the lymphocyte cultures.

Chromosome Aberrations in Human Lymphocyte Cultures Treated with FeF Cetyl Trimethyl Ammonium Bromide (CTAB) USP/NF in the Absence of S9-Mix in the First Cytogenetic Assay (3 H Exposure Time, 24 H Fixation Time) Conc Milli-Q 1 µg/mL 8 µg/mL 10 µg/mL MMC-C 0.5 µg/mL Culture  A    B A+B  A    B A+B  A    B A+B  A    B A+B  A    B A+B Mitotic Index (%) 100 84 66 58 56 No. of Cells scored 150    150 300 150    150 300 150    150 300 150    150 300 150    150 300 No. of Cells with aberrations (+ gaps) a) 1 1 2 2 0 2 0 0 0 0 2 2 39 38 ***^) 77   No. of Cells with aberrations (- gaps) 1 0 1 0 0 0 0 0 0 0 2 2 38 32 ***^) 70   g’       1                   2   g”   1   1                 1 5   b’ 1                   1   12 9   b”                     1   17 13   m’                         3 2   m”                         2 1   exch.                         7 9   dic                               d’                           1   misc.       endo 5 poly 10 poly 13 poly 7 poly     total aberr (+ gaps) 1 1   2 0   0 0   0 2   42 42   total aberr (- gaps) 1 0   0 0   0 0   0 2   41 35   a) Abbreviations used for various types of aberrations. g’ = chromatid gap; g” = chromosome gap; b’= chromatid break; b” = chromosome break; m’= minute; m” = double minutes; exch. = exchange figure; dic. = dicentric chromosome; d’= chromatid deletion; misc. = (miscellaneous) aberrations not belonging to the ones mentioned above; The numerical variations endoreduplication (endo) and polyploidy (poly) were not counted as an aberration. *) Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01 or *** P < 0.001. Chromosome Aberrations in Human Lymphocyte Cultures Treated with FeF Cetyl Trimethyl Ammonium Bromide (CTAB) USP/NF in the Presence of S9-Mix in the First Cytogenetic Assay (3 H Exposure Time, 24 H Fixation Time) Conc Milli-Q 1 µg/mL 8 µg/mL 10 µg/mL CP 10 µg/mL Culture  A    B A+B  A    B A+B  A    B A+B  A    B A+B  A    B A+B Mitotic Index (%) 100 77 71 63 39 No. of Cells scored 150    150 300 150    150 300 150    150 300 150    150 300 150    150 300 No. of Cells with aberrations (+ gaps) a) 0 0 0 1 0 1 1 0 1 0 1 1 26 28 ***^) 54   No. of Cells with aberrations (- gaps) 0 0 0 0 0 0 1 0 1 0 1 1 22 24 ***^) 47   g’                         1 1   g”       1                 6 5   b’                         9 8   b”             1           12 13   m’                         1     m”                     1         exch.                         1 2   dic                           1   d’                               misc.         poly 7 poly   3 poly endo     total aberr (+ gaps) 0 0   1 0   1 0   0 1   30 30   total aberr (- gaps) 0 0   0 0   1 0   0 1   23 24    a) Abbreviations used for various types of aberrations. g’ = chromatid gap; g” = chromosome gap; b’= chromatid break; b” = chromosome break; m’= minute; m” = double minutes; exch. = exchange figure; dic. = dicentric chromosome; d’= chromatid deletion; misc. = (miscellaneous) aberrations not belonging to the ones mentioned above; The numerical variations endoreduplication (endo) and polyploidy (poly) were not counted as an aberration. *) Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01 or *** P < 0.001. Chromosome Aberrations in Human Lymphocyte Cultures Treated with FeF Cetyl Trimethyl Ammonium Bromide (CTAB) USP/NF in the Absence of S9-Mix in the Second Cytogenetic Assay (24 H Exposure Time, 24 H Fixation Time) Conc Milli-Q 0.1 µg/mL 5 µg/mL 8 µg/mL MMC-C 0.2 µg/mL Culture  A    B A+B  A    B A+B  A    B A+B  A    B A+B  A    B A+B Mitotic Index (%) 100 91 76 50 39 No. of Cells scored 150    150 300 150    150 300 150    150 300 150    150 300 75      150 225 No. of Cells with aberrations (+ gaps) a) 1 0 1 0 0 0 1 0 1 0 1 1 39 14 ***^) 53   No. of Cells with aberrations (- gaps) 1 0 1 0 0 0 1 0 1 0 1 1 39 14 ***^) 53   g’                               g”                               b’ 1           1       1   32 9   b”                         9 5   m’                               m”                               exch.                         7 3   dic                               d’                               misc.       poly poly poly 3 poly 9 poly     total aberr (+ gaps) 1 0   0 0   1 0   0 1   48 17   total aberr (- gaps) 1 0   0 0   1 0   0 1   48 17    a) Abbreviations used for various types of aberrations. g’ = chromatid gap; g” = chromosome gap; b’= chromatid break; b” = chromosome break; m’= minute; m” = double minutes; exch. = exchange figure; dic. = dicentric chromosome; d’= chromatid deletion; misc. = (miscellaneous) aberrations not belonging to the ones mentioned above; The numerical variation polyploidy (poly) were not counted as an aberration. *) Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01 or *** P < 0.001. Chromosome Aberrations in Human Lymphocyte Cultures Treated with FeF Cetyl Trimethyl Ammonium Bromide (CTAB) USP/NF in the Absence of S9-Mix in the Second Cytogenetic Assay (48 H Exposure Time, 48 H Fixation Time) Conc DMSO (1.0% v/v) 0.1 µg/mL 2 µg/mL 3 µg/mL MMC-C 0.1 µg/mL Culture  A    B A+B  A    B A+B  A    B A+B  A    B A+B  A    B A+B Mitotic Index (%) 100 103 65 41 61 No. of Cells scored 150    150 300 150    150 300 150    150 300 150    150 300 150    150 300 No. of Cells with aberrations (+ gaps) a) 0 0 0 0 1 1 2 0 2 0 0 0 40 36 ***^) 76   No. of Cells with aberrations (- gaps) 0 0 0 0 0 0 1 0 1 0 0 0 40 36 ***^) 76   g’             1           1     g”         1               1     b’             1           16 11   b”                         14 11   m’                               m”                               exch.                         16 17   dic                               d’                               misc.                     total aberr (+ gaps) 0 0   0 1   2 0   0 0   48 39   total aberr (- gaps) 0 0   0 0   1 0   0 0   46 39    a) Abbreviations used for various types of aberrations. g’ = chromatid gap; g” = chromosome gap; b’= chromatid break; b” = chromosome break; m’= minute; m” = double minutes; exch. = exchange figure; dic. = dicentric chromosome; d’= chromatid deletion; misc. = (miscellaneous) aberrations not belonging to the ones mentioned above; *) Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

Conclusions:

In conclusion, this test is valid and FeF Cetyl Trimethyl Ammonium Bromide (CTAB) USP/NF is not clastogenic in human lymphocytes under the experimental conditions described in this study.

Executive summary:

The objective of this study was to evaluate FeF Cetyl Trimethyl Ammonium Bromide (CTAB) USP/NF 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 study procedures described in this report are in compliance with the most recent OECD and EPA guidelines.

The vehicle of the test item was milli-Q water.

In the first cytogenetic assay, the test item was tested up to 10 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-mix. The test item precipitated in the culture medium at this dose level.

In the second cytogenetic assay, the test item was tested up to 8 µg/mL for a 24 h continuous exposure time with a 24 h fixation time and up to 3 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 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. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations. 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.

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

It was noted that the test item increased the number of polyploid cells both in the absence and presence of S9-mix in a dose dependent manner. This may indicate that the test item has the potential to disturb mitotic processes.

In conclusion, this test is valid and FeF Cetyl Trimethyl Ammonium Bromide (CTAB) USP/NF is not clastogenic in human lymphocytes under the experimental conditions described in this study.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August- September 2019

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:

Adopted July 21, 1997

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

31 May 2008

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

White powder
Batch (Lot) Number: GX0B433
Expiry date: 31 December 2022
Purity: 100% according to certificate of analysis

Target gene:

Histidine locus in strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537)
Tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight). Each S9 batch was characterized with the mutagens benzo-(a)-pyrene (Sigma) and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively.
S9-mix contained per 10 mL: 30 mg NADP and 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 above solution was filter (0.22 μm)-sterilized. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix.

Test concentrations with justification for top dose:

Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate in the dose-range finding test. The highest concentration of the test item used in the subsequent mutation assays was the level at which the test item inhibited bacterial growth.
First experiment: Based on the results of the dose-range finding test, the test item was tested up to the dose level of 52 and 164 μg/plate in the absence and presence of S9-mix, respectively with the tester strains, TA1535, TA1537 and TA98.
Second experiment: Based on the results of the first mutation assay, the test item was tested up to the dose level of 52 and 164 μg/plate in the absence and presence of S9-mix, respectively.

Vehicle / solvent:

The vehicle of the test item was Milli-Q water (Millipore Corp., Bedford, MA., USA).

Untreated negative controls:

no

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

Details on test system and experimental conditions:

Dose-range Finding Test
Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and WP2uvrA, both with and without S9-mix. Eight concentrations,
1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate. The highest concentration of the test item used in the subsequent mutation assays was the
level at which the test item inhibited bacterial growth. At least five different doses (increasing with approximately half-log steps) of the test item were tested in triplicate in each
strain in the absence and presence of S9-mix. The first experiment was a direct plate assay and the second experiment was a pre-incubation assay.
First experiment: Direct Plate Assay
The above mentioned dose-range finding study with two tester strains is reported as a part of the direct plate assay. In the second part of this experiment, the test item was tested both in the absence and presence of S9-mix in the tester strains TA1535, TA1537 and TA98. Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (109 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). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.
Second experiment: Pre-Incubation Assay
The test item was tested both in the absence and presence of S9-mix in all tester strains. Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were pre-incubated for 30 ± 2 minutes by 70 rpm at 37 ± 1°C, 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), 0.1 mL of a fresh bacterial culture (109 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test item in Milli-Q Water. After the pre-incubation period the solutions were added to 3 mL molten top agar. The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

Rationale for test conditions:

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.
All results presented in the tables of the report are calculated using values as per the raw data
rounding procedure and may not be exactly reproduced from the individual data presented.

Evaluation criteria:

In addition to the criteria stated below, 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.
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.
c) The increases in the number of revertants are dose-related

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 examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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 examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 μg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test item did not precipitate on the plates at this dose level.

In the first mutation experiment, the test item was tested in the strains TA1535, TA1537 and TA98 up to concentrations of 52 and 164 μg/plate in the absence and presence of S9-mix, respectively. The test item did not precipitate on the plates at this dose level. Cytotoxicity, as
evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.

In the second mutation experiment the test item was tested in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. The test item was tested up to concentrations of 52 and 164 μg/plate in the absence and presence of S9-mix, respectively.
Precipitation was observed at the dose level of 164 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of
S9-mix.

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.

Direct Plate Assay

Dose (μg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains ofSalmonella typhimurium.
	TA1535	TA1537	TA98
	Without S9-mix
Positive control	964 ± 22	679 ± 59	863 ± 109
Solvent control	11 ± 1	5 ± 5	13 ± 3
0.18	12 ± 4	7 ± 3	18 ± 8
0.55	12 ± 5	4 ± 3	14 ± 1
1.7	8 ± 4	6 ± 1	10 ± 1
5.4	9 ± 4n	6 ± 2	15 ± 3
17	6 ± 3s	5 ± 2n	18 ± 4n
52	e NP MC	e NP MC	e NP MC
	With S9-mix
Positive control	391 ± 11	326 ± 10	1174 ± 154
Solvent control	10 ± 5	6 ± 4	16 ± 5
0.55	6 ± 2	5 ± 1	19 ± 6
1.7	8 ± 3	5 ± 4	10 ± 5
5.4	8 ± 3	3 ± 2	15 ± 4
17	11 ± 7	6 ± 3	11 ± 3
52	8 ± 4n	6 ± 2n	16 ± 7n
164	e NP MC	0 ± 0a NP	0 ± 0a NP

MC Microcolonies

NP No precipitate

a Bacterial background lawn absent

e Bacterial background lawn extremely reduced

n Normal bacterial background lawn

s Bacterial background lawn slightly reduced

Pre-incubation Assay

Dose (μg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains ofSalmonella typhimuriumand oneEscherichia colistrain.
	TA1535	TA1537	TA98	TA100	WP2uvrA
	Without S9-mix
Positive control	953 ± 40	151 ± 62	1161 ± 93	763 ± 52	1540 ± 140
Solvent control	8 ± 4	10 ± 4	13 ± 7	101 ± 12	15 ± 1
0.18	9 ± 2	9 ± 6	15 ± 2	113 ± 4	17 ± 2
0.55	14 ± 0	6 ± 4	17 ± 4n	125 ± 16	21 ± 4
1.7	7 ± 6n	3 ± 3n	10 ± 6s	95 ± 3n	23 ± 4n
5.4	8 ± 4m	e MC	10 ± 4m	73 ± 9m	17 ± 2s
17	e MC	e MC	e MC	e MC	13 ± 1e MC s
52	e NP MC	e NP MC	e NP MC	e NP MC	e NP MC
	With S9-mix
Positive control	259 ± 14	153 ± 17	577 ± 41	1610 ± 154	700 ± 57
Solvent control	10 ± 2	7 ± 3	13 ± 8	89 ± 11	22 ± 6
0.55	7 ± 1	5 ± 3	15 ± 4	100 ± 6	22 ± 8
1.7	10 ± 2	7 ± 4	15 ± 6	105 ± 5	30 ± 4
5.4	13 ± 3	8 ± 2	15 ± 5	104 ± 16	23 ± 6
17	7 ± 2n	12 ± 2n	15 ± 4n	90 ± 27n	30 ± 10
52	e NP MC	e NP MC	13 ± 7m NP	e NP MC	17 ± 5n NP
164	0 ± 0a SP	0 ± 0a SP	0 ± 0a SP	0 ± 0a SP	0 ± 0a SP

MC Microcolonies

NP No precipitate

SP Slight Precipitate

a Bacterial background lawn absent

e Bacterial background lawn extremely reduced

m Bacterial background lawn moderately reduced

n Normal bacterial background lawn

s Bacterial background lawn slightly reduced

Conclusions:

In conclusion, based on the results of this study it is concluded that FeF Cetyl Trimethyl Ammonium Bromide (CTAB) USP/NF is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The objective of this study was to determine the potential of FeF Cetyl Trimethyl Ammonium Bromide (CTAB) USP/NF 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 an exogenous mammalian metabolic activation system (S9).

The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay.

The study procedures described in this report were based on the most recent OECD and EC guidelines. The vehicle of the test item was Milli-Q water.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 μg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test item did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in both tester strains in the absence and presence of S9-mix. Results of this dose-range finding test were reported as part of the first mutation assay.

In the first mutation experiment, the test item was tested in the strains TA1535, TA1537 and TA98 up to concentrations of 52 and 164 μg/plate in the absence and presence of S9-mix, respectively. The test item did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.

In the second mutation experiment the test item was tested in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. The test item was tested up to concentrations of 52 and 164 μg/plate in the absence and presence of S9-mix, respectively. Precipitation was observed at the dose level of 164 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.

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.

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. These results were confirmed in a follow-up experiment.

In conclusion, based on the results of this study it is concluded that FeF Cetyl Trimethyl Ammonium Bromide (CTAB) USP/NF is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August-October 2019

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:

adopted 29 July 2016

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

August 1998

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

White powder
Batch (Lot) Number: GX0B433
Expiry date: 31 December 2022
Purity: 100% according to certificate of analysis

Target gene:

forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells. The TK mutational system detects base pair mutations, frame shift mutations and small deletions.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and was prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight).
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. The above 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. The concentration of the S9-fraction in the exposure medium was 4% (v/v).

Test concentrations with justification for top dose:

Eight doses of the test item were tested in the mutation assay. The test item was tested in the presence of S9-mix with a 3 hour treatment period and in the absence of S9-mix with 3 and
24 hour treatment periods. The highest doses that were tested gave a cell survival of approximately 10-20% and the survival in the lowest doses was approximately the same as the cell survival in the solvent control. Also, some intermediate doses were tested.
First mutagenicity test:
Based on the results of the dose-range finding test, the following dose-range was selected for the first mutagenicity test:
Without S9-mix: 0.01, 0.05, 0.1, 0.5, 1, 2, 4, 6, 8 and 10 μg/mL exposure medium.
With S9-mix: 0.01, 0.05, 0.1, 0.5, 1, 2, 4, 10, 15, 20, 25, 30 and 40 μg/mL exposure medium.
Second mutagenicity test:
Based on the results of the dose-range finding test and experiment 1, the following dose levels were selected for mutagenicity testing: 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 4, 6, 8 and 10 μg/mL exposure medium.

Vehicle / solvent:

The vehicle for the test item was Milli-Q water.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

Test system: L5178Y/TK+/--3.7.2C mouse lymphoma cells

Per culture 8 x 106 cells (106 cells/mL for 3 hour treatment) or 6 x 106 cells (1.25 x 105 cells/mL for 24 hour treatment) were used. The cell cultures for the 3 hour treatment were placed in sterile 30 mL centrifuge tubes, and incubated in a shaking incubator at 37.0 ± 1.0°C and 145 rpm. The cell cultures for the 24 hour treatment were placed in sterile 75 cm2 culture flasks at 37.0 ± 1.0°C. Solvent and positive controls were included and the solvent control was tested in duplicate.
In the first experiment, cell cultures were exposed for 3 hours to the test item in exposure medium in the absence and presence of S9-mix. In the second experiment, cell cultures were exposed to the test item in exposure medium for 24 hours in the absence of S9-mix.
For the 3 hour treatment, cell cultures were exposed to the test item in exposure medium in the absence as well as in the presence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 50 mL growth medium (R10-medium).
For the 24 hour treatment, cell cultures were exposed to the test item in exposure medium in the absence of S9-mix. After exposure, the cells were separated from the treatment solutions by 2 centrifugation steps (216 g, 5 min). The first centrifugation step was followed by removal of the supernatant and resuspension of the cells in Hanks’ balanced salt solution and after the second centrifugation step the cells were resuspended in 20 mL growth medium (R10-medium). The cells in the final suspension were counted with the coulter particle counter.

For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period. During this culture period at least 4 x 106 cells (where possible) were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test item the cells were plated for determination of the cloning efficiency (CEday2) and the mutation frequency (MF).

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.

Rationale for test conditions:

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 analyzed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 106 survivors and ≤ 170 per 106 survivors.
c) The suspension growth (SG) over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency, that is, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10-6. At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x10-6).

Evaluation criteria:

In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical
control data range.
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the 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: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

First mutagenicity test: No biologically relevant 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 test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.
The results were not all within the acceptability criteria as defined in paragraph 5, specifically the absolute cloning efficiency of the solvent controls (CEday2) in the presence of S9-mix which was not between 65 and 120%.
Second mutagenicity test: To obtain more information about the possible mutagenicity of the test item, a second mutation experiment was performed in the absence of S9-mix with a 24 hour treatment period. No biologically relevant 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.

Experiment 1: Cytotoxic and Mutagenic Response of FeF Cetyl Trimethyl Ammonium Bromide (CTAB) USP/NF in the Mouse Lymphoma L5178Y Test System

dose	RSG	CE day2	RCE	RTG	mutation frequency per 10^6survivors
(μg/ml)	(%)	(%)	(%)	(%)	total	small	large
Without metabolic activation – 3 hour treatment
SC1	100	78	100	100	86	12	72
SC2		93			53	14	38
0.05	111	77	90	100	78	10	67
0.1	109	135	158	172	94	17	72
1	95	83	97	92	57	6	50
2	62	84	98	61	73	9	63
4	37	61	72	26	86	19	65
6	29	76	89	26	79	17	60
8	18	55	64	11	51	8	43
MMS	79	45	52	41	684	384	250
With metabolic activation – 3 hour treatment
SC2	100	81	100	100	97	17	78
0.5	102	78	96	98	51	11	40
1	100	78	96	96	69	9	59
2	95	90	111	105	50	2	47
4	87	81	100	87	89	13	75
10	41	94	115	47	65	8	56
15	29	95	117	33	49	9	40
25	20	110	135	27	59	12	45
30	10	97	119	12	64	23	39
CP	50	31	38	19	1194	723	371

Note: all calculations were made without rounding off

RSG = Relative Suspension Growth; CE = Cloning Efficiency; RCE = Relative Cloning Efficiency

RTG = Relative Total Growth; SC = Solvent control = Milli-Q water; MMS = Methylmethanesulfonate;

CP = Cyclophosphamide

Experiment 2: Cytotoxic and Mutagenic Response of FeF Cetyl Trimethyl Ammonium Bromide (CTAB) USP/NF in the Mouse Lymphoma L5178Y Test System

dose	RSG	CE day2	RCE	RTG	mutation frequency per 10^6survivors
(μg/ml)	(%)	(%)	(%)	(%)	total	small	large
Without metabolic activation – 24 hour treatment
SC1	100	90	100	100	141	48	85
SC2		98			102	37	60
0.01	100	91	97	97	150	51	89
0.02	104	97	103	107	118	46	65
0.05	102	98	104	106	145	56	79
0.1	102	90	96	97	160	75	73
0.2	79	131	139	109	120	52	58
0.5	55	89	94	52	108	53	50
1	25	94	100	25	112	36	71
2	8	85	90	7	134	48	78
MMS	82	71	76	62	1168	442	489

Note: all calculations were made without rounding off

RSG = Relative Suspension Growth; CE = Cloning Efficiency; RCE = Relative Cloning Efficiency;

RTG = Relative Total Growth; SC = Solvent control = Milli-Q water; MMS = Methylmethanesulfonate

Conclusions:

In conclusion, FeF Cetyl Trimethyl Ammonium Bromide (CTAB) USP/NF is not mutagenic in the TK mutation test system under the experimental conditions described in this study.

Executive summary:

The objective of this study was to evaluate the mutagenic potential of FeF Cetyl Trimethyl

Ammonium Bromide (CTAB) USP/NF by testing its ability to induce forward mutations at

the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or

presence of a metabolic system (S9-mix). The TK mutational system detects base pair

mutations, frame shift mutations and small deletions.

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

the presence of S9-mix with a 3 hour treatment period.

The study procedures described in this report were based on the most recent OECD guideline.

The vehicle of the test item was Milli-Q water.

In the first experiment, the test item was tested up to concentrations of 8 and 30 μg/mL in the

absence and presence S9-mix, respectively. The incubation time was 3 hours. Relative total

growth (RTG) was reduced to 11 and 12% in the absence and presence of S9-mix,

respectively.

In the second experiment, the test item was tested up to concentrations of 2 μg/mL in the

absence of S9-mix. The incubation time was 24 hours. The RTG was reduced to 7%.

The mutation frequency found in the solvent control cultures was within the acceptability

criteria of this assay and within the 95% control limits of the distribution of the historical

negative control database.

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced

significant increases in the mutation frequency. In addition, the mutation frequency found in

the positive control cultures was within the 95% control limits of the distribution of the

historical positive control database. It was therefore concluded that the test conditions were

adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, the test item did not induce a biologically relevant increase in the

mutation frequency in the first experiment. This result was confirmed in an independent

experiment with modification in the duration of treatment.

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

mutation frequency.

In conclusion, FeF Cetyl Trimethyl Ammonium Bromide (CTAB) USP/NF is not mutagenic

in the mouse lymphoma L5178Y test system under the experimental conditions described in

this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Cetrimonium bromide has been shown to have no potential for gene mutations in baceria and ion mammalian cells or any potetial for clastogenic effects in mammalian cells. Thus, no concern for genotoxicity applies and the substances is not to be CLP classified in relation to mutagenicity.