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Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Cetrimonium bromide
EC Number:
200-311-3
EC Name:
Cetrimonium bromide
Cas Number:
57-09-0
Molecular formula:
C19H42N.Br
IUPAC Name:
hexadecyltrimethylazanium bromide
Test material form:
solid
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

Method

Species / strain
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.
Controls
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.

Results and discussion

Test results
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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.