Tetradonium bromide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

It is considered scientifically justified to read across to the target substance from the source substances with highly comparable chemical structures both in relation to toxicokinetic behaviour and in relation to type of toxicity end-points. See assessment report attached in Section 13 for hypothesis and justification for read-across of data on toxicological information.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1980, 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Read-across to tetradonium bromide from data on cetrimonium chloride (with data reliability value of 1).

Principles of method if other than guideline:

Reverse mutation assay
Study 1980 according to Ames et al 1975

GLP compliance:

yes

Remarks:

study 1983 - not stated

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: TA98 an TA100

Test concentrations with justification for top dose:

Study 1980: 0.05, 0.1, 0.5, 1.0, 5.0 and 10.0 μg/plate

Species / strain:

other: Salmonella typhimurium

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: strain/cell type: TA98 and TA100

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation

Cetrimonium chloride showed to be negative under the test conditions with a cytotoxic concentration of 5.0 μg/plate without metabolic activation.

Executive summary:

A US National Toxicology Programme report mentions two references of 1980 and 1983

describing bacterial reverse mutation assays with cetrimonium chloride in Salmonella

typhimurium TA98 and TA100, with and without S9 activation. Cetrimonium chloride

showed to be negative under the test conditions with a cytotoxic concentration of

5.0 μg/plate without metabolic activation.

Cross-reference 2

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Apr-Jul 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Read-across to tetradonium bromide from data on cetrimonium chloride (with data reliability value of 1).

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

- S9-mix: 0.1, 0.3, 0.6, 1.0, 3.0 and 6.0 μg/ml
+ S9-mix: 0.1, 0.5, 1.0, 3.0, 6.0 and 10.0 μg/ml

Vehicle / solvent:

Water

Positive controls:

yes

Remarks:

Ethylmethanesulfonate and cyclophosphamide in 18-hour cultures without and with S9-mix, respectively.

Details on test system and experimental conditions:

medium at concentrations of 0.1 to 6 μg/ml without S9-mix and at 0.1 to 10 μg/ml with S9-
mix (from Aroclor 1254 induced rats) for 4 hours. Cells were subsequently washed in
glucose-containing saline and cultured in normal medium for 7, 18 and 28 hours.
Ethylmethanesulfonate and cyclophosphamide were used as positive controls in 18-hour
cultures without and with S9-mix, respectively. Two hours (7 hour interval) or 2.5 hours (18
and 28 hours intervals) before the end of the incubation period, colcemid was added to the
cultures. The cells were put onto glass slides, treated with hypotonic potassium chloride
solution, fixed in methanol and acetic acid and stained with Giemsa solution. In each
experimental group two parallel cultures were set up. Per culture 100 metaphases were
scored for structural chromosomal aberrations (breaks, fragments, deletions, exchanges
and chromosomal disintegrations). Chromosomal gaps were recorded separately.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In concentration-finding pre-tests, cytotoxic effects were observed at 1 μg/ml without S9-
mix and at 6 μg/ml with S9-mix as a colony-forming ability below 20% of controls.
Evaluated dose levels were 1.0 μg/ml without S9-mix and 10.0 μg/ml with S9-mix for 7
hours; 0.3, 1.0 and 3.0 μg/ml without S9-mix and 1.0, 3.0 and 10.0 μg/ml with S9-mix for
18 hours; and 3.0 μg/ml without S9-mix and 10.0 μg/ml with S9-mix for 28 hours. There
were no biologically relevant and statistically significant increases in cells with structural
aberrations after treatment with the test substance at any fixation interval either with or
without metabolic activation. The reference mutagens used as positive controls showed
distinct increases in cells with structural chromosome aberrations.

Conclusions:

Based on the observations in the study it is concluded that cetrimonium chloride (24-26% in water) did not induce chromosomal aberrations in V79 cells either with or without S9-mix at up to 3.0 and 10.0 μg/ml respectively.

Executive summary:

The SCCS opinion document refers to a study with 24-26% cetrimonium chloride in water. The study was carried out according to OECD TG 473 in vitro mammalian chromosome aberration test with V79 Chinese hamster cells.

Based on the observations in the study it is concluded that cetrimonium chloride (24-26% in water) did not induce chromosomal aberrations in V79 cells either with or without S9-mix at up to 3.0 and 10.0 μg/ml respectively.

Cross-reference 3

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Read-across to tetradonium bromide from data on dodecyltrimethylammonium chloride (with data reliability value of 1).

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Principles of method if other than guideline:

Unscheduled DNA synthesis assay (UDS) with rat primary hepatocyte

GLP compliance:

yes

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Species / strain / cell type:

hepatocytes: rat

Metabolic activation:

not applicable

Test concentrations with justification for top dose:

0.004 to 0.1 μl/ml

Positive controls:

yes

Positive control substance:

other: Benzanthrazene

Details on test system and experimental conditions:

A preliminary assessment of toxicity was conducted to select dose levels for the UDS assay.
For the UDS assay, male rat hepatocytes were treated with the test substance at concentrations ranging from 0.004 μl/ml to 0.1 μl/ml.

Species / strain:

hepatocytes:

Metabolic activation:

not applicable

Genotoxicity:

other: inactive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

lethal at concentrations exceeding 0.048 µl/ml

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Concentrations below 0.048 μl/ml were nontoxic. Eight treatments ranging from 0.004 μl/ml to 0.048 μl/ml were selected for analysis of nuclear labeling. No indication of induction of UDS by the test substance was observed. Negative and positive controls had nuclear grain counts in the acceptable range.

Remarks on result:

other: strain/cell type: male rat primary hepatocytes

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
other: Genotoxicity was inactive

Dodecyltrimethylammonium chloride showed to be inactive under the test conditions and concentrations below 0.048 μl/ml were nontoxic.

Executive summary:

A US National Toxicology Programme report describes a study with dodecyltrimethylammonium chloride in an Unscheduled DNA synthesis assay (UDS). Dodecyltrimethylammonium chloride showed to be inactive under the test conditions and concentrations below 0.048 μl/ml were nontoxic.

Cross-reference 4

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Read-across to tetradonium bromide from data on dodecyltrimethylammonium chloride (with data reliability value of 2).

Principles of method if other than guideline:

Reverse mutation assay

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: TA98, TA100, TA1535, TA1537 and TA1538

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

0.004 - 0.4 μl per plate

Vehicle / solvent:

Dimethylsulphoxide

Details on test system and experimental conditions:

Plate incorporation assay was used. The test was run in triplicate.

Evaluation criteria:

A three-fold increase in back mutations was considered as the criterion for a positive test for mutagenicity.

Species / strain:

other: Salmonella typhimurium

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

with and without metabolic activation

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

0.1 µl/plate

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: strain/cell type: TA98, TA100, TA1535, TA1537 and TA1538

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

Dodecyltrimethylammonium chloride showed to be negative under the test conditions with a cytotoxic concentration of 0.1 μl/plate.

Executive summary:

A US National Toxicology Programme reports a reference of 1982 describing bacterial reverse mutation assays with dodecyltrimethylammonium chloride in Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA1538, with and without S9 activation. Dodecyltrimethylammonium chloride

showed to be negative under the test conditions with a cytotoxic concentration of 0.1 μl/plate.

Cross-reference 5

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Read-across to tetradonium bromide from data on dodecyltrimethylammonium chloride (with data reliability value of 1).

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

Mammalian cell forward mutation assay

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

0.0038 - 0.050 μl/ml (10 concentrations) without S9
0.012 - 0.16 μl/ml (10 concentrations) with S9

Details on test system and experimental conditions:

The cells were exposed to the test chemical, positive control and negative control for 4 hours. An expression time of 2 days was allowed with
cell population adjustment at 24 and 48 hours. At the end of the expression period, the cells were placed in cloning medium. Cell counts were made for each preparation and the appropriate number of cells were removed and plated. Total number of colonies per plate and the mutation frequency were determined .

Evaluation criteria:

The criteria for a positive test were: if there was a positive dose response and one or more of the three highest doses exhibited a mutant frequency which was two-fold greater than the background level. A two-fold increase without dose response was considered equivocal and the test was considered negative if no cultures exhibited a two-fold increase in mutant frequency.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

with and without metabolic activation

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

0.01 µl/ml without metabolic activation; 0.1 µl/ml with metabolic activation

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

Dodecyltrimethylammonium chloride showed to be negative under the test conditions with a cytotoxic concentration of 0.01 μl/ml without metabolic activation and 0.1 μl/ml with metabolic activation.

Executive summary:

A US National Toxicology Programme reports a reference of 1982 describing a L5178Y TK+/- Mouse lymphoma mutagenesis assay with and without S-9 activation with dodecyltrimethylammonium chloride. Dodecyltrimethylammonium chloride showed to be negative under the test conditions with a cytotoxic concentration of 0.01 μl/ml without metabolic activation and 0.1 μl/ml with metabolic activation.

Applicant's summary and conclusion

Conclusions:

Based on read-across from C12 TMAC and C16 TMAC, a lack of mutagenic potential can be concluded for tetradonium bromide.

Executive summary:

Due to the structural similarities and of the substances read-across for mutagenic effects is considered highly relevant.

Data on C16 TMAC from testing in salmonella typhimurium with and without S9 was negative.

Data on C12 TMAC from testing in salmonella typhimurium with and without S9 was negative.

Data on C16 TMAC from testing in in vitro chromosome aberration test (OECD 473) was negative.

Data on C12 TMAC from testing in an in vitro mouse lymphoma assay (comparable to OECD 476) with and without S9 was negative.

Data on C12 TMAC from testing in an in vitror at hepatocytes UDS assay (comparable to OECD 482) was negative. 

In vivo test with C12 TMAC rats in a bone marrow chromosome aberration test (comparable to OECD 475) was negative.

 

The bromide ion of the target substance is not considered to influence these results (see comparison of effects from the bromide ion and chloride ion in the assessment report attached in Section 13).

Thus, using read-across a lack of mutagenic potential can be concluded for tetradonium bromide as well.