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Genetic toxicity in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From December 07, 1994 to December 16, 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Reference:
Composition 1
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Specific details on test material used for the study:
- Code: 06 FHFR 555
- Batch no.: E06 1859561, dated May 31, 1994
- Purity: 79.8% (w/v)
- Stability and homogeneity: Stable for 4 h
- Storage condition: Dark at approximately 20⁰C
- Appearence: White to yellowish scales
- pH (in water): 5.1
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
0.8, 4, 20, 100, 500 and 2,500 µg/plate (for both with and without metabolic activation)
Vehicle:
Ethanol
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Tested for TA 100, TA 1535 in the absence of S9-mix
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Tested for TA 1537 in the absence of S9-mix
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Tested for TA 98 in the absence of S9-mix
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-amino anthracene
Remarks:
Tested for TA 100, TA 1535, TA 1537, TA 98 in presence of S9-mix
Details on test system and conditions:
METHOD OF APPLICATION: In agar (plate incorporation)
DURATION:
- Preincubation period: 48h

Evaluation criteria:
The test substance is classified as mutagenic if it has either of the following effects:
- 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn.
- Dose related increase in the mean number of revertants per plate of atleast one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in atleast two to three concentration of the test substance at complete bacterial background lawn.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
Solubility: Visible precipitation of the test substance on the plates was observed at 5,000 µg/plate in the first experiment.
Toxicity: The test substance was toxic to most of the bacterial strain at doses of 100 µg/plate and above. Thinning of the bacterial lawn and a reduction in the number of colonies were observed at this dose.
Conclusions:
Under the study conditions, the test substance was not mutagenic in Salmonella typhimurium strain TA97, TA98, TA100 or TA1535, with or without metabolic activation.
Executive summary:

A study was conducted to determine the in vitro genetic toxicity of the test substance according to a method similar to OECD Guideline 471, in compliance with GLP. Salmonella typhimurium strains TA97, TA98, TA100 and TA1535 were treated with the test substance using the Ames plate incorporation method. Tests were carried out in triplicate, both with and without the addition ofmetabolic activation (S9-mix). The concentration range of the test substance was 0.8 to 2,500 µg/plate. Cytotoxicity was observed at ≥100 µg/plate. No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains at any of the test substance concentrations tested, either with or without metabolic activation. Under the study conditions, the test substance was not mutagenic in Salmonella typhimurium strain TA97, TA98, TA100 or TA1535, with or without metabolic activation (Muller, 1995).

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From August 30, 2006 to March 12, 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
RA study
Justification for type of information:
Refer to the section 13 for details on the read across justification. The in vitro genetic toxicity study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Directive 2000/32/EC, L1362000, Annex 4E
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Test material information:
Composition 1
Specific details on test material used for the study:
- Physical state: Colourless liquid
- Analytical purity: 25%
- Composition of test material, percentage of components: 25% cetrimonium chloride, 75% water
- Lot/batch No.: CE61250001
- Stability under test conditions: Stable in water for at least 48 h
- Storage condition of test material: Room temperature
Target gene:
HPRT locus
Species / strain:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell lines (if applicable):
- Type and identity of media: MEM (minimal essential medium) supplemented with 10% foetal calf serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Experiment I:
without S9 mix: 0.2, 0.4, 0.8, 1.5 and 2.3 µg/mL
with S9 mix: 1.6, 3.1, 6.3, 12.5 and 18.8 µg/mL
Experiment II:
without S9 mix: 0.4, 0.8, 1.6, 3.1 and 4.7 µg/mL
Vehicle:
- Vehicle(s)/solvent(s) used: deionised water
Negative controls:
yes
Remarks:
Untreated control
Solvent controls:
yes
Remarks:
Deionised water
True negative controls:
other: Untreated cells were cultivated without interruption throughout the assay and without addition of test item, in order to obtain the initial spontaneous mutation rate at the beginning of the experiments.
Positive controls:
yes
Remarks:
Without metabolic activation
Positive control substance:
ethylmethanesulphonate
Negative controls:
yes
Remarks:
Untreated control
Solvent controls:
yes
Remarks:
Deionised water
True negative controls:
other: Untreated cells were cultivated without interruption throughout the assay and without addition of test item, in order to obtain the initial spontaneous mutation rate at the beginning of the experiments.
Positive controls:
yes
Remarks:
With metabolic activation
Positive control substance:
7,12-dimethylbenzanthracene
Details on test system and conditions:
METHOD OF APPLICATION: Seeded into plastic culture flasks
DURATION
- Preincubation period: 24h
- Exposure duration: 4h (in experiment I; with and without S9), 24h (in experiment II; without S9)
- Expression time (cells in growth medium): 7d
- Selection time (if incubation with a selection agent): Day 7

SELECTION AGENT (mutation assays): Thioguanine (6TG)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
The test item was considered positive if (a) It reproducibly induces with one of the test compound concentrations a mutation frequency that is three times higher than the spontaneous mutant frequency in this experiment. (b) There is a reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently from the enhancement factor for induced mutants. (c) Survival of the responding dose group is at least 30%. However, in a case by case evaluation both decisions depend on the level of the corresponding negative control data.
Statistics:
A linear regression was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT® statistics software.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No effects
- Effects of osmolality: No effects
- Precipitation: No precipitation or phase separation of the test substance was observed up to the maximum concentration in both main experiments.

RANGE-FINDING/SCREENING STUDIES: Two range finding pre-tests were performed in the presence (4h treatment) and absence (4h and 24h treatment) of S9. In the first pre-test test substance concentrations between 25 and 3200 µg/mL (active substance) were used to evaluate toxicity. In this first pretest strong toxic effects were noted at all concentrations with and without metabolic activation. Therefore, a second pre-test was performed using concentrations of 0.2 to 25 µg/mL (active substance).
Following 4h treatment without S9 mix strong toxicity occurred at 1.58 µg/mL. The cell growth was completely stopped at the next higher concentration of 3.13 µg/mL and above. In the presence of S9 mix (4h treatment) strong toxicity was determined at the highest concentration of 25 µg/mL. After 24h of treatment a relevant toxic effect occurred at 3.13 µg/mL. At all higher concentrations the cell growth was also completely inhibited.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

The sensitivity of the test system and efficacy of the S9 mix was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control substances.

Mutation results:

Main experiment:

- No relevant and reproducible increase of the mutation frequency occurred at any concentration with and without metabolic activation. All mutant frequencies remained well within the historical range of negative and solvent controls.

- A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in both cultures of experiment I with metabolic activation. However, a small increase of the mutation frequency at toxic concentrations is common in this assay system and does not indicate a possible mutagenic potential provided that the mutation frequency does not exceed the threshold of 3 times above the corresponding solvent control. Since the mutation frequency neither exceeded the historical range of negative and solvent controls nor the threshold as indicated above, the statistical results were considered as biologically irrelevant.

Conclusions:
Based on the results of the read across study, the test substance did not induce gene mutations in the HPRT locus in V79 Chinese hamster cells, either in the presence or absence of metabolic activation.
Executive summary:

A study was conducted to determine the in vitro genetic toxicity of the read across substance, cetrimonium chloride (C16 TMAC), according to OECD Guideline 476, in compliance with GLP. This experiment was performed at the HPRT locus in V79 Chinese hamster cells. All positive controls showed a distinct increase in the number of mutant colonies. No substantial and reproducible concentration-dependent increases in the mutation frequency at the HPRT locus, was seen in test substance-treatment cells either with or without metabolic activation. Based on the results of the read across study, the test substance was considered to be non-mutagenic both with and without metabolic activation (Wollny, 2007).

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From April 17, 1989 to September 15, 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
RA study
Justification for type of information:
Refer to the section 13 for details on the read across justification. The in vitro genetic toxicity study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: Toxicity test guideline, Japan 1984
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Test material information:
Composition 1
Specific details on test material used for the study:
- Name of test material (as cited in study report): Cetyltrimethylammoniumchlorid (24-26 % active ingredient in aqueous solution)
- Physical state: Clear transparent liquid
- Analytical purity: 24-26%
- Composition of test material, percentage of components: 24-26% cetrimonium chloride; 74-76% water
- Lot/batch No.: 3118322, 18.01.89
- Stability under test conditions: Stable for more than several h in water
- Storage condition of test material: Room temperature
Species / strain:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell lines (if applicable):
- Type and identity of media: MEM medium supplemented with 10% foetal calf Serum (FCS)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
without S9 mix: (7 hours: 1.0 µg/mL; 18 hours: 0.3, 1.0 and 3.0 µg/mL; 28 hours: 3.0 µg/mL)
with S9 mix: (7 hours: 10.0 µg/mL; 18 hours: 1.0, 3.0 and 10.0 µg/mL; 28 hours: 10.0 µg/mL)
Vehicle:
- Vehicle(s)/solvent(s) used: Water
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
ethylmethanesulphonate
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
cyclophosphamide
Details on test system and conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h
- Fixation time (start of exposure up to fixation or harvest of cells): 7h (high dose), 18h (low, medium and high dose), and 28h (high dose)

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (approx. 0.2 µg/mL/culture medium)

STAIN (for cytogenetic assays): Giemsa stains

NUMBER OF REPLICATIONS: Two

NUMBER OF CELLS EVALUATED: 100 cells of each cell culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
Evaluation criteria:
- A test article is classified as clastogenic if it induces either a significant dose-related increase in the number of structural chromosomal aberrations or a significant and reproducible positive response for at least one of the test points.
- A test article producing neither a significant dose-related increase in the number of structural chromosomal aberrations nor a significant and reproducible positive response at any one of the test points is considered non-clastogenic in this system.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
- In the pre-experiments for toxicity the colony forming ability of the V79 cells was totally reduced after treatment with 6.0 µg/mL. Accordingly, one (for 7 and 28h time point) and three concentrations (for 18h time point) were selected to evaluate metaphases for cytogenetic damage. Mitotic index was reduced after treatment with the highest dose levels in the absence and presence of S9 mix, in the main test.
- Mutation results:
There was no relevant increase in cells with structural aberrations after treatment with the test substance at any fixation interval either without or with metabolic activation by S9 mix. Positive controls showed distinct increases in cells with structural chromosome aberrations. The sensitivity of the test system and efficacy of the S9 mix was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control substances.
(COMPARISON WITH HISTORICAL CONTROL DATA: Yes)

Conclusions:
Based on the result of the read across study, the test substance did not induce structural chromosome aberrations in the V79 Chinese hamster cell line with and without metabolic activation.
Executive summary:

A study was conducted to determine the in vitro genetic toxicity of the read across substance, cetrimonium chloride (C16 TMAC), according to OECD Guideline 473 and EU Method B.10, in compliance with GLP. The experiment was performed in V79 Chinese hamster lung cells. The concentration range of the test substance was determined in a pre-experiment using the plating efficiency assay as an indicator of toxicity response. Cells were exposed for 7, 18 or 28 hours at concentrations levels of 0.0 to 10.0 µg/L test substance with or without metabolic activation. Treatment with 3.0 µg/mL and 10.0 µg/mL completely reduced the plating efficiency of the V79 cells. The mitotic index was reduced after treatment with the highest concentration at each fixation interval in the presence and absence of S9 mix. Positive controls showed a distinct increase in the number of cells with structural chromosome aberrations. There was no relevant increase in cells with structural aberrations after treatment with the test substance at any fixation interval either without or with S9 mix. Based on the results of the read across study, the test substance did not induce structural chromosome aberrations in the V79 Chinese hamster cell line with and without metabolic activation (Heidemann, 1989).

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

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
genetic toxicity in vivo, other
Data waiving:
other justification
Justification for data waiving:
other:
Reason / purpose:
data waiving: supporting information
Related information:
Composition 1

Additional information

In vitro:

Study 1. A study was conducted to determine the in vitr ogenetic toxicity of the test substance according to a method similar to OECD Guideline 471, in compliance with GLP. Salmonella typhimurium strains TA97, TA98, TA100 and TA1535 were treated with the test substance using the Ames plate incorporation method. Tests were carried out in triplicate, both with and without the addition of metabolic activation (S9-mix). The concentration range of the test substance was 0.8 to 2,500 µg/plate. Cytotoxicity was observed at ≥100 µg/plate. No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains at any of the test substance concentrations tested, either with or without metabolic activation.Under the study conditions, the test substance was not mutagenic in Salmonella typhimurium strain TA97, TA98, TA100 or TA1535, with or without metabolic activation (Muller, 1995).

Study 2.A study was conducted to determine the in vitro genetic toxicity of the read across substance, cetrimonium chloride (C16 TMAC), according to OECD Guideline 476, in compliance with GLP. This experiment was performed at the HPRT locus in V79 Chinese hamster cells. All positive controls showed a distinct increase in the number of mutant colonies. No substantial and reproducible concentration-dependent increases in the mutation frequency at the HPRT locus, was seen in test substance-treatment cells either with or without metabolic activation. Based on the results of the read across study, the test substance was considered to be non-mutagenic both with and without metabolic activation (Wollny, 2007).

Study 3. A study was conducted to determine the in vitro genetic toxicity of the read across substance, cetrimonium chloride (C16 TMAC), according to OECD Guideline 473 and EU Method B.10, in compliance with GLP. The experiment was performed in V79 Chinese hamster lung cells. The concentration range of the test substance was determined in a pre-experiment using the plating efficiency assay as an indicator of toxicity response. Cells were exposed for 7, 18 or 28 hours at concentrations levels of 0.0 to 10.0 µg/L test substance with or without metabolic activation. Treatment with 3.0 µg/mL and 10.0 µg/mL completely reduced the plating efficiency of the V79 cells. The mitotic index was reduced after treatment with the highest concentration at each fixation interval in the presence and absence of S9 mix. Positive controls showed a distinct increase in the number of cells with structural chromosome aberrations. There was no relevant increase in cells with structural aberrations after treatment with the test substance at any fixation interval either without or with S9 mix. Based on the results of the read across study, the test substance did not induce structural chromosome aberrations in the V79 Chinese hamster cell line with and without metabolic activation (Heidemann, 1989).

In vivo:

Data waiving since the test substances were negative inin vitroassays (i.e. bacterial and mammalian mutagenicity assays as well as a chromosome aberration assay). Therefore, an in vivo mutagenicity test is not required.

Justification for classification or non-classification

Based on the available negative in vitro genotoxicity data, classification of C18 TMAC for genotoxicity according to Directive 67/548/EEC and Regulation (EC) 1272/2008 is not required.