Benzododecinium chloride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 22, 2001 to September 25, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Quaternary ammonium compounds, benzyl C12-C16 (even numbered) -alkyldimethyl chlorides

Molecular formula:

C12-14H25-29-(CH3)2-C6H5-N.CL

IUPAC Name:

Quaternary ammonium compounds, benzyl C12-C16 (even numbered) -alkyldimethyl chlorides

Specific details on test material used for the study:

- Physical state: Extremely pale yellow, slightly viscous liquid with white precipitate
- Analytical purity: The purity of the a.s. is typically >93% ADBAC as described in Section 2; the a.s. is supplied in aqueous/alcohol solution of 50% or 80% a.s. Doses are based on a.s. (i.e. corrected for the dilution in alcohol/water)
- Lot/batch No.: DEGE001033
- Expiration date of the lot/batch: January 2002
- Stability under test conditions: The test substance is hydrolytically and photolytically stable under the conditions of this study and has been shown to be stable in aqueous, alcohol and alcohol/aqueous solutions for extended periods, e.g. at least five years under standard laboratory conditions.
- Storage condition of test material: Room temperature in darkness

Method

Species / strain

Species / strain / cell type:

lymphocytes: human

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbitone and ß-naphthoflavone - induced rat liver S9 fraction

Test concentrations with justification for top dose:

Preliminary toxicity test: 0, 19.5, 39, 78.1, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL (with and without activation)
Chromosome aberration test:
Experiment 1: 0, 4, 8, 16, 20 µg/mL (with and without activation)
Experiment 2: 0, 4, 8, 12, 16, 24 µg/mL (with and without activation)

Vehicle / solvent:

Eagle’s minimal essential medium with HEPES buffer (MEM)

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Negative (media) control

Negative solvent / vehicle controls:

yes

Remarks:

Eagle’s minimal essential medium with HEPES buffer (MEM)

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

(Without S9, at 0.4 and 0.25 µg/mL in Experiment 1 and 2 respectively)

Controls 2

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

(With S9, at 12.5 and 10.0 µg/mL in experiment 1 and 2 respectively)

Details on test system and experimental conditions:

Method of application: In Eagle’s minimal essential medium with HEPES buffer (MEM)
Duration:
- Exposure duration:
Without metabolic activation: 4 and 24h
With metabolic activation: 4h
- Exposure procedure: The cultures were incubated at 37˚C for 4 or 24h (as appropriate) in the presence of the test substance at predetermined concentrations/vehicle/positive controls with or without the S9 reaction mixture.
- Expression time: Approximately 20h after initiation of treatment
- Fixation time: 4h
Spindle inhibitor: Demecolcine (colcemid, 0.1 μg/mL) was added approx. 2 h prior to harvest timeSpindle
Stain: When the slides were dry they were stained in 5% Gurrs Giemsa solution for 5 minutes, rinsed, dried and coverslipped using mounting medium.
Number of replications: At least 2 slides/ flask
Number of cell evaluated: 100 consecutive well-spread metaphase cells (if possible), from each culture were counted, and if the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted.
Determination of the cytotoxicity: A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

Statistics:

The frequency of cells with aberrations (both including and excluding gaps) and the frequency of polyploid cells were compared, where necessary, with the concurrent vehicle control value using Fisher’s Exact test and chi-squared test.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slightly toxic at 20 µg/mL in experiment 1 (with S9 activation) and toxic at 16 µg/mL in experiment 1 and at 20 µg/mL in experiment 2 (without S9 activation))

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table 1. Results of chromosomal aberration in human lymphocytes (Experiment 1)

Treatment (μg/mL)	S9 Activation	Treatment Time	Mean Mitotic Index	Cells Scored	Total Number of Aberrations +Gaps - Gaps	Cells with Numerical Aberrations + Gaps(%)	Cells with Structural Aberrations - Gaps(%)
Vehicle	-	4	4.2	200	11    7	5.0	3.5
Test substance
4	-	4	3.2	200	6   1	3.0	0.5
8	-	4	2.7	200	2   1	1.0	0.5
16	-	4	1.5	200	7   5	3.0	2.0
Positive control (MMC) 0.4	-	4	2.2	200	83   64	32.0	26.0**
Vehicle	+	4	3.9	200	5    1	2.5	0.5
Test substance
8	+	4	2.3	200	4   1	2.0	0.5
16	+	4	2.1	200	7   4	2.5	1.0
20	+	4	1.6	200	7   3	3.5	1.5
Positive control (CP) 12.5	+	4	1.4	300	76   45	20.0	12.0**

Treatment: Cells from the 4-h treatment regimens were harvested 20h after the initiation of the treatments.

                         Frequency of Aberrant Cells: **, p ≤ 0.001

Table 2. Results of chromosomal aberration in human lymphocytes (Experiment 2)

Treatment (μg/mL)	S9 Activation	Treatment Time	Mean Mitotic Index	Cells Scored	Total Number of Aberrations +Gaps - Gaps	Cells with Numerical Aberrations + Gaps(%)	Cells with Structural Aberrations - Gaps(%)
Vehicle	-	4	7.0	200	4    1	2.0	0.5
Test substance
4	-	24	4.9	200	6   4	2.0	1.0
8	-	24	2.7	200	6   6	2.5	2.5
12	-	24	2.6	200	12   2	6.0	1.0
Positive control (MMC) 0.2	-	24	2.3	200	115   86	37.0	30.0**
Vehicle	+	4	5.9	200	8    4	2.5	0.5
Test substance
8	+	4	5.2	200	7   3	3.5	1.5
16	+	4	3.3	200	0   0	0	0
24	+	4	3.4	200	1   1	0.5	0.5
Positive control (CP) 12.5	+	4	1.4	200	108   79	33.5	27.0**

Treatment: Cells from both the 4-h and 24 h treatment regimens were harvested 20 h after the initiation of the treatments.

Frequency of Aberrant Cells: **, p ≤ 0.001

For further details, refer to the attachment under 'Attached background material'

Applicant's summary and conclusion

Conclusions:

Based on the results of the study, the test substance is not considered to be non-clastogenic to human lymphocytes with and without metabolic activation.

Executive summary:

A study was conducted to determine the in vitro genetic toxicity of the test substance, C12-16 ADBAC (50 -80% active) in chromosome aberration test, according to OECD Guideline 473 and EU Method B.10, in compliance with GLP. This experiment was performed in human lymphocyte cells. Duplicate cell cultures of human lymphocytes, treated with the test substance, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls (mitomycin-C (without S9) and cyclophosphamide (with S9)). Four treatment conditions were used for the study. Experiment 1 and 4 h exposure with and without metabolic activation was followed by a 20 h expression period. In Experiment 2, the 4 h exposure with metabolic activation was repeated while in the absence of metabolic activation the exposure time was increased to 24 h. The doses studied were 0, 4, 8, 16, 20 µg/mL (with and without activation) in Experiment 1 and 0, 4, 8, 12, 16, 24 µg/mL (with and without activation) in Experiment 2. The test substance was considered negative for chromosomal aberrations in human lymphocytes in vitro under the S9 metabolic activation and non-activation conditions of the assay. There was no indication of chromosomal ploidy changes in cultures exposed to the test substance in either the presence or absence of S9 mix. Mutant frequencies of all cultures treated with the test substance were within the acceptable range for background mutant frequencies. Based on the results of the study, the test substance is not considered to be non-clastogenic to human lymphocytes with and without metabolic activation (Durward, 2001).