Ethyldimethyl[2-[(2-methyl-1-oxoallyl)oxy]ethyl]ammonium ethyl sulphate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07/12/1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented report of a guideline study conducted to GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (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

Details on test material:

- Name of test material (as cited in study report): MADQUAT MC 75
- Physical state: liquid
- Analytical purity: 75.5%
- Impurities (identity and concentrations): water (24.5%)
- Composition of test material, percentage of components: 100%
- Purity test date: 06/09/1994
- Lot/batch No.: M 67 bis
- Stability under test conditions: stable
- Storage condition of test material: in the dark at room temperature

Method

Target gene:

Not applicable.

Species / strain

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Two healthy human volunteers

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix, prepared by rat liver enzyme induction by Aroclor 1254

Test concentrations with justification for top dose:

- With S9 mix:
. 0, 1000, 3000 and 5000 µg/ml for the 3-h exposure with a 17-h recovery (3+17)
. 0, 125, 250, 500, 1000, 3000 and 5000 µg/ml for the 3-h exposure with a 41-h recovery (3+41)
- Without S9 mix:
. 0, 500, 1000 and 3000 µg/ml for the 20-h exposure without recovery (20+0)
. 0, 500, 1000 and 3000 µg/ml for the 44-h exposure without recovery (44+0)

Vehicle / solvent:

distilled water

Controls

Untreated negative controls:

yes

Remarks:

distilled water

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: without S9-mix: Mitomycine C (MMC 0.2 µg/ml), . with S9-mix: Cyclophosphamide (CPA 50 µg/ml)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) incubation temperature 37°C
- Exposure / recovery:
- Experiment 1: 3/17 (with S9-mix) and 20/0 (without S9-mix)
- Experiment 2: 3/41 (with S9-mix) and 44/0 (without S9-mix)
DURATION
- Preincubation period: no
- Exposure duration: 1.5 h
- Expression time : Number of metaphases analyzed
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):
CELL CULTURE:
- Source: two healthy human volunteers
- Cell cycle length: 12-14h
- Number of passages: no data
- Method of maintenance of cell cultures:
RPMI 1640 medium containing 20% v/v foetal calf serum and 100 U/ml penicilline + 100 U/ml streptomycine + 0.25 µg/ml fungison + 2 mM L-glutamine + 3.6% phytohaemagglutinin. Incubation at 37°C for approximately 48 hours.
- Absence of mycoplasma: no data
METAPHASE ARREST
- Spindle poison used: colcemid
- Duration of exposure: 1.5 h
- Time before harvest: 1.5 h
CELL PROCESSING: After hypotonic treatment (0.075 M KCl), cells are fixed in a methanol/glacial acetic acid mixture (3/1, v/v).
Cells are then spread on slides and stained with Giemsa. Slides are rinsed, dried and mounted with coverslips.
NUMBER OF REPLICATIONS: 2
DETERMINATION OF CYTOTOXICITY : Cytotoxicity was evaluated using the mitotic index (which indicates whether an item induces mitotic
inhibition) at the following doses: 0, 125, 250, 500, 1000, 3000 and 5000 µg/ml
OTHER EXAMINATIONS:
Types of sought aberrations: gaps, chromatid and chromosome breaks and exchanges, and others (multiple aberrations and pulverizations).
In addition, the following numerical aberrations were recorded when encountered: polyploidy, hyperdiploidy and endoreduplication.

Evaluation criteria:

- Acceptance criteria:
- the frequency of cells with structural aberrations in the vehicle control was consistent with historical data
- the frequency of cells with structural chromosome aberrations in the positive control was significantly higher than that of the control and consistent with historical data
- Evaluation criteria:
A reproducible and statistically significant increase in the frequency of cells with structural chromosome aberrations (excluding gaps) for at least one of the dose-levels and one of the two harvest times was considered as a positive result. Reference to historical data was also taken into account in the evaluation of the findings.

Statistics:

Chi-square test

Results and discussion

Test results

Species / strain:

lymphocytes: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

doses: . 500, 1000 and 3000 µg/ml without S9 mix, . 1000, 3000 and 5000 µg/ml with S9 mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

CYTOTOXICITY:
The results of mitotic index were as follows:
- Without S9 mix:
- At the first harvest time, at 5000 µg/ml, the mitotic index was nil, at 1000 and 3000 µg/ml, it was reduced by approximately 60% to the controls; at 125, 250 and 500 µg/ml, by approximately 30% to the controls.
- At the second harvest time, at 5000 µg/ml, the mitotic index was nil, at 3000 µg/ml, it was reduced by approximately 60% to the controls; at 1000 µg/ml, by approximately 30% to the controls; at the other doses, it was similar to that of controls.
- With S9 mix:
- At the first harvest time, at 5000 µg/ml, the mitotic index was reduced by approximately 65% to the controls; at 1000 µg/ml, by approximately 40% to the controls; at the other doses, it was similar to that of controls.
- At the second harvest time, at 5000 µg/ml, the mitotic index was reduced by approximately 20% to the controls; at the other doses, it was similar to that of controls.
Consequently, the chromosome abnormalities were scored on the slides corresponding to the following doses:
- 500, 1000 and 3000 µg/ml without S9 mix,
- 1000, 3000 and 5000 µg/ml with S9 mix.
GENOTOXICITY:
The test substance did not induce any significant increase in aberrant cell frequency, with or without metabolic activation, for the two harvest times.
Negative control results were within the historical data range. Positive control results were significantly higher than those of negative control.

Remarks on result:

other: strain/cell type: human lymphocytes

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test substance did not induce any significant increase in aberrant cell frequency, with or without metabolic activation, for the two harvest times.
MADQUAT MC 75 is not geneotoxic in the human lymphocytes cytogenetic test