3-C12-18-(even numbered)-alkylamido-N,N-dimethylpropan-1-amino oxide

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:

2000-05-26 - 2000-09-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

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:

3-C12-18-(even numbered)-alkylamido-N,N-dimethylpropan-1-amino oxide

EC Number:

939-581-9

Cas Number:

1471314-81-4

Molecular formula:

Not applicable (UVCB substance)

IUPAC Name:

3-C12-18-(even numbered)-alkylamido-N,N-dimethylpropan-1-amino oxide

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

- Name: NINOX HCDO
- Purity: approximately 79%
- Lot/batch No.: 876 TK
- Physical state: off white paste
- Storage condition of test material: room temperature, in the dark
- Other: an allowance for test material purity was made prior to each days formulation.

Method

Species / strain

Species / strain / cell type:

lymphocytes: sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability.

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Preliminary Toxicity test: 0, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/ml
Experiment 1: with and without S9: 0, 9.77, 19.53, 39.06, 78.13, 156.25, 234.38 and 312.5 µg/ml
Experiment 2: without S9: 0, 4.89, 9.77, 19.53, 39.06, 78.13, 117.19 µg/ml
Experiment 2: with S9: 0, 19.53, 39.06, 78.13, 156.25, 234.38 and 312.5 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Eagle's minimal essential medium (MEM)

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

Eagle's minimal essential medium (MEM)

Controls 2

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without metabolic activation system

Controls 3

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation system

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium. 1.0 ml of the appropriate solution of vehicle, positive control or test material was added to 9.0 ml 0f each culture. 1 ml of 10% S9 (ie 1 % final concentration of S9) was used for the Preliminary Toxicity Test and Experiment 1, and 1 ml of 20% S9 (ie 2% final concentration of S9) for Experiment 2.
- Exposure duration and Expression time (cells in growth medium):
Experiment 1: with S9-mix was exposed 4 hours with a cell harvest after a 20-hour expression period.
Experiment 1: without S9-mix was exposed 4 hours with a cell harvest after a 20-hour expression period.
Experiment 2: with S9-mix was exposed 4 hours with a cell harvest after a 20-hour expression period.
Experiment 2: without S9-mix the exposure time was 24 hours.
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours, the mitosis was arrested two hours before the required harvest time

SELECTION AGENT (mutation assays): Colcemid
STAIN (for cytogenetic assays): the slides were stained in 5% Gurrs Giemsa for 5 minutes.

NUMBER OF REPLICATIONS: all cultures were set up in duplicate.

NUMBER OF CELLS EVALUATED: where possible the first 100 consecutive well-spread metaphases from each culture were counted

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index. 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.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

If the metaphase cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides. A statistically significant increase in the frequency of cells with chromosome aberrations (excluding gaps) is considered as a positive result.

Statistics:

The frequency of cells with aberrations (both including and excluding gaps) and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact 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:

In all cases test substance showed some toxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: there was no observable change in pH when the test substance was dosed into the media.
- Effects of osmolality: the osmolality did not increase (50 mOSM)
- Precipitation: there was no precipitate of the test material seen in the cultures.

COMPARISON WITH HISTORICAL CONTROL DATA: The vehicle control cultures had frequencies of cell with chromosome aberrations within the expected range and the positive control treatments gave statistically significant increases in the frequency of cells with aberrations.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Metaphase cells were present at dose levels up to 2500 µg/ml in the 4(20) hour treatment in the absence of metabolic activation (S9) and up to 156.25 µg/ml in the 4(20) hour treatment in the presence of S9. The maximum dose with metaphases present in the 24-hour continuous exposure was 78.13 µg/ml. In the 4 hour exposure group without S9, approximately 50% mitotic inhibition was achieved at 156.25 µg/ml and complete mitotic inhibition at 5000 µg/ml.

Applicant's summary and conclusion

Conclusions:

The test substance was shown to be non-clastogenic to human lymphocytes in vitro and therefore is not to be classified according to CLP Regulation.

Executive summary:

The In Vitro Mammalian Chromosome Aberration Test for the test substance was performed with human lymphocytes. A total of two experiments with four treatment conditions were used for the study, ie. 4 hours exposure with the addition of metabolic activation system (S9) with cell harvest after a 20-hour expression period and a 4-hour exposure in the absence of activation with a 20-hour expression period, this was Experiment 1. In the Experiment 2 the 4-hour exposure with addition of S9 was repeated (using a 2% final concentration of S9), whilst in the absence of activation the exposure time was increased to 24 hours. For the metaphase analysis the dose levels selected were: 39.06, 78.13, and 156.25 µg/ml in the Experiment 1; 39.06, 78.13 and 117.19 µg/ml in the Experiment 2, treatment time 24 hours, and 39.06, 78.13 and 156.25 µg/ml in the Experiment 2, treatment time 4 hours. The test substance showed some evidence of toxicity in all cases, a complete mitotic inhibition was observed in the 4 hour exposure group without S9, at 5000 µg/ml. The test substance did not induce a statistically significant increase in the frequency of cells with chromosome aberrations (excluding gaps) in either the presence or absence of a metabolic activation system in either of two separate experiments. The test substance was therefore considered to be non-clastogenic to human lymphocytes in vitro.