1-Octanamine, N,N-dimethyl-, N-oxide

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:

between 01 November 2007 and 28 February 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Schedule 1 (Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by 2004/0994))

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

N,N-dimethyl-N-octylhydroxylamine

EC Number:

607-854-9

Cas Number:

2605-78-9

Molecular formula:

CH3(CH2)7N(CH3)2O

IUPAC Name:

N,N-dimethyl-N-octylhydroxylamine

impurity 1

Reference substance name:

Dimethyl(octyl)amine

EC Number:

230-939-3

EC Name:

Dimethyl(octyl)amine

Cas Number:

7378-99-6

Molecular formula:

C10H23N

IUPAC Name:

N,N-dimethyloctan-1-amine

impurity 2

Reference substance name:

Methanol

EC Number:

200-659-6

EC Name:

Methanol

Cas Number:

67-56-1

Molecular formula:

CH4O

IUPAC Name:

Methanol

impurity 3

Test material form:

liquid: viscous

Specific details on test material used for the study:

- Analytical purity: 82.3%
- Purity test date: Not available
- Lot/batch No.: GN-8
- Expiration date of the lot/batch: Not available
- Appearance: Amber coloured, extremely viscous liquid
- Storage: In the dark at room temperature
- Additional information on test material used for the study: It was attempted to purify the substance to a purity of >80% by using an alternative synthetic route, as opposed to the usual commercial route of synthesis which results in a purity of the substance of approximately 40% in water. This alternative synthetic route generated the substance at 82.3% purity, however it also generated an impurity (methanol at 4.0%) which is not present in the commercial substance. Based on the classification and labelling of methanol, it is considered that this additional impurity would not have an influence on any of the endpoints discussed in this dossier other than the acute oral toxicity study. Therefore all studies (including the one covering this endpoint) contained in this dossier (other than the acute oral toxicity study) were conducted on the 82.3% pure substance containing the 4.0% methanol impurity. The acute oral toxicity study has been conducted on the commercially generated substance at a purity of approximately 40% in water.

Method

Target gene:

Total DNA

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction

Test concentrations with justification for top dose:

For the preliminary toxicity test, a range of concentrations of the test material were used, from 6.75 to 1730 µ/ml, also a set of parallel flasks were set up without whole blood to observe any precipitation within the concentration range. The concentrations of test material used in the main test were 108.13 to 1730 µg/ml.

Vehicle / solvent:

Dissolved in MEM (Minimal essential medium)

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Solvent control

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

in the absence of S9 dissolved in MME Migrated to IUCLID6: Experiment 1: 0.4µg/ml, Experiment 2: 0.2µg/ml

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Vehicle control

True negative controls:

no

Positive controls:

no

Positive control substance:

no

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Solvent control

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

In the presence of S9 dissolved in dimethyl sulphoxide Migrated to IUCLID6: 5µg/ml in both experiments 1 and 2

Details on test system and experimental conditions:

METHOD OF APPLICATION: dissolved in medium


DURATION
- Preincubation period: 48 hours for experiment 1 with and without metabolic activation.
- Exposure duration: Experiment 1: 4 hour exposure with and without S9 Experiment 2: 24 hour continuous exposure without S9 mix and a 4 hour exposure with S9 mix.
- Expression time (cells in growth medium): 20 hours
- Fixation time (start of exposure up to fixation or harvest of cells): Up to 24 hours

Selection agent: Not applicale for a chromosome aberration study

Spindle ihibitor: Demi-C

STAIN (for cytogenetic assays): 5% Gurrs Giesma


NUMBER OF REPLICATIONS: Duplicates cultures were used

NUMBER OF CELLS EVALUATED: 2000 per slide for mitotic index, 100 metaphases for aberrations

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; chromosome damage

Evaluation criteria:

A positive response was recorded if the % cells with aberrations, excluding gaps, markedly exceed that of the concurrent control either with or without a clear dose-relationship. For modest increases a dose response relationship is required along with appropriate statistical tests.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared using the recommendations of the UKEMS (Richardson et al 1989) and where necessary with the concurrent vehicle control value using Fisher's Exact test.

Results and discussion

Test results

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

other: not applicable to this study

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No effects to pH with addition of test substance
- Effects of osmolality: No significant change in osmolality, no increase above 50mOsm
- Precipitation: No precipitation occured


RANGE-FINDING/SCREENING STUDIES: A preliminary toxicity test was conducted on the cells, some toxicity was observed within the 24 hour continiual exposure with metabolic activation


COMPARISON WITH HISTORICAL CONTROL DATA: Comparison of positive controls with historical control data

Remarks on result:

other: other:

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Chormosome Aberration Test- Experiment 1 Without Metabolic Activation -S9 Treatment Group Replicate Mitotic index % Number of Cells Scored Total Number of Aberrations Frequency of Aberrant Cells % (+Gaps) (-Gaps) (+Gaps) (-Gaps) Vehicle Control A 5.15 100 1 0 1 0 B 3.10 100 2 2 2 2 Total (100) 200 3 2 3 (1.5) 2 (1.0) 432.5 µg/ml A 5.75 100 1 0 1 0 B 5.45 100 1 1 1 1 Total (136) 200 2 1 2 (1.0) 1 (0.5) 865 µg/ml A 2.90 100 1 0 1 0 B 1.25 100 0 0 0 0 Total (50) 200 1 0 1 (0.5) 0 (0.0) 1730 µg/ml A 3.25 100 3 2 3 2 B 4.20 100 1 1 1 1 Total (90) 200 4 3 4 (2.0) 3 (1.5) Positive Control MMC 0.4 µg/ml A 3.10 100 13 9 12 9 B 2.50 50a 27 20 21 16 Total (68) 150 40 29 33(22.0) 25 *** (16.7) Chormosome Aberration Test- Experiment 1 With Metabolic Activation +S9 Treatment Group Replicate Mitotic index % Number of Cells Scored Total Number of Aberrations Frequency of Aberrant Cells % (+Gaps) (-Gaps) (+Gaps) (-Gaps) Vehicle Control A 5.65 100 1 0 1 0 B 1.55 100 0 0 0 0 Total (100) 200 1 0 1 (0.5) 0 (0.0) 432.5 µg/ml A 3.15 100 2 2 1 1 B 5.20 100 4 1 3 1 Total (116) 200 6 3 4 (2.0) 2 (1.) 865 µg/ml A 3.25 100 4 4 2 2 B 3.80 100 1 0 1 0 Total (98) 200 5 4 3 (1.5) 2 (1.0) 1730 µg/ml A 3.35 100 2 2 2 2 B 3.25 100 1 1 1 1 Total (92) 200 3 3 3 (1.5) 3 (1.5) Positive Control CP 5 µg/ml A 1.55 100 24 19 14 12 B 1.35 100 34 28 27 21 Total (40) 200 58 47 41(20.5) 33 *** (16.5) Chormosome Aberration Test- Experiment 2 Without Metabolic Activation -S9 Treatment Group Replicate Mitotic index % Number of Cells Scored Total Number of Aberrations Frequency of Aberrant Cells % (+Gaps) (-Gaps) (+Gaps) (-Gaps) Vehicle Control A 6.05 100 1 0 1 0 B 7.15 100 1 1 1 1 Total (100) 200 2 1 2 (1.0) 1 (0.5) 432.5 µg/ml A 5.50 100 3 3 3 3 B 3.70 100 0 0 0 0 Total (70) 200 3 3 3 (1.5) 3 (1.5) 648.75 µg/ml A 3.20 100 1 0 1 0 B 4.80 100 2 1 1 1 Total (61) 200 3 1 2 (1.0) 1 (0.5) 865 µg/ml A 1.70 100 7 5 7 5 B 2.60 100 10 4 8 3 Total (33) 200 17 9 15 (7.5) 8* (4.0) Positive Control MMC 0.4 µg/ml A 3.25 50a 23 21 20 19 B 3.20 50a 28 24 21 20 Total (49) 100 51 45 41(41.0) 39 *** (39.0) Chormosome Aberration Test- Experiment 2 With Metabolic Activation +S9 Treatment Group Replicate Mitotic index % Number of Cells Scored Total Number of Aberrations Frequency of Aberrant Cells % (+Gaps) (-Gaps) (+Gaps) (-Gaps) Vehicle Control A 5.85 100 2 2 2 2 B 4.90 100 0 0 0 0 Total (100) 200 2 2 2 (1.0) 2 (0.0) 865 µg/ml A 4.00 100 0 0 0 0 B 5.00 100 3 1 3 1 Total (84) 200 3 1 2 (1.5) 1 (0.5) 1297.5 µg/ml A 4.05 100 2 0 2 0 B 4.70 100 0 0 0 0 Total (81) 200 2 0 2 (1.0) 0 (0.0) 1730 µg/ml A 3.65 100 4 3 3 2 B 4.65 100 3 3 3 3 Total (77) 200 7 6 6 (3.0) 5 (2.5) Positive Control CP 5 µg/ml A 1.35 100 34 26 20 15 B 2.15 50a 28 22 22 18 Total (33) 150 62 48 42 (28.0) 33 *** (22.0) Mean Frequency of Polyploid cells (%): Experiment 1 Dose level µg/ml Harvest Time 24 Hours 4 Hours Without S9 4 Hours With S9 0 0.0 0.0 432.5 0.0 0.0 865 0.0 0.5 1730 0.0 0.5 MMC 0.4 0.0 NA CP5 NA 0.5 Mean Frequency of Polyploid cells (%): Experiment 2 Dose level µg/ml Harvest Time 24 Hours 4 Hours Without S9 4 Hours With S9 0 0.0 0.0 432.5 0.0 - 648.75 0.0 NA 865 0.5 0.5 1297.5 - 0.0 1730 NA 0.0 MMC 0.2 0.0 NA CP 5 NA 0.5  Key for all tables: MMC = Mitomycin C CP = Cyclophosphamide NA = Not applicable - = Not assessed for mitotic index a = Slide evaluation terminated at 50 cells because approximately 50% cells with aberrations had been observed. *** = P0.001 * = P0.05 NM = No scorable metaphases

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

There were no significant toxicological increases in the frequency of cells with chromosome aberrations with or without activation. The test material is considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

This report describes the results of an in vitro study for the detection of structural chromosome aberrations in cultured mammalian cells. It supplements microbial systems in so far as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al, 1990). The method used followed that described in the OECD Guidelines for tetsing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome aberration Test" and Method B10 of Commission Directive 2000/32/EC. The study design also meets the requirements of the UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity.

Methods:

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. The test material dose levels used were selected using data from a preliminary toxicity test. Four treatment conditions were used for the study, i.e. In experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with a cell harvest after a 20 - hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20 -hour expression. In Experiment 2 , the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

Results:

All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material was only moderately toxic and did not induce any toxicologically significant increases in the frequency of cells with aberrations, in either of two separate experiments when tested up to the 10 mM limit dose.

Conclusion:

The test material was considered to be non-clastogenic to human lymphocytes in vitro.