Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay:

C12 -14 AO: Negative for S. typhimurium strains TA 100, TA 1535, TA 98, TA 1537 and TA 1538 +/- S9 mix (OECD 471)

C10 AO: Negative for S. typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 +/- S9 mix (OECD 471)

C14 AO: Negative for S. typhimurium strains TA 1535, TA 1537, TA 98 & TA 100 +/- S9 mix and E. coli WP2 uvr A +/- S9 mix (OECD 471)

In vitro mammalian cell micronucleus test:

C12 -14 AO: Negative for human peripheral blood lymphocytes +/- S9 mix (OECD 487)

In vitro mammalian cell gene mutation assay:

C12 -14 AO: Negative for Chinese hamster lung fibroblasts (V79) +/- S9 mix (EU Method B.17)

Examination of amine oxides for structural alerts indicating potential genotoxic or carcinogenic potential shows the absence of relevant structural moieties or fragments.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1989-01-31 to 1989-02-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine for Salmonella typhimurium
Species / strain / cell type:
other: S. typhimurium TA 100, TA 1535, TA 1537, TA 1538 and TA 98
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: All five strains are deficient in complete structure of their lipopolysaccharide layer and in DNA excision repair system
Metabolic activation:
with and without
Metabolic activation system:
S-9 rat liver induced with Aroclor 1254
Test concentrations with justification for top dose:
Zero to 10,000 microgram/plate (see Tables 1-10, attached)
Vehicle / solvent:
Compound was dissolved in 100 uL DMSO
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 mix Migrated to IUCLID6: TA 98, TA 100, TA 1535, TA 1537 and TA 1538
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene with TA 98, TA 100, TA 1535, TA 1537 and TA 1538
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without metabolic activation Migrated to IUCLID6: TA 100 and TA 1535
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without metabolic activation Migrated to IUCLID6: TA 1537
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Without metabolic activation Migrated to IUCLID6: TA 98 and TA 1538
Details on test system and experimental conditions:
Preparation and storage of liver homogenate fraction (S-9)

Male Sprague Dawley rats (200-300 g) received a single intraperitoneal injection of Aroclor 1254 (500 mg/kg bodyweight) 5 days before sacrifice. Preparation was performed at 0 to 4 degrees Centigrade using cold sterile solution and glassware. The livers from at least 5-6 animals were removed and pooled before washing in 150 mM KCl (approximately 1 mL/g wet livers). The washed livers were cut into small pieces and homogenized in three volumes of KCl. The homogenate was then centrifuged at 9000 g for 10 minutes. The supernatent (the S-9 fraction) was divided into small portions, frozen rapidly and stored at -80 degrees Centigrade for not longer than three months.

Preparation of S-9 Mix

Sufficient S-9 fraction was thawed immediately before each test at room temperature. One volume of S-9 fraction was mixed with 9 volumes of the S-9 cofactor solution and kept on ice until required. That preparation was termed the S-9 mix and the composition was defined as follows: 8mM magnesium chloride; 33 mM potassium chloride; 5 mM glucose-6-phosphate; 4 mM NADP+; 100 mM phosphate buffer pH 7.4.

Bacteria

Bacteria were grown overnight in nutrient broth (25 g Oxoid Nutrient Broth No 2/L) at 37 degrees Centigrade. A presence of a suitable amount of bacteria in the cell suspension was checked by nephelometry. For inoculation, stock cultures stored at -80 degrees Centigrade were used and the various bacterial strains were periodically identified.

Toxicity experiments and dose range finding

The first experiment was performed with all tester strains using three plates per dose to obtain information on mutagenicity and toxicity for calculation of an appropriate dose range. A reduced rate of spontaneously occurring colonies was used as an indicator for toxicity together with visible thinning of the bacterial lawn. Thinning of the bacterial lawn was controlled microscopically.

In combination with the second experiment, toxicity testing was performed as follows: 0.1 mL of the relevant test compound dilution was thoroughly mixed with 0.1 mL of 10E-6 dilution the overnight culture of TA 100 and plated onto histidine and biotin rich top agar (3 plates per dose).

Mutagenicity test

Top agar was prepared for the Salmonella strains by mixing 100 mL agar (0.6% agar, 0.5 % sodium chloride) with 10 mL of a 0.5 mM histidine-biotin solution. The following ingredients were added (in order) to 2 mL of molten top agar at 45 degrees Centigrade: 0.1 mL of an overnight nutrient broth culture of the bacterial tester strain; 0.1 mL test compound solution; 0.5 mL S-9 mix (if required) or buffer. After mixing, the liquid was poured into a petridish with minimal agar (1.5 % agar, Vogel-Bonner E medium with 2 % glucose).
Evaluation criteria:
The solvent control was compared with the number of colonies per plate in the presence of the test compound. Results were then presented as a ratio of these values (equal to surviving fraction). For the mutagenicity test, colonies (his+ revertants) were counted after incubation for 48 to 72 hours at 37 degrees Centigrade in the dark.
Statistics:
No data
Species / strain:
other: S. typhimurium TA 100, TA 1535, TA 1537, TA 1538 and TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Referring to Tables 1-5 (attached), the test compound proved to be toxic to the bacterial strains at a dose of 2,500 microgram per plate with metabolic activation and very toxic to the bacterial strains at a dose of 20 microgram/plate without metabolic activation. For mutagenicity testing, 2,500 microgram/plate was chosen as the highest dose in the second experiment.

The test compound did not cause a significant increase in the number of revertant colonies with any of the tester strains either in the the absence or presence of S-9 mix. No dose dependent effect is shown by the results presented in Tables 6-10 (attached).
Conclusions:
The test substance is not mutagenic in the presence or absence of an exogenous metabolizing system
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
Please refer to the Amine Oxide Category justification attached in Section 13

2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL
Please refer to the Amine Oxide Category justification attached in Section 13
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine for samlmonella and tryptophan for E.coli.
Salmonella: uvrB-
E.coli: uvrA-
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: incapable of DNA excision repair and deep rough mutation (rfa)
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: incapable of DNA excision repair and deep rough mutation (rfa)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S9
Test concentrations with justification for top dose:
preliminary study: 0, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
main study: 0, 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): WP2uvrA- , TA100 and TA1535; 9-aminoacridine (9AA) for TA1537 and 4-Niroquinoline-1-oxide (4NQO) for TA 98. +S9: 2-Aminoanthracene (2AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 10 hours
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: each test concentration in triplicate in two seperate experiments

DETERMINATION OF CYTOTOXICITY
- Method: growth of bacterial background lawn



Evaluation criteria:
Frequency of revertant colonies
For a substance to be considered positive in the test system it should induce a dose-related and statisically significant increase in the revertant count in one or more strains of bacteria in the presence and/or absence of S9 in both experiments at sub-toxic levels. To be considered negative, the number of revertants at each dose level should be less than two-fold that of the vehicle control frequency.
Statistics:
Kirkland DJ (Ed) (1989) Statitical evaluation of mutagenicity test data. UKEMS Sub-committee on guidlelines for mutagenicity testing, Report Part III, Cambridge University Press.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
500µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
The test substance is not mutagenic in the presence or absence of an exogenous metabolizing system
Executive summary:

In an OECD 471 in vitro gene mutation study in bacteria (Ames Test) the test substance was not mutagenic in either the presence or absence of arochlor-induced rat liver S9.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
2011-11-03 to 2012-01-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
Please refer to the Amine Oxide Category justification attached in Section 13

2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL
Please refer to the Amine Oxide Category justification attached in Section 13
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
his- → his+ reversion
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Arochlor 1254 induced rat liver S9
Test concentrations with justification for top dose:
3.16, 10.0, 31.6, 100, 316 and 1000 µg per plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: completely soluble in water and represents a standard solvent in the testing facility.
Untreated negative controls:
yes
Remarks:
DMSO (vehicle)
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
-S9: TA 98, TA 102, TA 1537 - 2-amino-anthracene in DMSO (2 µg/plate); TA 100, TA 1535 - cyclophosphamide in aqua ad iniectabilia (1500 µg/plate)
Positive control substance:
other: +S9: TA 1535 and TA 100 - sodium azide in aqua ad iniectabilia (10 µg/plate); TA 98 - 2-nitro-fluorene in DMSO (10 µg/plate); TA 1537 - 9-amino-acridine in ethanol, abs. (100 µg/plate); TA 102 - methyl methane sulfonate (MMS) in DMSO (1300 µg/pl
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 2nd test only: 20 minutes
- Exposure duration: 48 - 72 hours
- Expression time (cells in growth medium): 10 hours

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: 10E08

DETERMINATION OF CYTOTOXICITY
- Method: 50% reduction in colony number and scarce background lawn growth
Evaluation criteria:
The AMES test data are evaluated using the statistical method described below. A response is considered positive if
- the number of revertants is significantly increased (p =< 0.05, U-test according to MANN and WHITNEY) compared with the solvent control to at least 2-fold of the solvent control for TA 98, TA 100 and TA 102 and 3-fold of the solvent control for TA 1535 and TA 1537 in both independent experiments;
- in addition, a significant (p =< 0.05) concentration (log value)-related effect (Spearman’s rank correlation coefficient) is observed;
- positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.
The range of spontaneous reversion frequencies in our laboratory is generally:

TA 98: 20 - 60
TA 100: 100 - 200
TA 102: 240 - 320
TA 1535: 10 - 35
TA 1537: 3 - 20

The numbers may be slightly different on plates with S9 and may vary slightly from experiment to experiment.
Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the solvent control and/or a scarce background lawn.
Statistics:
See above.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
1000 µg/plate all test strains, with/without activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

See attached tables

Conclusions:
No mutagenic effect (no significant increase in revertant colony numbers as compared with control counts) was observed for N,N-dimethyldecylamine-N-oxide (solution), tested up to a cytotoxic dose level of 1000 µg N, N-dimethyl-decylamine-N-oxide/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).
Executive summary:

In an in vitro reverse gene mutation (Ames) study performed in accordance with OECD Guideline 471 and EC Method B.13/14, N,N-dimethyldecylamine-N-oxide (solution) was examined in the 5 Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test.

N,N-dimethyldecylamine-N-oxide (solution) was completely dissolved in dimethyl-sulfoxide (DMSO). A correction factor of 2.47 was used to correct for the content of active matter in the supplied solution (40.5%).

Ten concentrations ranging from 0.316 to 5000 µg N,N-dimethyldecylamine-N-oxide/plate were tested in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA 100. Pronounced cytotoxicity (scarce background lawn and reduction of the number of revertants by more than 50%) was noted in the plate incorporation test without and with metabolic activation at concentrations of 1000 µg/plate and higher.

Hence, 1000 µg N,N-dimethyldecylamine-N-oxide/plate was chosen as the maximum dose level for the main study in the plate incorporation test and in the preincubation test. Six concentrations ranging from 3.16 to 1000 µg N,N-dimethyldecylamine-N-oxide per plate were employed in two independent experiments each carried out without and with metabolic activation.

In two independent plate incorporation and preincubation tests, each carried out without and with metabolic activation, cytotoxicity (scarce background lawn and reduction of the number of revertants) was noted at the maximum dose level of 1000 µg/plate in all test strains

No mutagenic effect (no increase in revertant colony numbers as compared with control counts) was observed for the test material, tested up to the cytotoxic dose level of 1000 µg in any of the 5 test strains.

Under the present test conditions the test material tested up to a cytotoxic dose level of 1000 µg N,N-dimethyldecylamine-N-oxide/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 October 2017 - 18 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Lot # 5314-007-001
- Expiration date of the lot/batch: 04 July 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15-25°C, protected from light
Species / strain / cell type:
lymphocytes: human
Remarks:
peripheral blood lymphocytes
Metabolic activation:
with and without
Metabolic activation system:
S-9 prepared from male Sprague-Dawley rats induced with Aroclor 1254.
Test concentrations with justification for top dose:
Range-Finder: 18.14 - 5000 μg/mL
Micronucleus Experiment:
3+21 hrs, -S-9: 10-100 μg/mL
3+21 hrs, +S-9: 15-100 μg/mL
24+24 hrs, -S-9: 5-50 μg/mL
A maximum concentration of 5000 μg/mL was selected for the cytotoxicity Range-Finder Experiment, in order that treatments were performed up to the maximum recommended concentration, for test articles of this type, according to current regulatory test guidelines. Concentrations selected for the Micronucleus Experiment were based on the results of this cytotoxicity Range-Finder Experiment.
Test concentrations are based on the active content.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The test material is manufactured as an approximately 30% aqueous solution, therefore further dilution with water, as necessary, is appropriate.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Sterile purified water
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: vinblastine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours
- Exposure duration: 3 or 24 hours (-S9); 3 hours (+S9)
- Fixation time (start of exposure up to fixation or harvest of cells): 72 or 96 hours (-S9); 72 hours (+S9)

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B; 6 μg/mL

STAIN (for cytogenetic assays): Acridine Orange in phosphate buffered saline; 12.5 μg/mL

NUMBER OF REPLICATIONS: Duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Lymphocytes were kept in fixative at 2-8°C prior to slide preparation for a minimum
of 3 hours to ensure that cells were adequately fixed. Cells were centrifuged (approximately 1250 g, 2-3 minutes) and resuspended in a minimal amount of fresh fixative (if required) to give a milky suspension. Several drops of cell suspension were gently spread onto multiple clean, dry microscope slides. Slides were air-dried and stored protected from light at room temperature prior to staining. Slides were stained by immersion in 12.5 μg/mL Acridine Orange in phosphate buffered saline (PBS), pH 6.8 for approximately 10 minutes and washed with PBS (with agitation) for a few seconds. The quality of the staining was checked. Slides were air-dried and stored protected from light at room temperature. Immediately prior to analysis 1-2 drops of PBS were added to the slides before mounting with glass coverslips.

NUMBER OF CELLS EVALUATED: 1000 binucleate cells from each culture (2000 per concentration) were analysed for micronuclei, where possible.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Scoring was carried out using fluorescence microscopy. Binucleate cells were only included in the analysis if all of the following criteria were met:
1. The cytoplasm remained essentially intact, and
2. The daughter nuclei were of approximately equal size.
A micronucleus was only recorded if it met the following criteria:
1. The micronucleus had the same staining characteristics and a similar morphology to the main nuclei, and
2. Any micronucleus present was separate in the cytoplasm or only just touching a main nucleus, and
3. Micronuclei were smooth edged and smaller than approximately one third the diameter of the main nuclei.

DETERMINATION OF CYTOTOXICITY
- Method: Replication index
- Any supplementary information relevant to cytotoxicity: Slides from the cytotoxicity Range-Finder Experiment were examined, uncoded, for proportions of mono-, bi- and multinucleate cells, to a minimum of 200 cells per concentration. From these data the replication index (RI) was determined.
RI, which indicates the relative number of nuclei compared to vehicle controls was determined using the formula as follows:
RI = (number binucleate cells + 2 (number multinucleate cells))/total number of cells in treated cultures
Relative RI (expressed in terms of percentage) for each treated culture was calculated as follows:
Relative RI (%) = (RI of treated cultures/ RI of vehicle controls) x100
Cytotoxicity (%) is expressed as (100 – Relative RI).
A selection of random fields was observed from enough treatments to determine whether chemically induced cell cycle delay or cytotoxicity had occurred.

OTHER EXAMINATIONS:
- Determination of polyploidy: Not applicable
- Determination of endoreplication: Not applicable
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): Not applicable
Evaluation criteria:
For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed
3. A concentration-related increase in the proportion of MNBN cells was observed (positive trend test).
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result. Biological relevance was taken into account, for example consistency of response within and between
concentrations, or effects occurring only at very toxic concentrations.
Statistics:
The proportions of MNBN cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test.
The proportions of MNBN cells for each treatment condition were compared with the proportion in vehicle controls by using Fisher's exact test.
A Cochran-Armitage trend test was applied to each treatment condition. Probability values of p≤0.05 were accepted as significant.
Key result
Species / strain:
other: human peripheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Water solubility: no
- Precipitation: no
- Definition of acceptable cells for analysis: no
- Other confounding effects: none reported

RANGE-FINDING/SCREENING STUDIES:

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: refer to table below
- Negative (solvent/vehicle) historical control data: refer to table below

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Cytotoxicity (%) is expressed as (100 – Relative RI).

Summary of the findings

Treatment

Concentration (µg/mL)

Cytotoxicity $ (%)

Mean MNBN Cell Frequency (%)

Historical Control Range # (%)

Statistical Significance

3+21 hour -S-9

aVehicle

-

0.28

0.20 – 1.00

-

50

7

0.3

NS

80

30

0.35

NS

90

45

0.35

NS

*MMC, 0.30

27

7.1

p≤0.001

3+21 hour +S-9

aVehicle

-

0.35

0.20 – 1.07

-

60

11

0.45

NS

85

36

0.3

NS

100

58

0.53

NS

*CPA, 2.00

2

1.3

p≤0.001

*CPA, 3.00

10

0.85

p≤0.01

24+24 hour -S-9

aVehicle

-

0.15

0.10 – 0.90

-

5

8

0.4

NS

12.5

21

0.35

NS

20

44

0.6

p ≤0.05

25

55

0.25

NS

*VIN, 0.04

55

3.45

p ≤0.001

a

Vehicle control was water

*

Positive control

#

95thpercentile of the observed range

$

Based on replication index

NS

Not significant 

Conclusions:
It is concluded that the test item did not induce micronuclei in human peripheral blood lymphocytes when tested up to toxic concentrations for 3+21 hours in the absence and presence of a rat liver metabolic activation system (S-9) and for 24+24 hours in the absence of S-9 under the experimental conditions described.
Executive summary:

Amines, C12-14 (even numbered)-alkyldimethyl, N-oxides was tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of two female donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254-induced rats. The test article was formulated in water (purified water). The highest concentrations analysed in the Micronucleus Experiment were limited by toxicity and were determined following a preliminary cytotoxicity Range-Finder Experiment.

Treatments were conducted 48 hours following mitogen stimulation by phytohaemagglutinin (PHA). The test article concentrations for micronucleus analysis were selected by evaluating the effect of Amines, C12-14 (even numbered)-alkyldimethyl, N-oxides on the replication index (RI). Micronuclei were analysed at three or four concentrations. Appropriate negative (vehicle) control cultures were included in the test system under each treatment condition. The proportion of micronucleated binucleate (MNBN) cells in the cultures fell within the 95th percentile of the current observed historical vehicle control (normal) ranges. Mitomycin C (MMC) and Vinblastine (VIN) were employed as clastogenic and aneugenic positive control chemicals respectively in the absence of rat liver S-9. Cyclophosphamide (CPA) was employed as a clastogenic positive control chemical in the presence of rat liver S-9. Cells receiving these were sampled in the Micronucleus Experiment at 24 hours (CPA, MMC) or 48 hours (VIN) after the start of treatment. One concentration of all positive control compounds induced statistically significant increases in the proportion of cells with micronuclei.

All acceptance criteria were considered met and the study was accepted as valid.

Treatment of cells with Amines, C12-14 (even numbered)-alkyldimethyl, N-oxides for 3+21 hours in the absence and presence of S-9 and for 24+24 hours in the absence of S-9 resulted in frequencies of MNBN cells that were similar to those observed in the concurrent vehicle controls and which fell within the normal ranges at all concentrations analysed under each treatment condition. A statistically significant increase in MNBN cell frequency (p≤0.05) was observed at an intermediate concentration of 20 μg/mL for the 24+24 hour treatment in the absence of S-9 but the MNBN cell frequency values for both replicates at this concentration were within the normal range and there was no statistically significant linear trend, therefore this observation was considered not biologically relevant.

It is concluded that Amines, C12-14 (even numbered)-alkyldimethyl, N-oxides did not induce micronuclei in human peripheral blood lymphocytes when tested up to toxic concentrations for 3+21 hours in the absence and presence of a rat liver metabolic activation system (S-9) and for 24+24 hours in the absence of S-9 under the experimental conditions described.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 4 2010 - May 12-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
The preparation of the Aroclor 1254 -induced rat S9 fraction was carried out according to MARON & AMES.
Test concentrations with justification for top dose:
Main study
Five concentrations ranging from 0.313 to 5.0 or 0.625 to 10.0 µg Amine, C10-16-alkyldimethyl, N-oxide (AO-1270)/mL were selected for the experiments without and with metabolic activation, respectively.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: aqua ad iniectibilia
- Justification for choice of solvent/vehicle: substance is soluble in water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
aqua ad iniectabilia
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
aqua ad iniectabilia
True negative controls:
no
Positive controls:
yes
Positive control substance:
9,10-dimethylbenzanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Experiment 1: 4 hr with or without S9
Experiment 2: 4 hr with S9, 24 hr without S9
- Expression time (cells in growth medium): 8 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: 5

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: relative plating efficiency

Evaluation criteria:
If in both independent experiments solvent and positive controls show results within the norm and if the test item does not increase the mutation frequency 2-fold above the mean of the solvent controls under any condition, or if the mutation frequency is always lower than 40 x 10-6 and if at least 1000000 cells per condition have been evaluated, the item is considered as negative in the test.

In case of a dose-dependent increase of the mutation frequency in both independent experiments (at similar concentrations) to at least 2-fold solvent control and at least 40 x 10-6 both in the presence and/or absence of S9 mix, the item is considered as positive in the test.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
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: none noted
- Effects of osmolality: none noted
- Evaporation from medium: not applicable
- Water solubility: completely soluble in water
- Precipitation: none noted
- Other confounding effects:

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxicity in form of decreased plating efficiency (PE1) and (PE2) was noted in both experiments each carried out without and with metabolic activation at the top concentrations of 5.0 or 10.0 µg Amin, C10-16-alkyldimethyl, N-oxide (AO-1270)/mL medium, respectively.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Mutagenicity

Experiments without metabolic activation (4-h or 24-h exposure)

The mutation frequency of the negative control aqua ad iniectabilia was 6.67 and 5.10 x 10-6 clonable cells. Hence, the negative controls were well within the expected range (see below).

The mutation frequency of the cultures treated with concentrations of 0.313, 0.625, 1.25, 2.5 or 5.0 µg Amin, C10-16-alkyldimethyl, N-oxide (AO-1270)/mL culture medium ranged from 1.21 to 7.10 x 10-6 clonable cells. These results are within the normal range of the negative controls.

Experiments with metabolic activation (4-h exposure)

The mutation frequency of the negative controlaqua ad iniectabilia was 5.94 and 2.05 x 10-6clonable cells. Hence, the negative controls were well within the expected range (see below).

The mutation frequency of the cultures treated with concentrations of 0.625, 1.25, 2.5, 5.0 or 10.0 µg Amin, C10-16-alkyldimethyl, N-oxide (AO-1270)/mL culture medium ranged from 0.53 to 7.42 x 10-6 clonable cells. These results are within the normal range of the negative controls.

The positive controls EMS (ethyl methanesulfonate) in the direct test and DMBA (9,10-dimethyl-1,2-benzanthracene) a compound which requires metabolic activation caused a pronounced increase in the mutation frequencies ranging from250.98to1038.82x 10-6 clonable cells in the case of EMS and ranging from66.48to193.85x 10-6 clonable cells in the case of DMBA, indicating the validity of this test system.

The background mutation frequency at LPT ranges from 1.30 to 38.36 x 10-6clonable cells for the negative controls. The mutation frequency of the positive controls at LPT ranges from 112.1 to 1708.4 x 10-6clonable cells forand 130.0 to 2693.3 x 106clonable cells for DMBA.

No relevant change in pH value or osmolality was noted.

Tables of results are attached.

Conclusions:
Nagative, with and without S9 in this assay.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Rodent dominant lethal assay

C12 -14 AO: Negative for mice (C3D2F1/J) (OECD 478)

Link to relevant study records
Reference
Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1974-05-01 to 1975-10-01
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The test substance was evaluated in a rodent dominant lethal test using methods comparable to OECD Test Guideline 478. A repeat-dose treatment regimen (once daily for 5 days) was used; however, justification for this treatment regimen was not provided in the study report. Also, the age and weight of the test animals were not specified in the study report, and housing and feeding conditions of the animals were not reported. The source, purity and other characteristics of the test substance were not reported. A positive control was not concurrently run in the study. Observations of the animals during the experiment, including signs of toxicity, were not reported. The results did not include individual animal data for each mating (i.e., identity of each male and female mated). The mating interval, dose level for males, and the numbers of live implants and dead implants were not reported for each female; only summary results for each dose group were reported.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)
Deviations:
yes
Remarks:
See rationale for reliability above.
Principles of method if other than guideline:
N/A
GLP compliance:
no
Remarks:
N/A
Type of assay:
rodent dominant lethal assay
Species:
mouse
Strain:
other: C3D2F1/J
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Jackson Labs in Bar Harbor, Maine
- Age at study initiation: sexually mature
- Weight at study initiation: N/A
- Assigned to test groups randomly: yes; no data provided in the study on the randomization procedure
- Fasting period before study: N/A
- Housing: N/A
- Diet (e.g. ad libitum): N/A
- Water (e.g. ad libitum): N/A
- Acclimation period: 1 week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): N/A
- Humidity (%): N/A
- Air changes (per hr): N/A
- Photoperiod (hrs dark / hrs light): N/A


IN-LIFE DATES: From: N/A To: N/A
Route of administration:
oral: unspecified
Vehicle:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: N/A
- Concentration of test material in vehicle: N/A
- Amount of vehicle (if gavage or dermal): N/A
- Type and concentration of dispersant aid (if powder): N/A
- Lot/batch no. (if required): N/A
- Purity: N/A
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: N/A


DIET PREPARATION
- Rate of preparation of diet (frequency): N/A
- Mixing appropriate amounts with (Type of food): N/A
- Storage temperature of food: N/A
Duration of treatment / exposure:
The animals were administered the test substance for five consecutive days.
Frequency of treatment:
daily
Post exposure period:
N/A
Remarks:
Doses / Concentrations:
10, 100, and 1000 mg DDAO/kg BW
Basis:
other: No data was provided on whether the oral dose was through gavage, diet or drinking water.
No. of animals per sex per dose:
20
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Positive control(s):
none
- Justification for choice of positive control(s): N/A
- Route of administration: N/A
- Doses / concentrations:N/A
Tissues and cell types examined:
Total implantations, resorptions, and dead embryos were enumerated and recorded.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: N/A


TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Immediately after the males received their final treatment, each male was caged separately with two untreated virgin female mice of the same strain for a period of seven days. This mating procedure was continued weekly for an additional six weeks, thus encompassing the entire spermatogenic cycle of the mouse.
On day 13 or 14 of pregnancy (as measured from the mid-week of presumptive mating), the females were sacrificed. Total implantations, resorptions, and dead embryos were enumerated and recorded.


DETAILS OF SLIDE PREPARATION: N/A


METHOD OF ANALYSIS: The mutagenic potential of the test substance was detected using the following criteria: 1) increase in the frequency of dead implantations, 2) reduction in average number of living embryos, 3) reduction in average number of implantations, and 4) a reduction in the frequency of fertile matings. Corpora lutea counts which are laborious to do and are inaccurate in mice, were omitted. The numbers of total implants in test animals were compared with those in concurrent controls, thus affording a simple measure of preimplantion losses.
Mutagenic indices were calculated by the following equation:
(resorptions + dead embryos)/(total implantations) x 100 = mutagenic index


OTHER: N/A
Evaluation criteria:
N/A
Statistics:
Analysis of the data was carried out by use of the Chi-Square test (p=0.05), DF=1), which directly compares values obtained in each test group to the appropriate control group. Any significant differences between a control and test group in any of the biological parameters mentioned above were interpreted as a mutagenic response.
Sex:
male
Genotoxicity:
negative
Remarks:
N/A
Toxicity:
not specified
Remarks:
N/A
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not applicable
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: N/A
- Solubility: N/A
- Clinical signs of toxicity in test animals: N/A
- Evidence of cytotoxicity in tissue analyzed: N/A
- Rationale for exposure: N/A
- Harvest times: N/A
- High dose with and without activation: N/A
- Other: N/A


RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): N/A
- Induction of micronuclei (for Micronucleus assay): N/A
- Ratio of PCE/NCE (for Micronucleus assay): N/A
- Appropriateness of dose levels and route: N/A
- Statistical evaluation: N/A

The pregnancy rate, as a means of measuring antifertility effects, was not significantly affected by treatment when compared with both control groups.

Total implants (including live fetuses), early fetal deaths, and late fetal deaths, were scored individually for each pregnant mouse and weekly averages were determined for each experimental group. No significant reduction in the average number of total implants per pregnancy, which presumably reflects preimplantation losses of genetically damaged zygotes, was detected in any treated group.

No mutagenic effects, as measured by an increase in the average number of fetal deaths per pregnancy, were evident. Significant or consistent differences were not found in the incidence of early deaths for control and test substance treated mice in the study.

The data indicated no dose response or treatment related mutagenic effects when expressed as the mutagenic index. When it came to group data obtained from uterine examinations all group results were comparable in every respect.

Conclusions:
Three groups of twenty male mice (C3D2F1/J strain) were given daily oral doses of dodecyl dimethyl amine oxide in water for five consecutive days of 10, 100 or 1000 mg/kg BW to test for mutagenicity in the dominant lethal assay. The number of total implantations, resorptions and late deaths were enumerated and recorded. Under conditions of the assay the test substance did not induce a positive mutagenic response at any of the levels tested.
Executive summary:

Three groups of twenty male mice (C3D2F1/J strain) were given daily oral doses of test substance for five consecutive days of 10, 100 or 1000 mg/kg to test for mutagenicity in the dominant lethal assay.

After the last treatment each male was housed with two untreated females for one week. In order to study successive germ cell stages of the males, each male was housed with two additional females per week for a total of seven weeks. On day thirteen of gestation (measured from the mid-week of presumptive mating), the females were sacrificed and uterine examinations were made. The number of total implantations, resorptions and late deaths were enumerated and recorded. Systemic toxicity to the male rats was not noted in the report.

No significant reduction in the average number of total implants per pregnancy, which presumably reflects preimplantation losses of genetically damaged zygotes, was detected in any treated group.

No mutagenic effects, as measured by an increase in the average number of fetal deaths per pregnancy, were evident. Significant or consistent differences were not found in the incidence of early deaths for control and test substance treated mice in the study.

The data indicated no dose response or treatment related mutagenic effects when expressed as the mutagenic index. When it came to group data obtained from uterine examinations all group results were comparable in every respect.

Analysis of these data using the Chi-square test (p= 0.05, DF=1) indicated that the test substance did not induce a positive mutagenic response at any of the levels tested.

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

Additional information

Three bacterial reverse mutation assays are available for category members, all performed to OECD TG 471 under GLP.

C12-14 AO was tested at concentration levels of 0.8-2500 µg/plate against S. typhimurium strains TA100, TA1535, TA98, TA1537 and TA1538 with and without S9 mix [Muller W (1989)]. The concentration levels were selected based on toxicity seen in a preliminary test where the substance was very toxic to the bacterial strains at 20 ug/plate without metabolic activation and toxic at 2500 µg/plate with metabolic activation. Hence 2500 µg/plate was chosen as the highest dose in the second experiment. No precipitation was observed. The tests were negative, in both the presence and absence of S9 mix.

C10 AO was tested at concentration levels of 3.16-1000 µg/plate against S. typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 with and without S9 mix using both the plate incorporation and pre-incubation methods [Flugge C (2012)]. The concentration levels were selected based on toxicity seen in a preliminary plate incorporation test where the substance was toxic to TA 100 strain at 1000 µg/plate and above with or without metabolic activation. In the plate incorporation test and in the pre-incubation test cytotoxicity was observed at the maximum dose of 1000 µg/plate with or without metabolic activation in all strains. The tests were negative both in the presence and absence of S9 mix.

C14 AO was tested at concentration levels of 15-5000 µg/plate against S. typhimurium strains TA1535, TA1537, TA98 and TA 100 and Escherichia coli strain Wp2uvrA-with and without S9 mix [Thompson PW (1997)]. The concentration levels were selected based on toxicity seen in a preliminary test where the substance was toxic to the Salmonella strains and to E. coli at 500 µg/plate and 5000 µg/plate, respectively, without metabolic activation and at 1500 µg/plate and 5000 µg/plate, respectively, with metabolic activation. No precipitation was observed. The tests were negative both in the presence and absence of S9 mix.

C12 -14 AO was tested for mutagenic potential in an in vitro gene mutation assay in cultured mammalian cells (V79, genetic marker HPRT) with and without S9 mix according to EU Method B17 under GLP [Flugge C (2010)]. The duration of exposure was 4 or 24 hours in the experiments without S9 mix and 4 hours in the experiments with S9 mix. In a preliminary cytotoxicity experiment pronounced cytotoxicity was observed at concentrations of 5 or 10 µg AO/mL and above without and with metabolic activation, respectively. Hence high doses of 5 and 10 µg AO/mL were selected for the main tests without or with metabolic activation. Five concentrations ranging from 0.313 to 5.0 or 0.625 to 10.0 µg AO/mL were selected for the experiments without and with metabolic activation, respectively. Cytotoxicity in the form of decreased plating efficiency (PE1) and (PE2) was noted in both experiments each carried out without and with metabolic activation at the top concentrations of 5.0 or 10.0 µg AO/mL medium, respectively. In the experiments without metabolic activation (4-h or 24-h exposure) the mutation frequency of the cultures treated with AO were within the normal range of the controls. In the experiments with metabolic activation (4-h exposure) the mutation frequency of the cultures treated with AO were within the normal range of the controls. The mutation frequencies of the negative controls in both sets of experiments were well within the expected range. The positive controls also gave expected increases in mutation frequencies. It is concluded that the C12-14 AO was negative in this test, with and without metabolic activation.

C12-14 AO was tested in an in vitro micronucleus assay (OECD 487) using duplicate human lymphocyte cultures prepared from the pooled blood of two female donors in a single experiment [Lloyd (2018)]. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254-induced rats. The test article was formulated in water (purified water). The highest concentrations analysed in the Micronucleus Experiment were limited by toxicity and were determined following a preliminary cytotoxicity Range-Finder Experiment.

Treatments were conducted 48 hours following mitogen stimulation by phytohemagglutinin (PHA). The test article concentrations for micronucleus analysis were selected by evaluating the effect of C12-14 AO on the replication index (RI). Micronuclei were analysed at three or four concentrations.

Appropriate negative (vehicle) control cultures were included in the test system under each treatment condition. The proportion of micronucleated binucleate (MNBN) cells in the cultures fell within the 95th percentile of the current observed historical vehicle control (normal) ranges. Mitomycin C (MMC) and Vinblastine (VIN) were employed as clastogenic and aneugenic positive control chemicals respectively in the absence of rat liver S-9. Cyclophosphamide (CPA) was employed as a clastogenic positive control chemical in the presence of rat liver S-9. Cells receiving these were sampled in the Micronucleus Experiment at 24 hours (CPA, MMC) or 48 hours (VIN) after the start of treatment. One concentration of all positive control compounds induced statistically significant increases in the proportion of cells with micronuclei.

All acceptance criteria were considered met and the study was accepted as valid.

Treatment of cells with C12-14 AO for 3+21 hours in the absence and presence of S-9 and for 24+24 hours in the absence of S-9 resulted in frequencies of MNBN cells that were similar to those observed in the concurrent vehicle controls and which fell within the normal ranges at all concentrations analysed under each treatment condition. A statistically significant increase in MNBN cell frequency (p≤0.05) was observed at an intermediate concentration of 20 μg/mL for the 24+24 hour treatment in the absence of S-9 but the MNBN cell frequency values for both replicates at this concentration were within the normal range and there was no statistically significant linear trend, therefore this observation was considered not biologically relevant.

It is concluded that C12-14 AO did not induce micronuclei in human peripheral blood lymphocytes when tested up to toxic concentrations for 3+21 hours in the absence and presence of a rat liver metabolic activation system (S-9) and for 24+24 hours in the absence of S-9 under the experimental conditions described.

In a dominant lethal assay (OECD TG 478) three groups of twenty male mice (C3D2F1/J strain) were given daily oral doses of C12-14 AO for five consecutive days of 10, 100 or 1000 mg/kg. After the last treatment each male was housed with two untreated females for one week. In order to study successive germ cell stages of the males, each male was housed with two additional females per week for a total of seven weeks. On day thirteen of gestation (measured from the mid-week of presumptive mating), the females were sacrificed, and uterine examinations were made. The number of total implantations, resorptions and late deaths were enumerated and recorded. Systemic toxicity to the male rats was not noted in the report.

No significant reduction in the average number of total implants per pregnancy, which presumably reflects preimplantation losses of genetically damaged zygotes, was detected in any treated group.

No mutagenic effects, as measured by an increase in the average number of foetal deaths per pregnancy, were evident. Significant or consistent differences were not found in the incidence of early deaths for control and test substance treated mice in the study.

The data indicated no dose response or treatment related mutagenic effects when expressed as the mutagenic index. When it came to group data obtained from uterine examinations all group results were comparable in every respect.

Analysis of these data using the Chi-square test (p= 0.05, DF=1) indicated that the test substance did not induce a positive mutagenic response at any of the levels tested.

An oral carcinogenicity study performed using C12-14 AO is available (See CSR Section 5.8) in which rats (Charles River CD) were exposed via the oral route for 2 years to C12-14 AO at concentrations of 0.01, 0.1 or 0.2% in feed (equivalent to 4.5, 45 or 90 mg AO/kg bw/day). No neoplastic or non-neoplastic treatment related effects were identified [International Research and Development Corporation (1983)]. A dermal carcinogenicity study is also available [Hazelton Laboratories Inc. (1982)]. In this study 0.1 ml of an aqueous solution of C12-14 AO at 0.05%, 0.13%, and 0.26% (active ingredient) was applied to the dorsal skin of mice (ICR Swiss mice, CD-1), 3 times/week, for 2 years. The study did not result in any carcinogenic response on the exposed skin or systemically.

In addition to the toxicity data that are available, it is also of value to examine the general chemical structure of the amine oxides to determine whether they pose a potential carcinogenic or mutagenic risk. Lai & Woo (2001) identify ten general structural criteria of potential mutagenic and carcinogenic chemicals that are electrophiles or that may generate electrophiles after metabolic transformation. According to Lai and Woo these criteria were developed from a review of all structural classes of chemical carcinogens and SAR analysis of the effects of chemical reactivity, molecular geometry, and metabolism on carcinogenicity. These criteria are listed below:

1. Polycyclic structures with 3 to 6 aromatic rings that mimic the angular ring distribution of carcinogenic polycyclic aromatic hydrocarbons.

2. The presence of an amino, dimethylamino, nitroso or nitro group directly linked to a conjugated double bond system, particularly in instances where the amino or amine generating group is at the terminal end of the longest conjugated double bond system of the molecule.

3. Nitroso, hydrazo, aliphatic azo or aliphatic azoxy structures, 1-aryl-3,3-dialkyltriazenes and 1,1-diaryl-2-acetylenic carbamates.

4. The presence of sterically strained ring (eg epoxide, aziridine, gamma-lactones, and d-sultones) in any type of structure.

5. Low molecular weight carbamates, thiocarbamates and thiourea derivatives.

6.The presence of a haloalkyl (particularly 1,2-dihalo), haloalkenyl (both vinylic and allylic), alpha-haloether, alpha-haloalkanol or alpha-halocarbonyl grouping

7. Low molecular weight aliphatic structures containing conjugated double bonds or isolated double bonds situated at the terminal end of an aliphatic chain.

8. Low molecular weight aldehydes

9. Benzylic, allylic or pyrrolic esters if the acyloxy moiety is a good leaving group.

Amine oxides do not meet any of the above criteria used to classify electrophiles that are likely mutagens or carcinogens. The absence of these structural moieties or fragments on amine oxides provides additional evidence that amine oxides do not pose potential carcinogenic or mutagenic hazards.

 

Based on the in vitro and in vivo studies available of the C12-14 AO itself, the in vitro studies available on other category members, the carcinogenicity studies and the examination of structural alerts it can be concluded that the members of the Amine Oxide category, including C12-14 AO, are not genotoxic.

Justification for classification or non-classification

C10 AO, C14 AO and C12-14 AO were all negative with and without metabolic activation in bacterial reverse mutation assays performed to OECD TG 471.

C12-14 AO was negative with and without metabolic activation in an in vitro gene mutation assay in cultured mammalian cells (V79, genetic marker HPRT) [EU Method B17].

C12-14 AO was negative with and without metabolic activation in an in vitro micronucleus assay using duplicate human lymphocyte cultures [OECD TG 487].

C12-14 AO showed no mutagenic potential in a dominant lethal assay in mice [OECD TG 478].

Two carcinogenicity studies are available for C12-14 AO, one via the oral route in rats and the other via the dermal route in mice. Both were negative.

Examination of amine oxides for structural alerts indicating potential genotoxic or carcinogenic potential shows the absence of relevant structural moieties or fragments.

On the basis of all available evidence, it can be concluded that the members of the Amine Oxide category, including C12-14 AO are not genotoxic and, therefore, classification is not required.