Dodecyldimethylamine oxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No data are available for C12 AO.

C12 -14 AO was negative in a bacterial reverse mutation assay with multiple strains of Salmonella typhimurium [OECD TG 471] both with and without metabolic activation.

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

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

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

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

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

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

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

other: S. typhimurium TA 100, TA 1535, TA 1537, TA 1538 and TA 98

Metabolic activation:

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).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The test substance is not mutagenic in the presence or absence of an exogenous metabolizing system

Reference 2

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

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: Amines,
- Stability under test conditions: 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
#	95^thpercentile 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.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

March 4 2010 - May 12-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed to GLP

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

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 controlaqua ad iniectabiliawas 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 iniectabiliawas 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:

Interpretation of results (migrated information):
negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The available data for mutagenicity from studies performed using analogue susbtances are summarised below.

In a bacterial reverse mutation assay performed according to OECD TG 471 under GLP, C12 -14 AO was tested at concentration levels of 0-10000 µg/plate against Salmonella 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 µg/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.

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 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 performed using C12-14 AO male mice (20/group) were treated via the oral route with the substance at 10, 100 or 1000 mg AO/kg bw for five consecutive days. After the final treatment, the males were mated with untreated females for a period of seven days. Each male was then housed with two additional females for a period of seven weeks. Pregnant females were sacrificed on day 13 or 14 of pregnancy and total implantations, resorptions and dead embyos were counted and recorded. Pregnancy rate was not significantly affected by treatment of male mice with AO. There was no significant reduction in average number of implants per pregnancy, average number of resorptions or dead embryos detected in any treatment group. No mutagenic effects were detected in any treatment group [Procter & Gamble (1975c)].

In addition to the toxicity data that are available, it is also of value to examine the general chemical structure of amine oxides in general 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 carbaomates, thiocarbamates and thiourea derivatives.

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

On the basis of the in vitro and in vivo studies available for C12-14 AO and the examination of structural alerts it can be concluded that amine oxides, including C12 AO, are not genotoxic.

Justification for classification or non-classification

No data are available for C12 AO. C12 -14 AO was negative in a bacterial reverse mutation assay with multiple strains of Salmonella typhimurium [OECD TG 471] both 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 human lymphocyte cultures [OECD TG 487] C12 -14 AO was negative with and without metabolic activation. In vivo, C12-14 AO showed no mutagenic effects in a dominant lethal assay where male mice were dosed for five consecutive days with 10, 100 or 1000 mg AO/kg bw and mated with untreated females for seven days. Examination of amine oxides for structural alerts indicating potential genotoxic or carcinogenic potential shows the absence of relevant structural moieties or fragments.

It is concluded that amines oxides, including C12 AO are not genotoxic and classification is not required.