N,N-Bis(2-hydroxyethyl)-C12-18(even numbered, C18 unsaturated) alkyl-1-amine oxides

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phase of this study was performed between 09 September 2009 and 11 October 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: read-across

Justification for type of information:

The registration substance is a tertiary amine oxide and the read-across substance the corresponding tertiary amine so that an inter-convertibility of the registration substance and the proposed read-across substance can be presumed.

Reason / purpose for cross-reference:

assessment report

Remarks on result:

other: The assessment is based on the read-across

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Ethanol, 2,2'-iminobis-, N-coco alkyl derivs.

EC Number:

263-163-9

EC Name:

Ethanol, 2,2'-iminobis-, N-coco alkyl derivs.

Cas Number:

61791-31-9

IUPAC Name:

61791-31-9

Constituent 2

Reference substance name:

N,N-Bis(2-hydroxyethyl)-C12-18(even numbered)alkyl-amine

IUPAC Name:

N,N-Bis(2-hydroxyethyl)-C12-18(even numbered)alkyl-amine

Details on test material:

Sponsor's identification:Bis (2-hydroxyethyl) coco alkylamine (CAS Number 61791-31-9)
Description : Pale brown viscous liquid
Purity : 99.9%
Batch number : S-001016
Date received : 08 July 2009
Storage conditions: Approximately 4ºC in the dark under nitrogen

Method

Target gene:

Histidine for Salmonella.
Tryptophan for E.Coli

Species / strain

Species / strain 1

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:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta­naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment one: Salmonella strains (absence of S9): 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate.
Salmonella strains (presence of S9), E.coli strain WP2uvrA- (absence and presence of S9): 1.5, 5, 15, 50, 150, 500, 1500 µg/plate.
Experiment two: Salmonella strains (absence of S9): 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate.
Salmonella strains (presence of S9), E.coli strain WP2uvrA- (absence and presence of S9): 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide.
- Justification for choice of solvent/vehicle: The test material was fully miscible in dimethyl sulphoxide at 50 mg/ml in solubility checks performed in house. Distilled water was not evaluated as a potential vehicle in this test system as information provided by the sponsor suggested it was immiscible with the test material. Dimethyl sulphoxide was therefore selected as the vehicle.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 1 µg/plate

Remarks:

With S9 mix

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Controls 3

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Controls 4

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 10 µg/plate

Remarks:

With S9 mix

Controls 5

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix Migrated to IUCLID6: Benzo(a)pyrene: 5 µg/plate

Controls 6

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix Migrated to IUCLID6: 4-Nitroquinoline-1-oxide: 0.2 µg/plate

Controls 7

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix Migrated to IUCLID6: 9-Aminoacridine: 80 µg/plate

Controls 8

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 3 µg/plate

Controls 9

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate

Controls 10

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulphoxide

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 - 72 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:

Acceptance Criteria:

The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 109 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should not be an excessive loss of plates due to contamination.

Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Statistics:

Standard deviation
Dunnett's Linear Regression Analysis

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the first experiment (plate incorporation method) the test material caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially at 150 and 500 µg/plate in the absence and presence of S9, respective

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

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:

In the first experiment (plate incorporation method) the test material caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially at 150 and 500 µg/plate in the absence and presence of S9, respective

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility: The test material was fully miscible in dimethyl sulphoxide at 50 mg/ml in solubility checks performed in house. Distilled water was not evaluated as a potential vehicle in this test system as information provided by the sponsor suggested it was immiscible with the test material. Dimethyl sulphoxide was therefore selected as the vehicle.
- Precipitation: A precipitate (particulate in appearance) was initially noted at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The test material initially exhibited toxicity to the strains of bacteria used (TA100 and WP2uvrA-) from 150 and 500 µg/plate, respectively. The test material formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the first experiment (plate incorporation method) the test material caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially at 150 and 500 µg/plate in the absence and presence of S9, respectively. In the second experiment (pre-incubation method) the test material induced toxicity to the bacterial background lawns of all of the tester strains initially from 50 µg/plate. The test material was, therefore tested up to the toxic limit.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

 RESULTS

Preliminary Toxicity Test

The test material initially exhibited toxicity to the strains of bacteria used (TA100 and WP2uvrA^-) from 150 and 500 µg/plate, respectively.  The test material formulation and S9-mix used in this experiment were both shown to be sterile.

The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-) S9-mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	87	69	71	71	84	84	71	0V	0T	0T	0TP
+	TA100	73	86	95	77	80	62	83	77	0V	0T	0TP
-	WP2uvrA^-	32	33	24	28	31	25	25	17	0V	0T	0TP
+	WP2uvrA^-	30	33	28	26	27	21	20	31	15S	0T	0TP

S         Sparse bacterial background lawn
V         Very weak bacterial background lawn
T         Toxic, no bacterial background lawn

P         Precipitate

MutationTest

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.

Results for the negative controls (spontaneous mutation rates) are presented inTable 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation, are presented in Table 2 to Table 5.

In the first experiment (plate incorporation method) the test material caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially at 150 and 500 µg/plate in the absence and presence of S9, respectively. In the second experiment (pre-incubation method) the test material induced toxicity to the bacterial background lawns of all of the tester strains initially from 50 µg/plate. The test material was, therefore tested up to the toxic limit. A precipitate (particulate in appearance) was initially noted at and above 500 µg/plate, this observation did not prevent the scoring of revertant colonies.

No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. A small, statistically significant increase in TA100 revertant colony frequency was observed (presence of S9) at 5 µg/plate in Experiment 1. This response was considered not to be toxicologically significant because it was non-reproducible in three separate Experiments (including the Preliminary Toxicity Assay), the mean revertant count at 5 µg/plate was only 1.17 times the concurrent vehicle control value and individual revertant counts were within the acceptable in-house historical range for the bacterial tester strain.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

 

Table 1               Spontaneous Mutation Rates (Concurrent Negative Controls

Range-finding Test

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
123		11		22		26		10
98	(105)	15	(15)	18	(19)	16	(22)	12	(11)
95		18		18		24		11

Main Test

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
128		15		21		22		14
100	(108)	16	(16)	22	(22)	22	(22)	12	(12)
95		18		22		22		11

 

Table 2               Test Results: Range-Finding Test– Without Metabolic Activation

 

Test Period		From: 27 September 2009					To: 30 September 2009
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA^-			TA98		TA1537
-	0	109 97 89	(98) 10.1#	14 14 18	(15) 2.3	25 22 21		(23) 2.1	17 22 21	(20) 2.6	10 10 8	(9) 1.2
-	0.5	96 102 85	(94) 8.6	13 21 15	(16) 4.2	N/T			22 18 16	(19) 3.1	13 13 8	(11) 2.9
-	1.5	85 99 106	(97) 10.7	15 14 14	(14) 0.6	24 19 16		(20) 4.0	17 17 18	(17) 0.6	9 12 11	(11) 1.5
-	5	114 98 106	(106) 8.0	15 15 17	(16) 1.2	19 19 22		(20) 1.7	22 20 25	(22) 2.5	9 9 10	(9) 0.6
-	15	104 92 110	(102) 9.2	17 16 12	(15) 2.6	19 19 22		(20) 1.7	22 20 19	(20) 1.5	15 9 10	(11) 3.2
-	50	136 105 90	(110) 23.5	13 19 16	(16) 3.0	19 20 21		(20) 1.0	19 18 18	(18) 0.6	7 8 7	(7) 0.6
-	150	92 V 49 V 48 V	(63) 25.1	7 V 6 V 15 V	(9) 4.9	20 17 17		(18) 1.7	0 V 0 V 0 V	(0) 0.0	0 V 0 V 0 V	(0) 0.0
-	500	0 TP 0 TP 0 TP	(0) 0.0	0 TP 0 TP 0 TP	(0) 0.0	0 V 0 V 0 V		(0) 0.0	0 TP 0 TP 0 TP	(0) 0.0	0 TP 0 TP 0 TP	(0) 0.0
-	1500	N/T		N/T		0 TP 0 TP 0 TP		(0) 0.0	N/T		N/T
Positive controls   S9-Mix   -	Name Concentration (μg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO		9AA
		3		5		2			0.2		80
		383 293 381	(352) 51.4	205 276 243	(241) 35.5	719 761 668		(716) 46.6	191 106 126	(141) 44.4	1011 1003 913	(976) 54.4

 ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

N/T     Not tested at this dose level

P        Precipitate

T        Toxic, no bacterial background lawn

V        Very weak bacterial background lawn

#        Standard deviation

Table 3               Test Results: Range-Finding Test– With Metabolic Activation

Test Period		From: 27 September 2009					To: 30 September 2009
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA^-			TA98		TA1537
+	0	80 84 85	(83) 2.6#	15 11 12	(13) 2.1	22 19 20		(20) 1.5	24 24 24	(24) 0.0	8 7 11	(9) 2.1
+	1.5	82 90 91	(88) 4.9	11 10 11	(11) 0.6	21 21 20		(21) 0.6	25 24 25	(25) 0.6	13 10 13	(12) 1.7
+	5	101 101 90	$$$ (97) 6.4	10 8 12	(10) 2.0	22 19 19		(20) 1.7	21 25 18	(21) 3.5	7 12 7	(9) 2.9
+	15	82 90 81	(84) 4.9	11 7 10	(9) 2.1	20 21 24		(22) 2.1	21 17 17	(18) 2.3	10 9 9	(9) 0.6
+	50	89 88 80	(86) 4.9	8 9 8	(8) 0.6	19 19 22		(20) 1.7	24 27 22	(24) 2.5	9 8 8	(8) 0.6
+	150	80 79 80	(80) 0.6	8 9 8	(8) 0.6	17 18 20		(18) 1.5	21 20 15	(19) 3.2	12 11 10	(11) 1.0
+	500	0 V 0 V 0 V	(0) 0.0	0 T 0 T 0 T	(0) 0.0	12 S 9 S 11 S		(11) 1.5	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0
+	1500	0 TP 0 TP 0 TP	(0) 0.0	0 TP 0 TP 0 TP	(0) 0.0	0 TP 0 TP 0 TP		(0) 0.0	0 TP 0 TP 0 TP	(0) 0.0	0 TP 0 TP 0 TP	(0) 0.0
Positive controls   S9-Mix   +	Name Concentration (μg/plate) No. colonies per plate	2AA		2AA		2AA			BP		2AA
		1		2		10			5		2
		1510 2079 2379	(1989) 441.4	112 156 183	(150) 35.8	371 340 348		(353) 16.1	215 251 208	(225) 23.1	313 292 314	(306) 12.4

2AA    2-Aminoanthracene

BP      Benzo(a)pyrene

P        Precipitate

S        Sparse bacterial background lawn

T        Toxic, no bacterial background lawn

V        Very weak bacterial background lawn

$$$     p<0.005

#        Standard deviation

PLEASE SEE OVERALL REMARKS,) Tables 4 and 5 (Experiment 2)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The read-across supporting substance N,N-Bis(2-hydroxyethyl)-C12-18(even numbered)alkyl-amine did not induce any genotoxicity effect in Ames test (OECD 471). No significant mutagenicity can be derived for the registration substance.

Executive summary:

The genotoxicity of the registration substance is derived by use of data on the read-across supporting substance N,N-Bis(2-hydroxyethyl)-C12-18(even numbered)alkyl-amine.

N,N-Bis(2-hydroxyethyl)-C12-18(even numbered)alkyl-amine was investigated for its genotoxicity according to the OECD Guideline 471. No significant gentoxicity was found.

Likewise, no significant genotoxicity is expected for the registration substance.