1-(bis(2-(1,3-dimethylbutylideneamino)ethyl)amino)-3-phenoxypropan-2-ol

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:

26-06-2018 to 26-07-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

1-(bis(2-(1,3-dimethylbutylideneamino)ethyl)amino)-3-phenoxypropan-2-ol

EC Number:

271-340-7

EC Name:

1-(bis(2-(1,3-dimethylbutylideneamino)ethyl)amino)-3-phenoxypropan-2-ol

Cas Number:

68541-07-1

Molecular formula:

C25H43N3O2

IUPAC Name:

1-(bis(2-(1,3-dimethylbutylideneamino)ethyl)amino)-3-phenoxypropan-2-ol

Test material form:

liquid

Specific details on test material used for the study:

Identification: 1-(bis(2-(1,3-dimethylbutylideneamino) ethyl)amino)-3-phenoxypropan-2-ol
Appearance: Light yellow liquid
Batch: UL18401660
Purity/Composition: ca. 91.48%
Test item storage: At room temperature
Stable under storage conditions until: 15 April 2020 (expiry date)
Purity/Composition correction factor: Yes, correction factor is 1.09 according to purity.
Test item handling: No specific handling conditions required
Stability at higher temperatures: Not indicated

Method

Target gene:

S. typhimurium: Histidine locus
E.coli: Tryptophan operon

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other: rfa; gal;chl;bio;vvrB

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).

Test concentrations with justification for top dose:

expt 1: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate based on range finding study.
expt 2: 2.5, 5,10, 100, 500, 1500, 5000 µg/plate. The highest concentration of the test item used in the subsequent mutation assays was 5000 µg/plate or the level at which the test item inhibited bacterial growth.

Preparation of Test Item
A correction factor of 1.09 for the purity of the test item was applied in this study.
A solubility test was performed based on visual assessment. The test item formed a clear colourless solution in DMSO.
Test item concentrations were used within 2.5 hours after preparation.
Any residual volumes were discarded.

Vehicle / solvent:

The vehicle of the test item was dimethyl sulfoxide

Controls

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191: TA1537 without S9 2.5µg/plate; 2-aminoanthracene All strains with S9

Details on test system and experimental conditions:

Preparation of bacterial cultures
Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1°C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109 cells/mL). Freshly grown cultures of each strain were used for testing.

Agar plates
Agar plates (ø 9 cm) containing 25 mL glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Oxoid LTD) in Vogel-Bonner Medium E, 20 g glucose (Fresenius Kabi, Bad Homburg, Germany). The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 µg/plate biotin (Merck) and 15 µg/plate histidine (Sigma) and the agar plates for the test with the Escherichia coli strain contained
15 µg/plate tryptophan (Sigma).

Top agar
Milli-Q water containing 0.6% (w/v) bacteriological agar (Oxoid LTD) and 0.5% (w/v) sodium chloride (Merck) was heated to dissolve the agar. Samples of 3 mL top agar were transferred into 10 mL glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3°C.

Environmental conditions
All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C (actual range 34.9 - 39.2°C). The temperature was continuously monitored throughout the experiment. Due to addition of plates (which were at room temperature) to the incubator or due to opening and closing the incubator door, temporary deviations from the temperature may occur. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Metabolic Activation System
Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
Each S9 batch is characterized with the mutagens benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 µg/plate and 2.5 µg/plate, respectively.

Preparation of S9-Mix
S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per
10 mL: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 mL or 5.0 mL Milli-Q water (first or second experiment respectively) (Millipore Corp., Bedford, MA., USA); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution (Merck); 1 mL 0.33 M KCl solution (Merck). The above solution was filter (0.22 µm)-sterilized. To 9.5 mL of
S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the
S9-mix in the first experiment and to 9.0 mL of S9-mix components 1.0 mL S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment.

Experimental Design
Dose-range Finding Test
Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate. The highest concentration of the test item used in the subsequent mutation assays was 5000 µg/plate or the level at which the test item inhibited bacterial growth.

Mutation Assay
At least five different doses (increasing with approximately half-log steps) of the test item were tested in triplicate in each strain. The above mentioned dose-range finding study with the two tester strains TA100 and WP2uvrA, is reported as a part of the first mutation experiment. In the second part of this experiment, the test item was tested both in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. In a follow-up experiment with additional parameters, the test item was tested both in the absence and presence of 10% (v/v) S9-mix in all tester strains.
The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.
Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture
(109 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test item in DMSO and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

Colony Counting
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

Rationale for test conditions:

ACCEPTABILITY CRITERIA
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without
S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Evaluation criteria:

INTERPRETATION
No formal hypothesis testing was done.
In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.
 
A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

not applied

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

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

Test results 2

Species / strain:

S. typhimurium TA 1537

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

Test results 3

Species / strain:

S. typhimurium 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

Test results 4

Species / strain:

S. typhimurium TA 100

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

Test results 5

Species / strain:

E. coli WP2 uvr A

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

Any other information on results incl. tables

Dose-range Finding Test/First Mutation Experiment

The test item was tested in the tester strains TA100 and WP₂uvrAat concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate in the absence and presence of S9-mix. 

Based on the results of the dose-range finding test, the following dose-range was selected for the first mutation experiment with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 5.4, 17, 52, 164, 512 and 1600 μg/plate. Since no dose level with toxicity and/or precipitation was tested in tester strain TA1535 an additional experiment was performed to complete the data of the first mutation experiment. In this additional experiment, the test item was tested in tester strain TA1535 in the presence of
S9-mix at the concentration of 5000 µg/plate. This additional experiment is reported as part of the first mutation assay.

The results are shown in Table 1 and Table 2. 

Precipitate

Precipitation of the test item on the plates was only observed at the start of the incubation period in tester strains TA100 and WP₂uvrAat the highest tested concentration. At the end of the incubation period precipitation was only observed in tester strain WP₂uvrAin the presence of S9-mix at the highest tested concentration. 

Toxicity

To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.

Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix, except in tester strainWP₂uvrAin the presence of S9-mix.

Mutagenicity

No increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

Second Mutation Experiment

To obtain more information about the possible mutagenicity of the test item, a second mutation experiment was performed in the absence and presence of 10% (v/v) S9-mix. Based on the results of the first mutation assay, the test item was tested up to concentrations of
1500 µg/plate and 5000 µg/plate in all the tester strains in the absence and presence of
10% (v/v) S9-mix, respectively. Except in tester strain WP₂uvrAin the absence of S9-mix and tester strain TA100 in the presence of S9-mix, where the test item was tested up to concentrations of 5000 and 1500 µg/plate, respectively. 

Precipitate

Precipitation of 1-(bis(2-(1,3-dimethylbutylideneamino) ethyl)amino)-3-phenoxypropan-2-ol on the plates was not observed at the start or at the end of the incubation period. 

Toxicity

In the second mutation assay, cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.

Mutagenicity

In the second mutation assay, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

Table1          
Dose-Range Finding Test: Mutagenic Response of the test item in theSalmonella typhimuriumReverse Mutation Assay and in theEscherichia coliReverse Mutation Assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (±S.D.) with oneSalmonella typhimuriumand oneEscherichia colistrain.
	TA100	WP2uvrA

 

Without S9-mix

 

Positive control	1135	±	22		1474	±	155
Solvent control	141	±	18		25	±	1
1.7	131	±	16		28	±	11
5.4	131	±	26		33	±	6
17	142	±	29		29	±	9
52	132	±	16		29	±	8
164	127	±	9		28	±	7
512	22	±	10		27	±	4	^n
1600	3	±	2	^n	10	±	3	^s
5000	0	±	0	^a NP				^e MC NP

 

With S9-mix¹

 

Positive control	1195	±	77		469	±	26
Solvent control	130	±	4		40	±	6
1.7	130	±	14		34	±	12
5.4	124	±	23		40	±	5
17	125	±	14		45	±	10
52	133	±	22		34	±	4
164	105	±	7		34	±	6
512	101	±	19	^n	37	±	4
1600				^e MC	30	±	7	^NP
5000	0	±	0	^a NP	14	±	6	^n SP

 

^1	Plate incorporation assay (5% S9)
MC	Microcolonies
NP	No precipitate
SP	Slight Precipitate
a	Bacterial background lawn absent
e	Bacterial background lawn extremely reduced
n	Normal bacterial background lawn
s	Bacterial background lawn slightly reduced

Table2          
Experiment 1: Mutagenic Response of the test item in theSalmonella typhimuriumReverse Mutation Assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains ofSalmonella typhimurium.
	TA1535	TA1537	TA98

 

Without S9-mix

 

Positive control	619	±	41		936	±	38		733	±	145
Solvent control	8	±	2		6	±	4		14	±	2
5.4	8	±	3		4	±	1		9	±	2
17	9	±	3		6	±	2		10	±	2
52	7	±	4		5	±	3		9	±	3
164	7	±	4	^n	5	±	2	^n	11	±	6
512	6	±	3	^s	3	±	2	^s	4	±	2	^n
1600				^e MC NP				^e MC NP				^e MC NP

 

With S9-mix¹

 

Positive control	217	±	74		232	±	94		1043	±	171
Solvent control	9	±	3		6	±	5		11	±	2
5.4	11	±	1		4	±	1		13	±	4
17	10	±	6		5	±	4		14	±	3
52	7	±	4		8	±	3		15	±	4
164	10	±	3		6	±	3		14	±	5
512	10	±	9		5	±	4		9	±	4	^n
1600	8	±	1	^n NP	0	±	0	^n NP	2	±	2	^s NP
5000^2	0	±	0	^NP a	-				-

 

-	Not tested
MC	Microcolonies
NP	No precipitate
a	Bacterial background lawn absent
e	Bacterial background lawn extremely reduced
n	Normal bacterial background lawn
s	Bacterial background lawn slightly reduced
^1	Plate incorporation assay (5% S9)
^2	Data from additional experiment, positive control and negative control are presented in the table below:

 

With S9-mix

 

Positive control	283	±	55
Solvent control	7	±	3

Table3          
Experiment 2: Mutagenic Response of the test item in theSalmonella typhimuriumReverse Mutation Assay and in theEscherichia coliReverse Mutation Assay

 

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains ofSalmonella typhimuriumand oneEscherichia colistrain.
	TA1535	TA1537	TA98	TA100	WP2uvrA

 

Without S9-mix

 

Positive control	797	±	52		919	±	100		855	±	74		730		±	220		1145	±	122
Solvent control	8	±	2		6	±	5		10	±	2		113		±	7		18	±	4
2.5	10	±	1		10	±	0		14	±	4		131		±	18			-
5	9	±	6		8	±	2		11	±	6		119		±	9		18	±	3
10	7	±	3		4	±	3		16	±	6		132		±	11		19	±	7
100	17	±	6	^n	4	±	3	^n	9	±	5	^n	122		±	14	^n	18	±	4
500	8	±	3	^s	4	±	1	^m	9	±	2	^s	83		±	7	^s	22	±	5	^n
1500				^e MC NP	0	±	0	^a NP				^e MC NP		0	±	0	^a NP	19	±	1	^s
5000		-				-				-					-						^e MC NP

 

 

With S9-mix¹

 

Positive control	191	±	70		378	±	117		676	±		26		1209	±	256		252	±	32
Solvent control	9	±	3		8	±	2		17	±		3		102	±	26		27	±	7
2.5	-				-				-					93	±	10			-
5	14	±	6		6	±	1		19	±		4		107	±	9		28	±	8
10	10	±	6		9	±	3		17	±		6		106	±	9		26	±	5
100	10	±	9		3	±	3		15	±		10		90	±	12		24	±	10
500	12	±	3		6	±	2		10	±		2		83	±	17	^n	23	±	2
1500	12	±	4	^n	2	±	1	^n	10	±		3	^n	8	±	12	^s NP	19	±	3	^n
5000	0	±	0	^a NP	0	±	0	^a NP	0	±		0	^a NP		-			10	±	2	^s NP

 

 

-	Not tested
^1	Plate incorporation assay (10% S9)
MC	Microcolonies
NP	No precipitate
a	Bacterial background lawn absent
e	Bacterial background lawn extremely reduced
m	Bacterial background lawn moderately reduced
n	Normal bacterial background lawn
s	Bacterial background lawn slightly reduced

 

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results of this study it is concluded that 1-(bis(2-(1,3-dimethylbutylideneamino) ethyl)amino)-3-phenoxypropan-2-ol is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The objective of this study was to determine the potential of 1-(bis(2-(1,3-dimethylbutylideneamino) ethyl)amino)-3-phenoxypropan-2-ol and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP₂uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9). 

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch UL18401660 of the test item was a light yellow liquid. A correction factor of 1.09 was used to correct for the purity (91.48%). The vehicle of the test item was dimethyl sulfoxide.

In the dose-range finding test, the test item was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP₂uvrA. The test item precipitated only on the plates at the highest dose level in tester strain WP₂uvrA. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in both tester strains in the absence and presence of S9-mix, except in tester strainWP₂uvrAin the presence of S9-mix. Results of this dose-range finding test were reported as part of the first mutation assay.

Based on the results of the dose-range finding test, the test item was tested in the first mutation assay up to concentrations of 1600 µg/plate in all tester strains. Except in tester strain TA1535 in the presence of S9-mix, where the test item was tested up to concentrations of 5000 µg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.

In a follow-up experiment of the assay with additional parameters, the test item was tested up to concentrations of 1500 µg/plate and 5000 µg/plate in all the tester strains in the absence and presence of 10% (v/v) S9-mix, respectively. Except in tester strain WP₂uvrAin absence of S9-mix and tester strain TA100 in presence of S9-mix the test item was tested up to concentrations of 5000 and 1500 µg/plate, respectively. The test item did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix. 

The test item did not induce a significant dose-related increase in the number of revertant (His⁺) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp⁺) colonies in the tester strain WP₂uvrAboth in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

In conclusion, based on the results of this study it is concluded that 1-(bis(2-(1,3-dimethylbutylideneamino) ethyl)amino)-3-phenoxypropan-2-ol is not mutagenic in theSalmonella typhimuriumreverse mutation assay and in theEscherichia colireverse mutation assay.