4-(piperidin-4-yl)morpholine

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:

10 - 27 Sep 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted Jul 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

This study was conducted in accordance with "Standards for Toxicity Investigations" (Ministry of Labor, Notification No. 77, September 1, 1988 and Notification No. 67, June 2, 1997), and "Procedures of Mutagenicity Test Using Microorganisms and Evaluation of Test Results" (Ministry of Labor, Official Notification, February 8, 1999) and "III Mutagenicity test" of "Reverse-Mutation Assay in Bacteria" prescribed in "Testing Methods Relating to the New Chemical Substances" (Notification No. 1121002 of the Pharmaceutical and Food Safety Bureau, MHLW, No. 2 (2003.11.13) of the Manufacturing Industries Bureau, METI, No. 031121002 of the Environmental Health Department, MOB (November 21, 2003).

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-(piperidin-4-yl)morpholine

EC Number:

627-580-3

Cas Number:

53617-35-9

Molecular formula:

C9H18N2O

IUPAC Name:

4-(piperidin-4-yl)morpholine

Method

Target gene:

his operon, trp operon

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with a combination of phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

First experiment (range finding test): 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate with and without metabolic activation
Second experiment (main test): 156, 313, 625, 1250 and 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: The test substance solution of 50.0 mg/mL prepared with distilled water was considered to be stable, nased on the facts that there were no change in color, exothermic reaction nor gas generation at room temperature within 2 h after preparation

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: sodium azide (0.5 µg/plate) for TA1535; furylfuramide (0.01 or 0.1 µg/plate) for TA100, TA98 and WP2uvrA; ICR-191 (0.5 µg/plate) for TA1537; +S9: 2 aminoanthracene (0.5, 1, 2 or 10 µg/plate) for all strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 2 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: bacterial growth inhibition

Evaluation criteria:

The substance was judged to be positive when the number of revertant colonies increased to twice or more than that in the negative control and when the responses were dose-related and/or reproducible. The other cases were judged to be negative.

Statistics:

Mean values were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test substance was not observed.

RANGE-FINDING/SCREENING STUDIES: The pre-experiment served as Experiment I (all strains were tested in the pre-experiment).

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

Any other information on results incl. tables

Table 1: Results of the main test

With or without S9-Mix	Test substance concentration µg/plate	Mean number of revertant colonies per plate average of 2 plates
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2uvrA	TA98	TA1537
–	0	102	15	26	19	8
–	156	119	11	25	23	14
–	313	114	19	30	28	8
–	625	119	10	28	20	11
–	1250	132	17	27	21	8
	2500	123	17	18	23	8
–	5000*	97	6	28	10	3
Positive controls, –S9	Name	AF-2	NaN3	AF-2	AF-2	ICR-191
	Concentrations (μg/plate)	0.01	0.5	0.01	0.1	0.5
	Mean No. of colonies/plate (average of 2)	777	253	331	360	1387
+	0	111	12	28	24	20
+	156	114	18	30	24	18
+	313	107	22	36	30	15
+	625	107	17	21	27	10
+	1250	121	12	33	28	18
+	2500	122	13	30	30	10
	5000*	105	8	34	23	12
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	1	2	10	0.5	2
	Mean No. of colonies/plate (average of 2)	1433	285	379	314	484

AF-2 = Furylfuramide

NaN3: Sodium azide

2AA = 2-Aminoanthracene

* = bacterial growth inhibition was observed

Table 2: Historical control data (Mar - Aug 2010)

Negative control	-S9 mix					+S9 mix
	TA100	TA1535	WP2uvrA	TA98	TA1537	TA100	TA1535	WP2uvrA	TA98	TA1537
Mean	109	11	24	20	9	114	10	29	29	18
3xSD	16	4	6	6	4	17	4	7	7	6
Max	157	23	42	38	21	165	22	50	50	36
Min	61	1	6	2	1	63	1	8	8	1
Positive control	-S9 mix					+S9 mix
	TA100	TA1535	WP2uvrA	TA98	TA1537	TA100	TA1535	WP2uvrA	TA98	TA1537
Chemical	AF-2	NaN3	AF-2	AF-2	ICR-191	2AA	2AA	2AA	2AA	2AA
Dose	0.01	0.5	0.01	0.1	0.5	1	2	10	0.5	2
Mean	712	311	353	440	1617	1027	257	541	304	300
3xSD	70	50	57	79	268	145	46	89	34	76
Max	922	461	524	677	2421	1462	395	808	406	528
Min	502	161	182	203	813	592	119	274	202	72

AF-2 = Furylfuramide

NaN3: Sodium azide

2AA = 2-Aminoanthracene