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:

1981-06-30

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1981

Report date:

1981

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Method: Ames et al. (1975), Mutation Research 31, 347-364

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Morpholine

EC Number:

203-815-1

EC Name:

Morpholine

Cas Number:

110-91-8

Molecular formula:

C4H9NO

IUPAC Name:

morpholine

Test material form:

liquid

Method

Target gene:

In the Salmonella typhimurium strains (TA 1535, TA 100, TA 1537, TA 98) the amino acid histidine locus is the target gene, in which induced back mutations will transform the histidine auxotrophy (his-) to histidine prototrophy (his+).
The Salmonella typhimurium histidine (his) reversion system measures his- => his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations.

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:

Type and composition of metabolic activation system: S9 mix
- source of S9
Male Sprague Dawley rats (200-300 g) received a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight, dissolved in sunflower oil 1:5 v/v) 6 days before sacrifice. The liver was homogenized in three volumes of sterile, cold 150 mM KCl buffered with 10 mM Na phosphate at pH 7.4 . The homogenate was centrifuged at 10,000 g for 10 minutes. One volume of the resulting supernatant fraction was mixed with one volume of 24 mM MgCl2 containing 100 mM KCl and one volume of a solution which contained 12 mM NADP, 15 mM glucose-6-phosphate, and 150 mM Na phosphate buffer, pH 7.4 .

Test concentrations with justification for top dose:

0, 15.8, 50, 158, 500, 1580, 5000, 15800, 31,600 and 50000 µg/plate. Higher doses were tested but were too cytotoxic.
Desiccator method: 0.5, 1.0, 1.5 and 2.5 mg/20 L

Vehicle / solvent:

Vehicle used: water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine

Remarks:

without S9 mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: benzo(a)pyrene 4,5-oxide

Remarks:

without S9 mix

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

with S9 mix

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9 mix

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9 mix

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: benzo(e)pyrene

Remarks:

with S9 mix

Details on test system and experimental conditions:

UMBER OF REPLICATIONS:
- Number of cultures per concentration duplicate
- Number of independent experiments: 4

METHOD OF TREATMENT/ EXPOSURE:
- Test substance in preincubation and desiccator method.

DURATION
- Exposure duration: 2-3 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

Evaluation criteria:

No data

Statistics:

Not indicated

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Remarks:

slight increase at high doses

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Remarks:

slight increase at high doses

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Remarks:

slight increase at high doses

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Remarks:

slight increase at high doses

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Remarks:

slight increase at high doses

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

Plate incorporation with S-9 mix: experiment 1.

Dose (nL/per plate)	TA 100	TA 1537	TA 1537	TA 98	WP2 uvrA
0	146	15	29	23	29
15.8	136	16	33	20	35
50	144	16	37	25	27
158	128	12	31	25	29
500	140	19	41	28	24
1580	142	16	39	29	30
5000	162	16	40	27	26
15800	136	14	34	27	25
50000	167x	14	27	24	60x
10 µg B(a)P	1020	25	172	365	105
50 µg B(e)P	303	15	66	61	41
10 µg 2-AA	4200	371	181	3050	428
90 µg 3-MC	4000	10	114	1385	39

Plate incorporation without S-9 mix: experiment 1.

Dose (nL/per plate)	TA 100	TA 1537	TA 1537	TA 98	WP2 uvrA
0	94	13	14	15	21
15.8	83	9	9	15	30
50	96	10	13	18	24
158	107	9	14	15	29
500	109	15	11	14	32
1580	104	13	14	16	28
5000	97	10	13	17	28
15800	112	11	13	16	31
50000	148x	16	4	12	29
1 µg B(a)P	3300	11	689	3200	93
10 µg MNNG	25500	32500	98	48	441

Plate incorporation with S-9 mix: experiment 2.

Dose (nL/per plate)	TA 100	TA 1537	TA 1537	TA 98	WP2 uvrA
0	113	19	21	33	30
15800	129	17	16	34	26
50000	204	16	13	23	38x
100000	29	4	0	3	12
200000	0	0	0	0	0
10 µg B(a)P	1189	30	189	565	74
50 µg B(e)P	256	35	42	81	33
10 µg 2-AA	2500	284	137	3150	363
90 µg 3-MC	3150	10	107	2700	43

Plate incorporation without S-9 mix: experiment 2.

Dose (nL/per plate)	TA 100	TA 1537	TA 1537	TA 98	WP2 uvrA
0	85	12	8	20	24
15800	108x	15	7	22	28
50000	154x	14	8	18	28
100000	0	0	0	0	5
200000	0	0	0	0	0
1 µg B(a)P	1310	27	439	1550	48
10 µg MNNG	22000	32500	91	41	380

Plate incorporation with S-9 mix: experiment 3.

Dose (nL/per plate)	TA 100	TA 1537	WP2 uvrA
0	146	12	37
15800	172	12	34
31600	194	12	55x
50000	237	13	71x
10 µg B(a)P	1315	27	65
50 µg BPO	352	14	40
10 µg 2-AA	2600	338	398
90 µg 3-MC	2950	6	57

Desiccator method:

Dose (ml/per 20 L)	TA 100	TA 100	TA 1535	TA1535	TA1537	TA1537	TA 98	TA 98	WP2 uvrA	WP2 uvrA
	+ S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9
0	182	145	21	16	20	7	29	16	42	25
0.5	152	133	30	17	26	8	28	17	40	20
1	166	131	27	24	18	9	39	24	46	24
1.5	159	136	26	22	15	13	36	23	36	20
2.5	156	39	27	18	22	9	30	14	44	23
10 µg B(a)P	1030	-	40	-	136	-	384	-	95	-
10 µg 2-AA	3200	-	256	-	185	-	2600	-	423	-
90 µg 3-MC	3300	-	9	-	111	-	1700	-	32	-
1 µg BP0	-	1950	-	18	-	327	-	2250	-	93
10 µg MNNG	-	46500	-	38500	-	34	-	76	-	412

B(a)P: Benzo(a)pyrene

2-AA: 2-Aminoanthracene

3-MC: 3-Methylcholanthrene

BPO: Benzo(a)pyrene 4,5- oxide

MNNGG: N-Methyl-N'-nitro-N-nitrosoguanidine .

In the 1st experiment, a slight increase in the number of revertants was found at the 50000 µg dose in TA 100 without metabolic activation (increased by a factor of 1.57) and was reproduced in the 2nd experiment (factor: 1.8). Using metabolic activation, a slight increase in WP2 uvrA revertants was seen (factor: 2) in the 1st experiment; it was not reproduced in the 2nd experiment, but in a 3rd experiment it was only marginally increased (factor: 1.9) at the 50000 µg dose. Results from the dessicator method showed no increase in revertants.