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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Morpholine was not mutagenic in several key or supporting in vitro mutagenicity studies (Huntsman, 1980; Huntsman, 1982). However, ambiguous results or borderline mutagenicity were noted in a key Ames test (BASF AG, 1981) and a key mouse lymphoma mutation assay (Huntsman, 1979), respectively, at high and/or cytotoxic doses.

Link to relevant study records

Referenceopen allclose all

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
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
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 / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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
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
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
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
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
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
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
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
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
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
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
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

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.

 


 

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02-03-1982 to 25-06-1982
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
GLP compliance:
no
Type of assay:
other: Unscheduled DNA Synthesis
Target gene:
No target gene.
Species / strain / cell type:
hepatocytes: from adult male F344 rats
Metabolic activation:
without
Test concentrations with justification for top dose:
0.00001, 0.0001, 0.001, 0.01, 0.1 and 1%
Vehicle / solvent:
- Solvent used: DMSO
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
yes
Remarks:
Pyrene
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 1

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 18-20 hours (10 µCi/mL tritiated thymidine ([3H]-TdR), 60-80 Ci/mM added during exposure)

NUMBER OF REPLICATIONS: triplicate coverslips

NUMBER OF CELLS EVALUATED: Between 5 to 20 cells randomly selected from each quadrant of the coverslips were counted.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity of the test substance was identified by the absence of S phase cells in the autoradiograph and by general morphology.

OTHER: After exposure, each coverslip was rinsed in three successive washes of WME. Coverslips were then immersed with the cell surface up in 1 % sodium citrate for 10 min. Finally, the cells were fixed in three 30-minute changes of ethanol-glacial acetic acid (3:1), air dried, and mounted cell surface up on glass slides with Permount. Slides were placed in holders and dipped, in total darkness, into NTB-2 emulsion, prewarmed for 1 hour. Slides were dried overnight in a light tight box. Slides were stored for 7 days after which autoradiographs were developed.

The HPC/DNA repair test was performed according to the methods developed by Williams (2,3). Immediately after washing the cells, the test chemical and 10 µCi/mL tritiated thymidine ([3H]-TdR), 60-80 Ci/mM habe been added to the culture.
Evaluation criteria:
The test substance was reported positive when the minimum net grain count of 5 per nucleus was consistently observed in triplicate coverslips throughout the experiment. Where possible, a dose response profile was developed for each species in which the test substance was positive. The test substance was reported negative in the assay if the net nuclear count was less than 5 at the highest non toxic dose.
Statistics:
Not indicated.
Key result
Species / strain:
hepatocytes: rat
Metabolic activation:
without
Genotoxicity:
negative
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
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY AND UDS:
When the two sets of slides exposed to the two highest concentrations of the test substance were examined under the microscope, after they had been processed for autoradiography, cytotoxicity was observed in all of the slides in the two sets. Cytotoxicity was identified by a general absence of S-phase cells, and absence of grains in the few remaining hepatocytes and presence of hepatocytes with non-swollen nuclei. Counting of slides thus began with the slides exposed at 0.01 % and lower. The mean net nuclear grain counts of the slides exposed at 0.01 % and lower did not exceed 5. Therefore, it was concluded that the test substance was not genotoxic to the hepatocytes in this HPC/DNA repair assay. Parallel run DMSO and cell culture controls gave a mean net nuclear grain count of 0.3 ± 0.3 and 0.2 ± 0.4 respectively. The mean nuclear grain count of parallel run positive control was 20 ± 11.3 and negative control (pyrene) was 1.2 ± 1.1.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 5, 1979 - June 11, 1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
GLP compliance:
no
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
thymidine kinase locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: The cells were maintained in Fischer's mouse leukemia medium supplemented with L-glutamine, sodium pyruvate, and horse serum (10 % by volume). Cloning medium consisted of the preceding growth medium with the addition of agar to a final concentration of 0.35 % to achieve a semisolid state. Section medium was cloning medium containing 100 µg/mL of BrdU or 3 µg/mL of TFT.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system: S9 mix
- method of preparation of S9 mix
Fischer 344 male rats are normally used as the source of hepatic microsomes. Induction with Aroclor 1254 or other agents is performed by injections five days prior to sacrifice. After decapitation and bleeding, the liver is immediately dissected from the animal using aseptic technique and placed in ice cold 0.25M sucrose buffered with Tris at pH 7.4. When an adequate number of livers is obtained, the collection is washed twice with fresh buffered sucrose and completely homogenized. The homogenate is centrifuged for 10 minutes at 9,000x g in a refrigerated centrifuge and the supernatant (S9)- from this centrifuged sample is retained and frozen at -80°C until used in the activation system. The S9 fraction may be obtained from induced or noninduced rats or other species, as requested
Test concentrations with justification for top dose:
Without metabolic activation:
Experiment 1: 0.078, 0.500, 0.625, 1.000, 1.250 µL/mL
Experiment 2: 0.500, 0.625, 0.750, 1.000, 1.250 µL/mL
Experiment 3: 0.750, 1.000, 1.200 µL/mL

Metabolic activation:
Experiment 1: 0.156, 0.500, 0.625, 1.000, 1.250 µL/mL
Experiment 2: 0.500, 0.625, 0.750, 1.000, 1.250 µL/mL
Experiment 3: 0.750, 1.000 µL/mL
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent/vehicle: due to a technical error in the initial solubility testing, the test substance was described as insoluble at 100 uL/mL in deionized water. The substance was, in fact, soluble and water would have been the preferred solvent.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation, 0.5 µL/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: dimethylnitrosamine
Remarks:
with metabolic activation, 0.3 µL/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 2-3 days
- Selection time (if incubation with a selection agent): 10 days
- Fixation time (start of exposure up to fixation or harvest of cells): cells were harvested at the end of the selection time.

SELECTION AGENT: 5-bromo-2'deoxyuridine or 5-trifluorothymidine

NUMBER OF REPLICATIONS: 1-2

NUMBER OF CELLS EVALUATED: Mutation frequency was based on 10E6 cells.

DETERMINATION OF CYTOTOXICITY
- Method: % relative total growth
Evaluation criteria:
The test substance was considered mutagenic in the assay if:
- A dose response relationship was observed over 3 of the 5 dose levels employed.
- The minimum increase at the low level of the dose response curve was at least 2.5 times greater than the solvent and/or negative control values.
- The solvent and negative control data were within the normal range of the spontaneous background for the TK locus.
All evaluations of mutagenic activity were based on consideration of the concurrent solvent and negative control values run with the experiment in question. Positive control values were not used as reference points, but were included to ensure that the current cell population responded to direct and promutagens under the appropriate treatment conditions.
Occasionally, a single point within a concentration range was expected to show an increase 2.5 times greater than the spontaneous background. If the increase was at the high dose, was reproducible, and if an additional higher dose level was not feasible because of toxicity, the substance was possibly considered mutatgenic. If the increase was internal within the dose range and was not reproducible, the increase was considered aberrant. If the internal increase was reproducible, several doses clustered around the positive concentration were examined to either confirm or reject the reliability of the effect.
Statistics:
Not indicated.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
positive
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
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
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
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The test substance was tested for cytotoxicity at an applied dose range of 0.01-5 µL/mL. After an exposure time of four hours, the cells were washed and a viable cell count was obtained the next day. Relative cytotoxicities expressed as the reduction in growth compared to the growth of untreated cells were used to select seven to ten doses that covered the range from 0 to 50-90 % reduction in 24 hour growth. These selected doses were subsequently applied to cell cultures prepared for mutagenicity testing, but only four or five of the doses would be carried through the mutant selection process. The test substance remained soluble in the growth medium, but an alkaline pH was obtained at concentrations above about 0.3 µL/mL. Twenty four hours after treatment, the cell count was greatly reduced at 1.25 µL/mL and doses of 2.5 µL/mL and higher were completely lethal.

Four trials of the mutation assay were initiated, but the first trial became contaminated before any mutagenesis data could be collected. Under the nonactivation conditions, the test substance was lethal at 2.0 µL/mL and five treatments from 1.25 -0.078 µL/mL were assayed for mutant induction. The 1.25 µL/mL treatment was highly toxic (9.4 % relative growth) and did not induce a significant increase in mutant frequency. A 2.5 fold increase over the background was considered the minimum criterion for demonstrating mutagenesis at any given dose level. The other four treatments were moderately toxic and also yielded mutant frequencies similar to the background, except for the 1 µL/mL treatment. In this case, a 2.8 fold increase was observed. An increase of this small magnitude that is not followed by a similar or larger increase for more toxic treatments is normally regarded as spurious. Nevertheless, a repeat assay was performed to test the repeatability of the response.

In the second nonactivation assay, the toxicity of the test substance was more variable as a function of the applied concentration. Three of the assayed treatments were highly toxic and all of these induced mutant frequency increases near the 2.5 fold criterion used for evaluation as mutagenic. Thus, 2.2 fold, 2.4 fold and 2.7 fold increases were obtained for 0.625, 1 and 1.25 µL/mL, respectively. These results suggested that the test substance may be weakly mutagenic at the limit of detectability for this assay.

A third nonactivation assay was performed which yielded similar behavior. One treatment with 0.75 µL/mL and another at 1.2 µL/mL induced 2.5 and 2.7 fold increases in the mutant frequency. However, duplicate treatments at these two doses, both of which were highly toxic, caused no significant change from the background frequency. The results from all three trials therefore indicated that the test substance can be weakly mutagenic for highly toxic treatments in the 0.625 to 1.25 µL/mL dose range. The lack of mutagenic activity for some toxic treatments in this dose range suggested competition between toxic and mutagenic modes of action. In the presence of metabolic activation, the test substance was not as toxic, indicating a reaction with the activating system. Only moderately toxic treatments were achieved in the first trial for applied concentrations up to 1.25 µL/mL (35.5 % relative growth). The mutant frequencies in the treated cultures remained similar to the background. The next highest applied concentration of 2 µL/mL was completely lethal to the cells. A repeat assay gave similar results and confirmed the lack of detectable mutagenesis for moderately toxic treatments obtained with concentrations up to 1.25 µL/mL. In this assay, the next highest dose was 1.5 µL/mL, which was completely lethal. A third activation assay was performed in which most of the applied concentrations (from 1.2 to 2 µL/mL) were lethal and only two dose levels (in duplicate) were available for analysis. One of the 0.75 µL/mL treatments was moderately toxic (37.3 % relative growth), whereas the other treatments had no apparent effect on the cells. The mutant frequencies remained similar to the one solvent negative control that was not contaminated. Thus, the test substance had little to moderate toxicity in the presence of activation until a threshold concentration was reached where lethality suddenly became excessive. This threshold occurred between 1.25 and 1.5 µL/mL in trial 2 and between 1.0 and 1.2 µL/mL in trial 3.

Since the test substance was alkaline in the culture medium at these concentrations, the sharp increase in lethality may be pH related rather than test substance induced. Any mutagenic activity that might be associated with highly toxic treatments would therefore occur over a very narrow concentration range just preceding complete lethality. Such information was probably not biologically meaningful, so further attempts to achieve this toxicity range did not seem warranted. The average cloning efficiencies for the solvent and untreated negative control varied from 105 to 108% without activation and from 106 to 124 % with activation, which demonstrated excellent culturing conditions for the assays. The negative control mutant frequencies were all in the normal range, and the positive control substance yielded normal mutant frequencies that were greatly in excess of the background.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 16, 1979 - November 29, 1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)
Deviations:
not specified
GLP compliance:
no
Type of assay:
sister chromatid exchange assay in mammalian cells
Target gene:
No target gene
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Cells were grown and treated in McCoy's 5a medium supplemented with 10 % foetal calf serum.
- Properly maintained: yes
- average cycle time of 12-14 h
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system: S9 mix
- method of preparation of S9 mix
Fischer male rats normally were used as the source of hepatic microsomes. Induction with Arochlor 1254 was performed by injection 5 days prior to kill. After decapitation and bleeding, the liver was immediately dissected from the animal using aseptic technique and placed in ice-cold 0.25M sucrose buffered with Tris at pH 7.4. When an adequate number of livers had been obtained, the collectionwas washed twice with fresh buffered sucrose and completely homogenized. The homogenate was centrifuged for 10 minutes at 9,000 x in a refrigerated centrifuge, and the supernatant (S9) from this centrifuged sample was retained and frozen at -80°C until used in the activation system. This S9 fraction was added to a' core" reaction-mixture to form the activation system
Test concentrations with justification for top dose:
3.13, 6.25, 12.50, 25.00, 50.00 and 100.00 nL/mL
Vehicle / solvent:
- Solvent used: DMSO (1 %)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
triethylenemelamine
Remarks:
without metabolic activation, 0.2 µg/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation, 0.001 M
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: single
- Number of independent experiments : 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: approximately 3 x 10E6 cells
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 2 hours
- Expression time (cells in growth medium): 24 hours (5-bromo-2'-deoxyurdine, 20 mM, added during expression period)
- Fixation time (start of exposure up to fixation or harvest of cells): cells were harvested 26 hours after exposure, with colcemid present during the final 2 hours (final concentration 2x10E-7M)

SPINDLE INHIBITOR: colcemid

STAIN: slides were stained for 10 minutes with Hoechst 33258 (5 µg/mL) in phosphate buffer (pH 6.8), mounted in the same buffer and exposed to ultraviolet (UV) light from a mercury lamp or, at 60 degrees C from a 15 watt "black light tube" for the amount of time required for sister chromatid differentiation. Following UV exposure, the slides were stained with 10 % Giemsa for 10 minutes and air-dried.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: mitotic index (MI)
Evaluation criteria:
If an increase in sister chromatid exchange was observed, one of the following criteria had normally be met to assess the test substance as positive.
1. Two-fold increase: approximately a doubling in sister chromatid exchange frequency over the "background" (solvent and negative control) levels, at a minimum of three doses.
2. Dose response: a positive assessment may have been made in the absence of a doubling if there was a statistically significant increase at a minimum of three doses and evidence for a positive dose response.
In some cases, statistically significant increases may have been observed with neither a doubling or a dose response. These results were assessed according to repeatability, the magnitude of the response, and the proportion of the dose levels affected.
Statistics:
Student's t-test
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The solubility, toxicity and doses for the test substance were determined in the assay on a different batch of test substance (7H-4892/LOS-0575). The doses covered three orders of magnitude, and all doses that yielded sufficient numbers of scorable metaphase cells were considered in the analysis. No further information was provided in the report.

STUDY RESULTS:
Without metabolic activation, a small increase in SCE is evident at the two highest doses of morpholine, 50 nL/mL and 100 nL/mL. There is a slight suggestion of a dose response, but the overall result is not convincingly positive. In the presence of the
activation system, there is again a slight increase at three of the six doses, but no clear dose response. The maximum increases in SCE are only 25% and 22% with and without S9 mix, respectively, and in the absence of a positive dose response
these results indicate a very weak, or negative, response. Morpholine does not cause a meaningful increase in sister chromatid exchange under the conditions of this assay .

Genetic toxicity in vivo

Description of key information

Morpholine was not mutagenic in in a key in vivo mutagenicity study (Inui et al., 1979).

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Hamster embryos were exposed in utero to the action of sodium nitrite and Morpholine or Morpholine alone administered by stomach tube to the mothers on the 11th or 12th day of pregnancy. Embryo cells were examined for chromosomal aberrations, micronucleus formation, morphological or malignant transformation and drug resistance mutations. For detection of induced mutations, the embryo cells were cultured in normal medium for 72 h and then transferred to medium containing 10 or 20 µg/mL of 8-azaguanine or 1 mM ouabain.
GLP compliance:
not specified
Type of assay:
other: chromosomal aberration and micronucleus assay
Species:
hamster
Strain:
other: Syrian golden hamster
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 150 ± 30 g
- Age at study initiation: young adult (based on body weight)
- Housing: closed colony in a clean barrier system room
- Diet: sterilized standard laboratory chow (Japan Clea Ltd., Tokyo, Japan), ad libitum
- Water: ad libitum
Route of administration:
oral: gavage
Vehicle:
- Vehicle used: water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Morpholine was dissolved at a concentration of 500 mg/mL in distilled water immediately before use.
Duration of treatment / exposure:
single treatment on the 11th or 12th day of pregnancy
Frequency of treatment:
once
Post exposure period:
Twenty-four hours after treatment, hamster embryos were excised.
Dose / conc.:
500 mg/kg bw (total dose)
No. of animals per sex per dose:
no numbers given; all foetuses from each litter were used
Control animals:
yes, concurrent vehicle
Positive control(s):
N-nitrosomorpholine (Dr. Okada, Tokyo Biochemical Institute, Tokyo, Japan)
- Justification for choice of positive control: active compound
- Route of administration: oral gavage
- Doses / concentrations: 100 or 200 mg/kg bw; 500 mg/mL
Tissues and cell types examined:
Primary cultures of trypsinized cells
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION:

Preparation of samples for examination of chromosomes and micronucleus formation:
For preparation of chromosomes, some cells were treated with 0.3 µg/mL of colcemid for 3 h, within 24 h of initiation of the primary culture, so that mitotic cells could then be studied in the first cell cycle in culture. Chromosome preparations were made by the usual air-dry method with a slight modification (Rothfels & Simonovitch, 1958; Yoshida et al., 1970) and stained with Giemsa. The numbers and types of chromosomal aberrations were assessed by 200 well-spread metaphase plates. For examination of micronucleus formation, samples were made by a modification of the method of Schmid (1975); 36 h after initiation of cell cultures, the cells were collected with 0.1 % trypsin, smeared on glass slides and fixed with methanol for 30 min. The slides were dried and stained with Giemsa. Per experiment, over 5000 resting nuclei were examined for micronucleus formation.

Selection of resistant mutant cells:
For selection of drug-resistant mutant cells, standard-incubated primary cultures were inoculated into medium containing 10 or 20 µg/mL of 8-azaguanine (8AG) or 1 mM ouabain (Oua) in 60 mm plastic dishes. At least five independent experiments were made. For selection of 8AG-resistant mutations, the medium containing 8AG was changed every day for the first 3 days and then, when cells that could not produce colonies had died, the medium was changed once every 3 days. For selection of Oua-resistant mutants, the medium containing 1 mM Oua was changed after 1 week. After total cultivation periods of 15 to 20 days (8AG) and 30 days (Oua), dishes were fixed and stained, and the number of resistant colonies was investigated. Embryonic cells were obtained from mothers treated with Morpholine; embryonic cells from untreated mothers were used as controls. Control cells were cultured and selected in the same way as those in experimental group.
Evaluation criteria:
Not indicated
Statistics:
Statistically significant differences between treatment and control groups were determined at p<0.05 and p<0.01. The statistical test method chosen was not specified.
Key result
Sex:
female
Genotoxicity:
negative
Toxicity:
not examined
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Table 1: Results Chromosome Aberration Test

   Dose (mg/kg bw)  No. of metaphases observed  Cells with aberrations  Normal diploid  cells with over 44 chromosomes (%)
 control  0  200  4 (2%)  89.0%  4.0
 morpholine  500  200  3 (1.5%)  89.0%  3.0

Table 1: Results Micronucleus Test

   Dose (mg/kg bw)  No. of cells observed No. of cells with micronucleus % of cells with micronucleus Induced ratio*
 control  0  5000 21 0.42  -
 morpholine  500  5000 27 0.52  x 1.29

* The induced ratio means the ratio of the induced number to the number in control cells

Treatment with Morpholine at 500 mg/kg bw induced no increases in chromosomal aberrations or frequency of micronuclei in primary embryonic cell cultures.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro:

Genetic toxicity testing of Morpholine produced mostly negative findings as well as borderline positive findings.

Morpholine was tested for mutagenicity in vitro in the Ames test with and without metabolic activation. In the key reverse gene mutation assay (BASF AG, 1981), Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 were exposed to 0, 15.8, 50, 158, 500, 1580, 5000, 15800, 31600 or 50000 µg/plate in the standard plate test (plate incorporation method). Higher doses were tested, but were too cytotoxic. Slight increases in the number of revertants were observed at 50000 µg/plate with TA100 in the presence and absence of S9 mix and with WP2 uvrA only with metabolic activation. Morpholine exposure at 0.5, 1.0, 1.5 or 2.5 mL/20 L by means of the desiccator method did not reveal mutagenicity of Morpholine at any dose. Morpholine (or possibly an impurity) was weakly mutagenic in the Ames test at a concentration of 50000 µg/plate. However, the observed increase in the number of mutants at this relatively high dose was not more than 2-fold. Therefore, this test result is considered as ambiguous. The positive controls induced the appropriate responses in the corresponding strains. An Ames test reported by Haworth et al. (1983) was considered as supporting study as well as a test by Huntsman (1979). Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 were exposed to Morpholine at concentrations of 0, 109, 363, 1090, 3633 or 10900 µg/plate in the preincubation assay. Morpholine was tested up to slightly cytotoxic concentrations (10900 µg/plate). The positive controls induced the appropriate responses in the corresponding strains. In this study (and in the study by Huntsman, 1979), there was no evidence of a concentration-related positive response of induced mutant colonies over background.

In a key mouse lymphoma mutation assay with L5176Y TK +/- cells (Huntsman, 1979), the mutagenic potential of Morpholine was assessed in vitro at concentrations up to 1.25 µL/mL in the presence and absence of metabolic activation (S9 mix). This assay was repeated and in an additional assay concentrations from 1.2 to 2 µL/mL were tested. The positive controls induced the appropriate responses. The test material induced small increases in the mutation frequency over the applied concentration range of 0.625 to 1.25 µL/mL under non-activation conditions. These treatments were highly toxic and the mutant frequency increases (approximately 2.5 -fold) were at the limit of detectability for this assay with microsomal activation; concentrations up to 1.0 - 1.25 µL/mL were not very toxic and not detectably mutagenic; concentrations from 1.2 - 1.5 µL/mL were excessively lethal. Under the conditions of this study, Morpholine was considered to be very weakly mutagenic in the assay without metabolic activation. In a supporting study (Huntsman, 1979), Morpholine is considered to be active in the BALB/3T3 in vitro transformation assay.

In a key in vitro sister chromatid exchange assay (Huntsman, 1980), Chinese hamster ovary cells were treated with Morpholine at concentrations of 3.13, 6.25, 12.50, 25.00, 50.00 or 100.00 nL/mL in the presence and absence of mammalian metabolic activation (S9 mix). Positive control items induced the appropriate responses. The maximum increases in sister chromatid exchange noted were only 25 % and 22 % with and without S9 mix, respectively. In the absence of a positive dose response these results indicated a very weak, or negative, response. Morpholine did not cause a meaningful increase in sister chromatid exchange under the conditions of this assay.

Morpholine was tested in vitro in a rat hepatocyte primary culture (HPC/DNA Repair Assay) at concentrations of 0.00001, 0.0001, 0.001, 0.01, 0.1 or 1% (Huntsman, 1982). Positive control items induced the appropriate responses in this key study. Under the conditions of this assay, Morpholine did not induce DNA repair over background at the highest non-toxic doses and lower doses.

In this study identified as supporting study (Huntsman, 1979), Morpholine was evaluated for cell transformation in the BALB/3T3 in vitro transformation assay performed in accordance with EU guideline B21. Test concentrations were 0.000122, 0.00195, 0.0156, 0.125, 0.250 µL Morpholine/mL. The frequency of transformed foci was increased by all treatments expect the lowest (0.000122 µL/mL). The increases were dose-related and became significantly different from the negative control for the 0.125 µL/mL dose (95% confidence level) and the 0.250 µL/mL dose (99% confidence level). Morpholine induced an increase in transformed foci over the applied concentration range of 0.125 µL/mL to 0.250 µL/mL. Concentrations as low as 0.00195 µL/mL were possibly weakly transforming. The test material is considered to be active in the BALB/3T3 in vitro transformation assay. The positive control, 3-methyl-cholanthrene, did induce the appropriate response. A further cell transformation assay did not induce significant increases in transformed foci over the tested concentration ranges of 15.0 to 1400.0 nL/mL and 17.5 to 700.0 nL/mL in the non-activation and activation assay, respectively. The tested concentrations corresponded to approximately 20% to nearly 100% survival in the non-activation and activation preliminary cytotoxicity tests- Therefore, the test material is considered to be inactive in the Balb/3T3 and Balb/3T3 rat liver cell-mediated in vitro cell transformation assays (BASF AG, 1982).

Genetic toxicity in vivo:

In an in vivo transplacental mutagenesis study (Inui et al., 1979), sodium nitrite with Morpholine or Morpholine (500 mg/kg bw) alone were administered by single oral gavage to pregnant Syrian golden hamsters on day 11 or 12 of pregnancy. Twenty-four hours after treatment, the hamster embryos were excised and examined for chromosomal aberrations, micronucleus formation, morphological or malignant transformation and drug resistance mutation. Cells exposed in utero to Morpholine showed no increases in the numbers of chromosomal aberrations, micronuclei, 8-azaguanine- or ouabain-resistant mutants, or transformation rates. The number of resistant colonies was markedly increased after administration of sodium nitrite together with Morpholine only, showing that Morpholine alone displayed no mutagenic activity in vivo under the conditions of this study.

Conclusion

Morpholine was not mutagenic in several key or supporting in vitro mutagenicity studies (Hawoth et al., 1983; Huntsman, 1980; Huntsman, 1982) and in a key in vivo mutagenicity study (Inui et al., 1979). However, ambiguous results or borderline mutagenicity were noted in a key Ames test (BASF AG, 1981) and a key mouse lymphoma mutation assay (Huntsman, 1979), respectively, at high and/or cytotoxic doses. Taking the overall evidence into account and with special regard to the partially equivocal findings at high doses in vitro, Morpholine is not considered to be mutagenic.

 

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No 1272/2008. Based on the provided information, the test item is considered not to be classified for mutagenicity under Regulation (EC) No.1272/2008, as amended for the fifteenth time in Regulation (EU) 2020/1182.