4-(1-oxo-2-propenyl)-morpholine

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 19 May to 28 September 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

4-(1-oxo-2-propenyl)-morpholine

EC Number:

418-140-1

EC Name:

4-(1-oxo-2-propenyl)-morpholine

Cas Number:

5117-12-4

Molecular formula:

C7H11NO2

IUPAC Name:

1-(morpholin-4-yl)prop-2-en-1-one

Test material form:

liquid

Specific details on test material used for the study:

Lot No.: 0620451-LI
Purity: 99.95%

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: The Jackson Laboratory Japan, Inc.
- Age at study initiation: 6 weeks old
- Weight at study initiation: 233.60 g to 261.84 g
- Assigned to test groups randomly: stratified-by-weight randomization method
- Fasting period before study:
- Housing: Rat and mouse room (2124), Polycarbonate cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Actual range: 21.8 to 22.4°C (acceptable range: 19.0 to 25.0°C)
- Humidity (%): Actual range: 49.9 to 60.9% (acceptable range: 35.0 to 75.0%)
- Air changes (per hr): 6 to 20 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours per day (7:00 to 19:00)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water

Details on exposure:

Dose Methods:
A 3-mL disposable syringe attached with a gastric tube for rats was used for administration to each group. The test article formulations were taken into the syringe while being stirred with a magnetic stirrer.

Dose volume: 10 mL/kg

Duration of treatment / exposure:

24 hours

Frequency of treatment:

Twice (at a 24-hour interval)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

62.5 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

125 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 males

Control animals:

yes, concurrent vehicle

Positive control(s):

Ethyl methanesulfonate
- Route of administration: oral gavage
- Doses / concentrations: 200 mg/kg

Examinations

Tissues and cell types examined:

Liver, glandular stomach and duodenum were evaluated for the comet assay.
The liver is the major metabolism organ. The glandular stomach and duodenum are the first organ to be contacted by a test chemical when the animal is administered by oral route.

Details of tissue and slide preparation:

Preparation of specimens:
All animals were used for the specimen preparation at 3 hours after the final administration as the following procedures.
(1) Each rat was anesthetized with thiopental sodium by intraperitoneal injection. The animal was euthanized by exsanguination from the abdominal aorta.
(2) The liver, stomach and duodenum were removed from each animal and the single cells were isolated from these organs and prepared the comet samples as the following procedures.
(3) A corresponding sheet was made for each organ between the animal numbers and coding ones to prepare the sampling tubes including the cell suspensions coded.
(4) Each sample (40 µL of the cell suspension) was mixed with 0.5 w/v% low melting agarose gel (360 µL), and the mixture (40 µL) was placed onto each well of a comet slide.
(5) Three slides (3 wells/sample/animal) were assigned to each sample.

Preparation of the liver samples:
(1) The removed liver was examined macroscopically.
(2) A portion of the liver, around the central part of the left lateral lobe, was cut into a cube (approximately 5 mm).
(3) The residual parts of the left lateral lobe were fixed with 10% phosphate buffered formalin for the histopathological examinations.
(4) The liver cube was washed with a cold mincing buffer, and was minced with scissors about 100 times to isolate the hepatocytes.
(5) The isolated hepatocytes were suspended in a 3 mL of the cold mincing buffer by gently pipetting about 15 times. The cell suspension was filtered through a cell strainer (pore size: 40 µm).
(6) The filtered cell suspension was prepared at approximately 2.0E+05 cells/mL with the cold mincing buffer and the resultant cell suspension was used as the liver sample for the comet assay.

Preparation of the glandular stomach samples:
(1) The removed stomach was cut open along its greater curvature, washed twice with cold PBS (-) and examined macroscopically.
(2) A half of the stomach was used for the comet assay and another half was fixed with 10% phosphate buffered formalin for the histopathological examinations.
(3) The stomach tissue for the comet assay was washed with the cold mincing buffer and the forestomach was removed away if necessary. The glandular stomach was immersed into the fresh cold mincing buffer for 15 to 19 min actually (as prescribed for 15 to 30 min).
(4) The surface epithelium of the glandular stomach was scraped and exfoliated with a scraper and then washed well away with the cold mincing buffer.
(5) The mucous epithelium of the glandular stomach was scraped 5 times or more with a spatula to release the stomach cells. The cells were suspended in a 3 mL of the cold mincing buffer by gently pipetting about 15 times. The cell suspension was filtered through a cell strainer (pore size: 40 µm).
(6) The filtered cell suspension was prepared at approximately 2.0E+05 cells/mL with the cold mincing buffer and the resultant cell suspension was used as the glandular stomach sample for the comet assay.

6.6.5.2.3 Preparation of the duodenum samples
(1) The small intestine corresponding to the duodenum (proximal side) was cut open in the longitudinal direction along the mesenteric attachment side, this duodenum was used for the comet assay, and washed well with cold PBS(-). The duodenum was examined macroscopically.
(2) Residual duodenum (distal side) was fixed with 10% phosphate buffered formalin for the histopathological examinations.
(3) The duodenum for the comet assay was washed with the cold mincing buffer, and then immersed into the fresh cold mincing buffer for 15 to 21 minutes actually (as prescribed for 15 to 30 minutes).
(4) The mucosal surface of the duodenum was scraped and exfoliated well with a scraper and then washed well away with cold mincing buffer.
(5) The epithelial cells were exfoliated with a spatula to release the cells. The cells were suspended with a 3 mL of the cold mincing buffer by gently pipetting about 15 times. The cell suspension was filtered through a cell strainer (pore size: 40 µm).
(6) The filtered cell suspension was prepared at approximately 2.0E+05 cells/mL with the cold mincing buffer. The resultant cell suspension was used as the duodenum sample for the comet assay.

Evaluation criteria:

If the % tail DNA statistically increased in the test article group(s), the study result(s) would be evaluated whether the tissue damage and/or cytotoxic effects were related to the significance based on the frequencies of hedgehog cells and histopathological examinations (if those were done). If the test article induced significant increase(s) in the % tail DNA apart from the tissue damage or cytotoxicity, biological relevance should be comprehensively considered for the comet assay on the basis of the laboratory historical data, distribution and variation of individual data and so on. If the % tail DNA increased in the test article group(s) with biological and toxicological significances, the assay results should be judged positive.

Statistics:

EXSUS statistical software system (ver. 8.1.0, EPS Corporation) was used for the statistical analyses.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

ambiguous

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: negative in liver and glandular stomach, equivocal in duodenum

Additional information on results:

Liver:
The group means (± SD) of the median % tail DNA were 1.69 ± 0.36%, 1.71 ± 0.86%, 1.89 ± 0.32% and 1.72 ± 0.13% at 0 (negative control), 62.5, 125 and 250 mg/kg, respectively. No statistically significant increases in the median % tail DNA were detected in any of the test article groups compared to the negative control group. In contrast, the group mean of the median % tail DNA in the positive control group (24.23 ± 2.22%) was significantly higher than those in the negative control group.
The frequencies (%) of hedgehogs were 2.7% or lower in all the animals and the group mean frequencies were 1.5% or lower in all the groups.

Glandular stomach:
The group means (± SD) of the median % tail DNA were 5.50 ± 1.54%, 3.79 ± 0.41%, 4.74 ± 1.70% and 4.65 ± 1.90% at 0 (negative control), 62.5, 125 and 250 mg/kg, respectively. No statistically significant increases in the median % tail DNA were detected in any of the test article groups compared to the negative control group. In contrast, the group mean of the median % tail DNA in the positive control group (32.34 ± 3.28%) was significantly higher than those in the negative control group.
The frequencies (%) of hedgehogs were 6.7 % or lower in all the animals and the group mean frequencies were 5.5% or lower in all the groups.

Duodenum:
The group means (± SD) of the median % tail DNA were 1.00 ± 0.34%, 1.85 ± 0.23%, 1.98 ± 0.23% and 1.61 ± 0.33% at 0 (negative control), 62.5, 125 and 250 mg/kg, respectively. Statistically significant increases in the median % tail DNA were detected in all of the test article groups compared to the negative control group but there was no dose-dependency. The group mean of the median % tail DNA in the positive control group (17.63 ± 3.03%) was significantly higher than those in the negative control group.
The frequencies (%) of hedgehogs were 8.0 % or lower in all the animals and the group mean frequencies were 6.0% or lower in all the groups.

No abnormal findings were macroscopically observed in either the liver, stomach or duodenum of any animals.
No abnormal findings were observed in all animals.

Applicant's summary and conclusion

Conclusions:

Based on the results, ACMO was judged equivocal in the comet assay of the duodenum in rats. Therefore, it could not be completely denied that ACMO had potential to induce DNA damage in the duodenum. It was also concluded that ACMO did not have a potential to induce DNA damage in the liver and glandular stomach under the conditions of this study.

Executive summary:

A comet assay of ACMO was conducted in rats to assess the in vivo genotoxic potential to induce DNA damage by analyzing the DNA fragmentation of cellular nuclei of the liver,  glandular stomach and duodenum in rats, according to OECD Guideline 489.

The test article was suspended in a vehicle (water for injection) and administrated orally by gavage to male Crl:CD(SD) rats (5 animals per group, 7 weeks old at dosing) at dose levels of 0 (vehicle alone; negative control group), 62.5, 125 and 250 mg/kg for two consecutive days at a 24-hour interval.  The positive control, ethyl methanesulfonate (EMS), was treated at 200 mg/kg in the same manner with the test article.

As a result, there were no significant increases in the median % tail DNA in the liver or glandular stomach in any of the test article groups compared to the negative control group.  In contrast, there were significant increases in the median % tail DNA in the duodenum in all of the test article groups compared to the negative control group but there was no dose-dependency.  There was no clear evidence of tissue damage or cytotoxic effect in the scoring of hedgehogs and gross necropsy in all organs, and histopathological examination in the duodenum.  In the duodenum, each individual median % tail DNA in the negative control group was lower than the minimum limit of the laboratory historical data, and the median % tail DNA of the test article groups were comparable to the laboratory historical data (mean ± 2SD).  Therefore, it would be considered that a statistically increase was detected in all the test article groups due to lower values of the negative control group.

 

The liver and glandular stomach comet assay were judged to have been conducted under the appropriate conditions based on the results obtained from the control groups.  On the other hand, the negative and positive control groups in the duodenum comet assay did not satisfy the acceptance criteria of the study.

 

Based on the results described above, ACMO was judged equivocal in the comet assay of the duodenum in rats.  Therefore, it could not be completely denied that ACMO had potential to induce DNA damage in the duodenum.  It was also concluded that ACMO did not have a potential to induce DNA damage in the liver and glandular stomach under the conditions of this study.