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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro genetic toxicity tests are available:
Ames test:
Bacterial reverse mutation assay was performed according to OECD 471. Result: negative with/ without metabolic activation.

In vitro chromosome aberration study:
An in vitro cytogenicity / chromosome aberration study in mammalian cells was performed according to EG B.10 / OECD 473. Result: positive with/ without metabolic activation.

In Vitro Mammalian Cell Gene Mutation Test:
An in vitro gene mutation study in mammalian cells was performed to evaluate test item using the thymidine kinase gene capability to cause gene mutations in mammalian cells (L5178Y) which were cultured in vitro, after being exposed for 3 hours with and without metabolic activation respectively, and exposed for 24 hours without metabolic activation based on OECD 490. The test item is non-mutagenic under the conditions of this study.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 2020-11-09 to 2020-11-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

July 29, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

Batch No.: 06007352
Purity: 99.7%

Target gene:

Thymidine Kinase Gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
Mouse lymphoma L5178Y (TK+/- 3.7.2C) cell line from ATCC®CRL-9518

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
L5178Y cells were cultured at 36.2~39.0℃ in the humidified atmosphere of 4.9~5.0% CO2.
The culture media used were RPMI 1640 (RPMI in short), and three types of 0%, 10% and 20% FBSRPMI media (RPMI 0, RPMI 10 and RPMI 20) were prepared by adding FBS (0% , 10% or 20%) and 10000 U/mL Penicillin-Streptomycin (1%), Sodium pyruvate (2%).

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: prepared in-house, prepared from the livers of the rats induced with Aroclor 1254
- method of preparation of S9 mix:
10% S9 mix was prepared according to the recipe below immediately before being used and placed in the mixture of water and ice. The remaining S9 mix was discarded.

Phosphate buffer (0.2 mol/L) 40000 μL
MgCl2(0.4mol/L)-KCl(1.65 mol/L) solution 1600 μL
G-6-P-Na2 solution (140 mg/mL) 800 μL
NADP-Na2 solution (300 mg/mL) 800 μL
S9 8000 μL
Sterile distilled water 28800 μL

- concentration or volume of S9 mix and S9 in the final culture medium: 4 mL/ 20 mL
- quality controls of S9: the batch of S9 had been tested for its sterility, its protein content (not higher than 40 mg/mL), and its capability to activate known mutagens in Ames test. The results of the test indicated that each index of S9 was in the expected range to meet the test requirements.

Test concentrations with justification for top dose:

2000, 1000, 500, 250, 125, 62.5 and 31.25 μg/mL in the treatment conditions of exposure for 3 hours with and without metabolic activation respectively and for 24 hours without metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI1640

- Justification for choice of solvent/vehicle: chose based on the results of test item solubility

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 3E+5 cells/mL
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 hours with and without metabolic activation, 24 hours without metabolic activation

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 days
- Selection of mutant cells:
After expression period, the cells were suspended in media with and without the selective agent triflurothymidine (TFT) for the determination of the number of mutants (selection plates =TFT plates) and for cloning/planting efficiency (viability plates = PE plates), respectively.
According to the cell density on the day 2, the cells were adjusted to 1E+4 cells/mL in the volume of 50 mL as cell culture B. Then part of cell culture B was taken out and diluted twice as the table below to 25mL of 8 cells/mL as cell culture D. The mutant selective agent, trifluorothymidine (TFT), was added in the remaining cells of the culture B, and the final concentration of TFT in cell culture was 3 μg/mL.
For each cells culture, two 96-microwell plates were prepared with cell culture B as TFT plates, and one 96-microwell plate was prepared with cell culture D as PE plate. For all plates, 0.2 mL of the cells culture was added into each well.
- Criteria for small (slow growing) and large (fast growing) colonies:
1) Large colony:
Size: the diameter was not less than a quarter of diameter of the well;
Morphology: thin and dispersive
2) Small colony:
Size: the diameter was less than a quarter of diameter of the well;
Morphology: compact and dense massive structure


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative total growth (RTG)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The plates exposed for 3 hours with and without metabolic activation were incubated for 12 days. The plates exposed for 24 hours without metabolic activation were incubated for 11 days.
After the incubation, cell colonies in the plates were observed and counted.
For PE plate, empty wells and total wells were counted respectively.
For TFT plate, wells with viable colony and total wells were counted respectively. In addition, for TFT plate of the analyzable highest concentrations, solvent and positive controls, large colony and small colony were counted respectively.

Evaluation criteria:

1) When IMF(s) in one or several doses are more than GEF* (GEF= 126E-6), and the increase is concentration-related and/or replicated, the result is evaluated as positive.
2) The result is evaluated as negative if none of the above criteria is met.
3) In case the criteria above are not met at the same time, the repeated test should be performed using modified experimental conditions, and the biological relevance of the result should be considered. If the result is still not clear, the result is concluded to be equivocal.
*GEF = Global Evaluation Factor, established for the MLA making use of the version of microtiter plates as average background mutation frequency of negative controls.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The results of test item precipitate: There was no test item precipitate was observed in the cell cultures of all exposure concentrations at the beginning and end of the test of all the treatment condition.
The results of cytotoxicity: the RTG of the cells exposed for 3h in the absence of S9 at the doses of 2000, 1000, 500, 250, 125, 62.5 and 31.25 μg/mL were 0.60%, 16.30%, 25.99%, 46.57%, 50.36%, 55.88% and 90.23%; The RTG of the cells exposed for 3 h in the presence of S9 at the doses of 2000, 1000, 500, 250, 125, 62.5 and 31.25 μg/mL were 0.39%, 11.68%, 25.80%, 35.41%, 46.28%, 67.46% and 77.17%; the RTG of the cells exposed for 24 h in the absence of S9 at the doses of 500, 250, 125, 62.5 and 31.25 μg/mL were 0.43%, 15.76%, 39.39%, 71.43% and 84.92%.
The results of mutant frequency: The IMFs was less than GEF( GEF=126E-6) at all analyzable design doses under each treatment condition.

Negative and positive control results: In this study, the results of the solvent and positive controls met all criteria of the test validity, so the sensitivity of the test and the efficacy of the S9 mix were validated.

Statistic data of the treated cultures:

Treatment condition	Dose level /Control (μg/mL)	PE (%)	RPE (%)	SG	RSG (%)	RTG (%)	MF (1E-6)	IMF (1E-6)	MFsc (1E-6)	IMFsc (1E-6)
3h without S9	RPMI1640	95.06	100.00	9.31	100.00	100.00	116.00	0.00	71.80	0.00
	31.25	90.69	95.41	8.80	94.57	90.23	103.97	-12.03	—	—
	62.5	71.67	75.40	6.90	74.12	55.88	140.40	24.39	—	—
	125	60.45	63.59	7.37	79.19	50.36	174.41	58.41	—	—
	250	67.70	71.22	6.09	65.39	46.57	150.99	34.98	—	—
	500	52.42	55.14	4.39	47.13	25.99	204.19	88.18	—	—
	1000	44.40	46.71	3.25	34.90	16.30	160.48	44.48	54.07	-17.74
	2000	18.60	19.57	0.29	3.09	0.60	303.51	187.51	—	—
	MMS (5.0)	50.93	53.58	7.37	79.14	42.40	829.24	713.24	222.90	151.10
3h with S9	RPMI1640	96.66	100.00	9.49	100.00	100.00	120.84	0.00	40.62	0.00
	31.25	78.15	80.84	9.06	95.46	77.17	118.66	-2.18	—	—
	62.5	65.97	68.25	9.38	98.84	67.46	182.05	61.21	—	—
	125	63.09	65.27	6.73	70.91	46.28	182.55	61.71	—	—
	250	53.98	55.85	6.02	63.40	35.41	192.32	71.48	—	—
	500	54.74	56.63	4.32	45.57	25.80	195.55	74.71	—	—
	1000	40.18	41.56	2.67	28.11	11.68	230.80	109.96	83.78	43.16
	2000	11.47	11.87	0.31	3.25	0.39	479.38	358.54	—	—
	CP (3.0)	40.97	42.39	7.66	80.72	34.21	871.59	750.75	372.45	331.84
24h without S9	RPMI1640	99.64	100.00	50.09	100.00	100.00	100.99	0.00	30.99	0.00
	31.25	92.08	92.41	46.03	91.89	84.92	100.70	-0.29	—	—
	62.5	87.97	88.29	40.53	80.91	71.43	119.84	18.85	—	—
	125	56.48	56.69	34.81	69.49	39.39	183.80	82.81	—	—
	250	51.69	51.87	15.22	30.38	15.76	182.44	81.46	51.74	20.75
	500	23.09	23.17	0.94	1.87	0.43	308.57	207.59	—	—
	1000	—	—	0.00	0.00	—	—	—	—	—
	2000	—	—	0.00	0.00	—	—	—	—	—
	MMS (5.0)	58.80	59.01	29.92	59.73	35.25	620.99	520.00	290.11	259.12

Conclusions:

In this study, the results were negative under three treatment conditions. Thus the test item was considered to be non-mutagenic to the mammalian cells cultured in vitro under the conditions of this study.

Executive summary:

The study was performed to evaluate test item using the thymidine kinase gene capability to cause gene mutations in mammalian cells which were cultured in vitro, after being exposed for 3 hours with and without metabolic activation respectively, and exposed for 24 hours without metabolic activation based on OECD 490.

In this test, the L5178Y (TK^+/--3.7.2C) mouse lymphoma cell, generally called L5178Y were exposed at 7 exposure concentrations including 2000, 1000, 500, 250, 125, 62.5 and 31.25 μg/mL cell cultures in three treatment conditions above. The solvent (RPMI1640) and positive controls were included in each condition at the same time.

There was no test item precipitate was observed in the cell cultures of all exposure concentrations at the beginning and end of the test of all the treatment condition.

The results of mutant frequency showed that the induced mutant frequencies (IMF of all cultures exposed for 3h and 24h were consistently less than the average background mutant frequency of 126E-6.

The result of this study is negative, so it is concluded that the test item, using the thymidine kinase gene, is non-mutagenic to the L5178Y mouse lymphoma cells under the test conditions of this study.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 06 January to 26 April 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Batch number: 60513F07
Purity: 99.81%

Species / strain / cell type:

mammalian cell line, other: Human Lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from liver of Aroclor 1254 treated rats

Test concentrations with justification for top dose:

First test: 9.8, 19.5, 39.1, 78.1, 156.3, 312.5, 625, 1250, 2500, 5000 μg/mL
Second test (without S9): 19.5, 39.1, 78.1, 156.3, 200, 250, 312.5, 500, 625, 1000 μg/mL
Second test (without S9 repeat): 37.5, 75, 150, 200, 250, 300, 350, 400, 450, 500, 600, 1000 mg/mL
Second test (with S9): 156.3, 312.5, 625, 750, 1000, 1250, 1500, 2000, 2500 μg/mL

Vehicle / solvent:

Water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

in absence of S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

in presence of S9 mix

Details on test system and experimental conditions:

Exposure period (with metabolic activation): 3 hours
Exposure period (without metabolic activation): 18 hours
First test: without S9-mix 18 h culture
First test: with S9-mix 18 h culture
Second test: without S9-mix 18 h culture
Second test: with S9-mix 18 h culture

Key result

Species / strain:

mammalian cell line, other: Human Lymphocytes

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1000 μg/ml

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

mammalian cell line, other: Human Lymphocytes

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

312.5 μg/ml

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

First test: with and without S9-mix the test substance led to a statistical significant increase (p < 0.001) in the part of the aberriating cells in the highest tested concentrations. These increases to 9 % at 312,5 μg/ml
without S9-mix and to 17.5 % at 1250 μg/ml mit S9-mix are not within the historical controlls and indicate a clstogene activity.
Second test: The substance led to a statistic signifcant increase in the par of the metaphasis with chromosome aberration in all three concentrations without S9-mix and at 625 and 1000 μg/ml with the mix. The increasis with the S9-mix and at 300 μg/ml without S9-mix are not within the historical controlls and indicate a clastogene activity.

Conclusions:

The substance has shown evidence of clastogenic activity in both absence and presence of S9 mix, in this in vitro cytogenetic test system.

Executive summary:

The study is performed to assess ability of the substance to cause chromosomal aberrations in human lymphocytes cultured in vitro, according to OECD Guideline 473.

The substance has shown evidence of clastogenic activity in both absence and presence of S9 mix, in this in vitro cytogenetic test system.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 09 to 22 January 1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from Aroclor 1254 treated rat livers

Test concentrations with justification for top dose:

50, 150, 500, 1500, 5000 μg/plate

Vehicle / solvent:

Water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

for TA1535 and TA100 in absence of S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

for TA1537 in absence of S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

for TA1538 and TA98 in absence of S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

for all strains in presence of S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): 3
- Number of independent experiments: 2 tests

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance: in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 72 hours
- Harvest time after the end of treatment (sampling/recovery times):

Evaluation criteria:

A compound is deemed to provide evidence of mutagenic potential if:
1) a statistically significant dose-related increase in the number of revertant colonies is obtained in two separate experiments, and
2) the increase in the number of revertant colonies is at least twice the concurrent solvent control value.

Key result

Species / strain:

S. typhimurium, other: TA 98, 100, 1535, 1537, 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

The substance has no evidence of mutagenic potential in the bacterial test system with and without metabolic activation.

Executive summary:

The study is performed to assess the mutagenic potential of the substance, using strains of S. typhimurium TA 98, 100, 1535, 1537, 1538 according to OECD Guideline 471.

The substance has no evidence of mutagenic potential in the bacterial test system with and without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo micronucleus test:
A study is performed to assess the potential induction of micronuclei by the substance in bone marrow cells of mice, according to OECD Guideline 474.
The substance did not show any evidence of causing chromosome damage when administered by intraperitoneal injection in the in vivo test.

 

Comet assay:

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.

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.

Link to relevant study records

Reference

Reference 1

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

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

Specific details on test material used for the study:

Lot No.: 0620451-LI
Purity: 99.95%

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)

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)

Dose / conc.:

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

Dose / conc.:

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

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

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.

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.

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.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 15 January to 06 March 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

Lot No.: 06508520
Purity: 99.9%

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles river UK Limited, England
- Age at study initiation: 35 days
- Weight at study initiation: 22-24 g
- Assigned to test groups randomly: yes
- Housing: randonly assigned to groups, each group was kept, with sexes separated, in plastic disposable cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 4-5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-22°C
- Humidity (%): 26-64%
- Air changes (per hr): 20 air changes per hour
- Photoperiod (hrs dark / hrs light): artificial light for 12 hours per day

Route of administration:

intraperitoneal

Vehicle:

Water

Details on exposure:

Treatment volume: 20 mL/kg bodyweight

Duration of treatment / exposure:

single intraperitoneal injection

Frequency of treatment:

single intraperitoneal injection

Post exposure period:

48 hours

Dose / conc.:

125 mg/kg bw/day

Dose / conc.:

250 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

No. of animals per sex per dose:

5 males and 5 females for 125 and 250 mg/kg dose; 10 males and 10 females for 500 mg/kg dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin C, 5 males and 5 females, dose 12 mg/kg

Tissues and cell types examined:

The stained smears were examined by light microscopy to determine the incidence of micronucleated cells per 1000 polychromatic erythrocytes per animal.

Evaluation criteria:

A positive response is normally indicated by a substantial, statistically significant increase in the incidence of micronucleated polychromatic erythrocytes compared to incidence for concurrent vehicle control group for at least one of sampling times.

Statistics:

Non-parametric statistical methods based on rank are chosen for analysis of results.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No mortalities and no adverse clinical signs were obtained for treated animals over the duration of test.
The substance did not cause any statistically significant increases in number of micronucleated polychromatic erythrocytes at either sampling time 24 hours and 48 hours.

Conclusions:

The substance did not show any evidence of causing chromosome damage when administered by intraperitoneal injection in the in vivo test.

Executive summary:

The study is performed to assess the potential induction of micronuclei by the substance in bone marrow cells of mice, according to OECD Guideline 474.
The substance did not show any evidence of causing chromosome damage when administered by intraperitoneal injection in the in vivo test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Available data:
Ames test, OECD 471: negative with/without metabolic activation;
In vitro chromosome aberration study, OECD 473: positive with/without metabolic activation;
In vitro Mammalian Cell Gene Mutation Test, OECD 490: non-mutagenic to mouse lymphoma;
In vivo micronucleus test, OECD 474: did not show evidence of causing chromosome damage to mouse bone marrow cells;

In vivo Comet assay, OECD 489: maybe induce DNA damage in duodenum of rat, no potential to induce DNA damage in liver and glandular stomach of rat.

Therefore in accordance with Regulation (EC) No. 1272/2008 Table 3.5.1, given negative result in in-vivo micronucleus test and inconclusive result in comet assay, the classification of this substance cannot be concluded for the mutagenicity endpoint.