Registration Dossier

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 19, 2013 to February 04, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Clear colorless liquid
Details on test material:
Name: DEGMEE
CAS No.: 1002-67- 1
Lot No.: 20130625
Storage condition: Room temperature [(1~30)°C],
Expiration date: 2014- 06- 25
Appearance: Clear colorless liquid
Purity: 99.98 %
Molecular formula and MW: C2H5O)CH2CH2O)2CH3 / 148.20
Specific gravity: 0.918 - 0.928
Water solubility: Soluble
Treatment after end of study: Dispose

Test animals

Species:
mouse
Strain:
ICR
Sex:
male
Details on test animals and environmental conditions:
Animal species (strain): CrljOri:CD1 (ICR) Mouse, SPFSex and age: Male, 6 weeks of age at receptionSupplierName: Orient Bio, IncorporationAddress: 699-13, Mokdong- Ri, Buk-Myeon, Gapyoung-Gun, Gyeonggi- do, KoreaNumber of animals and range of weightNumber of animals at the time of receipt: Dose range-finding test 20 mice; Main test: 28 miceA range of body weight at the time of receipt: Dose range-finding test (28.08 - 32.08) g; Main test (27.22 - 32 .07) gNumber of animals at the time of administration: Dose range-finding test 18 mice; Main test: 25 miceA range of body weight at the time of administration (1st / 2nd): Dose range - finding test (31.83 ~ 35.01 / 31.51 ~ 35.54) g; Main test (30.89 ~ 36.31 / 30.40 ~ 35.08) gReason for selection: The animals used in this test have been widely used for micronucleus tests and was chosen for this study since there were a lot of accumulated fundamental data capable of comparison to this results.Testing area and conditions for housing animalsTesting area: This test was accomplished in the 3rd clean room of animal care room in KTR Health Care Research Laboratory of Korea Testing & Research Institute (KTR).Conditions of animal care roomTemperature: (22 ± 3) °CHumidity: (50 ± 20) %Light intensity: (150 ~ 300) LuxLight cycle: 12 hours lighting up (AM 08:00 ~ PM 08:00); 12 hours lighting out (PM 08:00 ~ AM 08:00)Ventilation frequency: (10 ~ 15) times/hourHousing in the cage: Dose range-finding test - 3 animals in 1 polysulfonate cage; Main test - 5 animals in 1 polysulfonate cage (Polysulfonate cage : [180(W) x 300(0) x 140(H) mm, Three Shine, Korea])Feed and water supply: Pelleted food was obtained from Cargill Agri Purina Korea, Inc. (Jeollabuk-do, Gunsan-si, Soryong-d ong, 56-4), and UV sterilized water available ad libitum.Bedding: Bedding (TAPVEI) was obtained from WOOJUNG BSC, Inc, and that was autoclaved at 121°C for 15 min before use.Analysis of contamination for food and water: The absence of contamination was confirmed by the food COA issued and supplied by food manufacturer. The absence of contamination in water was confirmed by the routine tests performed periodically according to the KTR SOPs.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Name: Sterile distilled water (SDW)Supplier: DAI HAN PHARM. CO., LTD.Lot No.: J8LBK21Storage condition: Room temperature [(1 ~ 30) °C]Reason for selection of the vehicle: The test substance was soluble in sterile distilled water. Therefore, sterile distilled water was chosen as a vehicle and it was used for preparation of test substance.
Details on exposure:
Preparation of test substance and positive control substance: The test substance was weighed and dissolved in sterile distilled water to each dose level, and positive control (CPA) was dissolved in sterile distilled water. After preparation, the test substance and positive control were immediately used.
Duration of treatment / exposure:
48 hours
Frequency of treatment:
Daily
Post exposure period:
24 hours
Doses / concentrationsopen allclose all
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
Doses / Concentrations:500, 1000, 2000 mg/kg bwBasis:nominal conc.
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 study groups consisting of 3 test groups, 1 negative control group and 1 positive control group were selected, and each group contained 5 mice.
Control animals:
yes
Positive control(s):
Name: Cyclophosphamide monohydrate (CPA)Supplier: Sigma- Aldrich, Inc.CAS No.: 6055- 19- 2Lot No.: SLBC0666VPurity: 98 %Storage condition: (2~8)°CReason for se lection of the vehicle and positive control: The chemical that is described in OECD guideline was chosen as positive control substance.Preparation of positive control substance: The positive control (CPA) was dissolved in sterile distilled water. After preparation, positive control was immediately used.

Examinations

Tissues and cell types examined:
The number of micronucleated polychromatic erythrocyte (MNPCE) of more than 2000 polychromatic erythrocytes (PCEs) per a mouse was counted .
Details of tissue and slide preparation:
Time of specimen preparation: The specimens were prepared after about 24 hours from final administration.Method of slide preparation: The animals were moved to the autopsy room. After cervical dislocation, the femur was removed with as little blood as possible. The bone marrow was flushed with FBS. The cells were collected to the centrifuge tube and centrifuged at 1000 rpm for 5 minutes. Supernatant was discarded, and concentrated cells were suspended with a little fresh serum. The cell suspension was smeared on a clean slide. The smeared slide was air-dried and thereafter fixed for 5 minutes in 99.9% methanol.Staining slide: For the observation, 40 μg/ml acridine orange was dropped on the methanol-fixed slide, and the reafter cover-glass was placed on.ObservationObservation method: The observation was done with blind method. The slide was observed under the fluorescent microscope at the magnification of over 400X.
Evaluation criteria:
Criteria for judgement-Discrimination of polychromatic erythrocytes Polychromatic erythrocytes are determined by appearance of orange- fluorescent light without nuclei.-Discrimination of nonchromatic erythrocytes Nonchromatic erythrocytes are determined by appearance of only their black shadows without fluorescent light.Criteria of micronucleus-Size: From the smallest distinguishable one to the one as large as a half the diameter of erythrocytes.-Shape: Mainly round and included the form of donut shape, half-moon shape and so on.-Color: Same color with near cell nucleus; green fluorescent in acridine orange.Observation of specimen: Within good smeared space, micronucleated polychromatic erythrocytes (MNPCE) were counted in more than 2000 polychromatic erythrocytes (PCE) per animal. In addition, PCE frequency in more than 200 whole erythrocytes (PCE+NCE) were calculated.
Statistics:
The following statistic analyses were done using a SPSS program (Ver. 19). The result of the statistical evaluation was regarded significantly when the P value was less than 0.05.Test for differences of numbers of MNPCEs between treated and negative control group: Kruskai- Wallis' H-test.Test for differences of numbers of MNPCEs between positive and negative control group: Mann-Whitnes's U-test.Test for differences of PCE/(PCE+NCE) ratio between treated (Including positive control group) and negative control group: value of each data was subjected to ANOVA and Dunnett's test.Test for differences of PCE/(PCE+NCE) ratio between treated (Including positive control group) and negative control group: value of each data was subjected to Student's t-test (Lovell, et. al. 1989).For comparison of body weight of animals at the time of sacrifice, ANOVA and Dunnett's test were performed.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Dose range- finding testIn all groups tested, there were no dead animals after treatment of test substanceBased on dose range-finding test, the highest dose of test substance in main test was determined at 2000 mg/kg B.W.Conditions of animals (main test)After 7 days of quarantine and acclimation, grouping for healthy 28 male animals were performed. At grouping, the range of weights were (30.89 ~ 36.31) g.Result of main testBased on dose range-finding test, total 5 groups including 3 dose levels (500, 1000, 2000 mg/kg B.W.) of test substance, negative control and positive control were tested in the main test. In all groups of main test, there was no dead animal and also no statistical significance in body weights compared with the negative control.As a result of observation in more than 2000 PCEs, the frequencies of MNPCE were (0.07 ± 0.03) % in negative control, (0.07 ± 0.03) % in 500 mg/kg B. W., (0.06 ± 0.04) % in 1000 mg/kg B.W., (0.07 ± 0.04) % in 2000 mg/kg B.W., and (4.96 ± 0.38) % in positive control. In other words, there was no statistical significance at any dose of the test substance. The positive control group showed statistical significance as anticipated (P(0.01 ).The ratio of PCEs to total erythrocytes as an index of bone marrow toxicity was (52.55 ± 3.46) %, (53.47 ± 3.87) %, (52.69 ± 2.71) %, (51.48 ± 3.98) % and (39.90 ± 1.95) % in the same order as described above. There was no statistical significance shown at any dose of the test substance. The positive control group showed statistical significance as anticipated (P(0.01).

Any other information on results incl. tables

Micronucleus test in ICR mice (Group summary)

Sex

Chemical treated

Does

 (mg/kg B.W.)

No. of animal

MNPCE/PCEs (Mean±S.D., %)

PCE/(PCE+NCE) (Mean±S.D., %)

Male

Negative control (SDW)

0

5

0.07 ± 0.03

52.55 ± 3.46

Test substance

500

5

0.07 ± 0.03

53.47 ± 3.87

Test substance

1000

5

0.06 ± 0.04

52.69 ± 2.71

Test substance

2000

5

0.07 ± 0.04

51.48 ± 3.98

Positive control (CPA)

70

5

4.96 ± 0.38

39.90 ± 1.95*

* P(0.01)

SDW: Sterile distilled water

MNPCE: PCE with one or more micronuclei

PCE: Polychromatic erythrocyte

NCE: Normochromatic erythrocyte

MC: Methyl cellulose

CPA: Cyclophosphamide monohydrate (positive control)

 

Clinical signs and mortalities (Group summary)

Sex

Chemical treated

Dose

 (mg/kg B.W.)

Clinical signs

Mortality

(dead/total)

Male

Negative control

(SDW)

0

N

0%

(0/5)a

Test substance

500

N

0%

(0/5)a

Test substance

1000

N

0%

(0/5)a

Test substance

2000

N

0%

(0/5)a

Positive control

(CPA)

70

N

0%

(0/5)a

N: Normal,a: No. of dead animals/No. of tested animals

SDW: Sterile distilled water, CPA: Cyclophosphamide monohydrate

 

Body weight of animals (Group summary)

Sex

Chemical treated

Dose

(mg/kg B.W.)

Body weight (mean ± S.D. g) at the time of

Administration (No. of animal)

Sacrifice (No. of animal)

1st

2nd

Male

Negative control

(SDW)

0

33.49 ± 1.15 (5)

32.80 ± 1.24 (5)

32.79 ± 1.40 (5)

Test substance

500

33.03 ± 1.22 (5)

32.67 ± 1.34 (5)

32.49 ± 1.31 (5)

Test substance

1000

32.94 ± 1.54 (5)

32.32 ± 1.78 (5)

32.10 ± 1.79 (5)

Test substance

2000

34.01 ± 1.57 (5)

33.27 ± 1.13 (5)

33.08 ± 1.12 (5)

Positive control

(CPA)

70

32.75 ± 1.70 (5)

32.59 ± 1.69 (5)

32.49 ± 1.90 (5)

SDW: Sterile distilled water, CPA: Cyclophosphamide monohydrate

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negativeThe test substance, DEGMEE, was determined not to induce an increased frequency of micronuclei in the bone marrow cells of male ICR mice under the present experimental condition.
Executive summary:

The test substance, DEGMEE, was evaluated for the potential to cause genotoxic changes by micronucleus test in bone marrow cells of male ICR mice. Male ICR mice used in the test have been widely used for micronucleus test and were chosen because the accumulated data related to the micronucleus test were abundant to analyze the result of test.

This study was conducted in compliance with the methods described in the OECD guidelines for the testing of chemicals, Section4, TG. No. 474 'Mammalian Erythrocyte Micronucleus Test' (1997-07-21).

 

To evaluate the genotoxicity of DEGMEE, a micronucleus test was performed using the bone marrow cells of specific pathogen free (SPF) male ICR mice. On the basis of dose range-finding test, the highest dose was determined at 2000mg/kg B.W. After 1 week acclimation of 6 -week old male mice, the test substance was orally administered twice at 24 -hour intervals at a dose of 0, 500, 1000 and 2000mg/kg B.W., and 5animals per each group were used.The positive control was administered intraperitoneally once at dose of 70mg/kg B.W. To evaluate the frequency of micronucleus and bone marrow toxicity, bone marrow cells were collected after about 24 hours of final administration.

 

The number of micronucleated polychromatic erythrocyte (MNPCE) of more than 2000 polychromatic erythrocytes (PCEs) per a mouse was counted. As a result, there was no increase of MNPCE at any dose of test substance compared to the negative control group, and also statistical significance was not observed. Moreover, no statistical significance was observed in the value for the ratio of PCE to total erythrocytes [PCE/(PCE+NCE)] between the test substance-dosed group and negative control group. As expected, there was a statistical significance in number of MNPCEs in the positive control group.

Therefore, the test substance, DEGMEE, was determined not to induce an increased frequency of micronuclei in the bone marrow cells of male ICR mice under the present experimental condition.