N,N'-bis(5,5-dimethyl-1,3,2-dioxaphosphinane-2-oxide-2-yl)ethane-1,2-diamine

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 19, 2013 to October 2, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

"OECD Guidelines for the Testing of Chemicals, No.474, Mammalian Erythrocyte Micronucleus Test " (Adopted: July 21, 1997)

Deviations:

yes

Remarks:

See "Any other information" for details

Test guideline 2

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

"Micronucleus test in rodents" of "Mutagenicity Test" stipulated in the "Test Methods of the New Chemical Substances etc." (March 31, 2011, Yakushokuhatsu 0331 No.7, Heisei 23.03.29 Seikyoku No.5, Kanpokihatsu No.110331009, partly amended by Yakushokuhatsu 0402 No.1, Heisei 24.03.28 Seikyoku No.2, Kanpokihatsu No. 120402001 on April 2, 2012)

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

2-({2-[(5,5-dimethyl-2-oxo-1,3,2λ⁵-dioxaphosphinan-2-yl)amino]ethyl}amino)-5,5-dimethyl-1,3,2λ⁵-dioxaphosphinan-2-one

EC Number:

835-272-7

Cas Number:

256374-76-2

Molecular formula:

C12H26N2O6P2

IUPAC Name:

2-({2-[(5,5-dimethyl-2-oxo-1,3,2λ⁵-dioxaphosphinan-2-yl)amino]ethyl}amino)-5,5-dimethyl-1,3,2λ⁵-dioxaphosphinan-2-one

Test material form:

solid: particulate/powder

Details on test material:

Name
Name: Reaction products of ethane-1,2-diamine, phosphoryl=trichloride and 2,2-dimethylpropane-1,3-diol which makes N,N'-bis(5,5-dimethyl-1,3,2-dioxaphosphinane=2-oxide-2-yl)ethane-1,2-diamine as a main component
Other name: SH-0850
CAS number: 256374-76-2 (main component)

Structural formula
Molecular formula: C12H26N2O6P2 (main component)
Molecular weight: 356.29 (main component)

Provided sample
Purity of the test substance: 100%
Lot number: SK-241002

Physical-chemical properties
Solubility in water: Less than 0.03% (w/w) by visual observation
Melting point: 277 °C
Appearance at normal temperatures: White powder

Storage condition
The test substance was stored in a dark place at room temperature.

Precaution for handling
Protective gloves, mask, glasses and clothes were put on in order to avoid contacts with skin or eyes and inhalation.

Specific details on test material used for the study:

No further details specified in the study report.

Test animals

Species:

mouse

Strain:

other: Crlj:CD1(ICR), SPF,

Details on species / strain selection:

Mouse is commonly used for micronucleus test because it is easy to observe micronuclei and plenty of background data are available.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species, strain, sex and supplier: Mouse, Crlj:CD1(ICR), SPF, male and female, Charles River Laboratories Japan, Inc. (Hino Breeding Center)

Weeks of age on arrival and number of animals purchased
Preliminary test: 6 weeks old, 24 males, 24 females
Micronucleus test: 6 weeks old, 35 males

Weeks of age and body weight range at administration
Preliminary test: 7 weeks old, body weight range of within +/-20% of mean value
Micronucleus test: 7 weeks old, body weight range of within +/-20% of mean value

Quarantine, acclimatization method and period
Appearance of the animals was observed on receipt and animals which were judged to be healthy were subjected to quarantine and acclimatization for more than 5 days. During this period, clinical condition was observed once daily. Body weight was measured on receipt and at the end of quarantine.

Allocation
Animals which grew steadily during quarantine and acclimatization period and were judged to be healthy by the administrator of experimental animals were allocated to the groups by random sampling based on body weight level on the day before the start of administration. The animals were allocated to 7 groups of 3 males and females, respectively, in the preliminary test, and to 5 groups of 6 animals in the micronucleus test.
The animals were administered at 1 day after group allocation. Excessive animals which were not used for test were excluded from the study after allocation and euthanized by cervical dislocation under ether anesthesia.

Identification method
Animal
On receipt, animal receipt numbers were assigned randomly. 5 or less animals were housed in each cage in ascending order of the number. However, for animals No. 21 - 24 used in the preliminary test 4 males and 4 females were housed respectively in each cage. 4 animals which were assigned with a smaller number in each cage were marked on their tails by oil ink (the 5th animals had no marking) for identification.
After allocation, the new animal numbers were assigned to the animals and 3 or less animals were housed in each cage (preliminary test: 1 or 2 cages/dose, micronucleus test: 2 cages/dose). The animals were identified on dorsal hair by marking of the number 1 - 3 in the preliminary test and 1 - 6 in the nucleus test in ascending order of the animal number for each dose level. For marking after allocation, water based ink (AnimalMarker FG-2000, Fuchigami-Kikai) was used and different colors were used for each dose level.

Cage
Cages were identified by labels which showed the study number, sex, animal receipt number, cage number, receipt date, weeks of age at receipt, species, strain and name of the study director before allocation, and by labels which showed the study number, cage number, rearing period (the date of allocation to the end date of rearing), strain, species, sex, group, dose, animal number and the name of the study director after allocation.

Weeks of age of the animals at the start of administration, number of animals used in the test and body weight at the initial administration
Preliminary test: 7 weeks old, 21 males, body weight at the initial administration: 27.8 - 33.0 g, mean: 31.1 g; 7 weeks old, 21 females, body weight at the initial administration: 23.1 - 28.7 g, mean: 25.7 g
Micronucleus test: 7 weeks old, 30 males, body weight at the initial administration: 32.0 – 37.4 g, mean: 35.3 g (positive control group is excluded from the values of body weight at the initial administration and mean.)

Husbandry
Environmental conditions
Animal room: During quarantine period: Quarantine room 2; After the end of quarantine: Animal room 4
After allocation for the preliminary test, the animals were maintained in Safety Clean Lack NCR-05 (Shibata Scientific Technology Ltd.,) which was located in Animal room.
Temperature: 21 - 25ºC (measured value: 22.8 - 24.3ºC)
Relative humidity: 40 - 70% (measured value: 47.9 - 60.9%)
Frequency of ventilation: 10 - 15 times/hour (55 - 72 times/hour when Safety Clean Rack was used.)
Lighting cycle: 12 hours light (7:00 – 19:00) / 12 hours dark (19:00 – 7:00)
Type and size of cage: Polycarbonate flat floor cage (210 x 320 x 130 mm, W x D x H)
Number of animals: 5 or less animals/cage
Frequency for change of housing equipment: Cage, cage lid, bedding, enrichment and watering bottle were changed at the end of quarantine. Cage and bedding were also changed on transfer to the necropsy room.

Feeding
Food and feeding method: Autoclaved Pelleted Food MF for experimental animal was given ad libitum by feeder of stainless cage.
Manufacturer and lot number
Manufacturer: Oriental Yeast Co., Ltd.
Lot number: 130515
Confirmation for quality: Based on data of analysis of contaminants at Eurofin which was provided by the manufacturer and “Limit for Contaminants in Feed and Medium” under Toxic Substances Control Act by EPA, US (1979), feed which was confirmed to satisfy the standard of Hita Laboratory was used.

Drinking water
Water and supplying method: Tap water of Hita city to which sodium hypochlorite (Purelox) was added to adjust chlorine concentration between 3 and 5 ppm was given ad libitum by watering bottle.
Confirmation for quality: Water quality was inspected in accordance with “Ministerial Ordinance for Water Quality Standard” (Ordinance by Ministry of Health, Labour and Welfare, No. 101) by the Ministry of Health, Labour and Welfare twice a year. It was confirmed that the latest result of the inspection which had been acquired before animal receipt fulfilled the standard of the Ministry of Health, Labour and Welfare.

Bedding and enrichment
Type: Bedding: Sunflake; Enrichment: Woodbite
Manufacturer and lot number
Manufacturer: Charles River Laboratories Japan, Inc.
Lot number: 130212 (Sunflake); 130219 (Woodbite)
Confirmation for quality: Based on data of analysis for contaminants at Eurofin which was provided by manufacturer and “Limit for Contaminants in Feed and Medium” in Toxic Substances Control Act by EPA (1979), bedding and enrichment which were confirmed to be within standard values of Hita Laboratory were used.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Negative control substance (vehicle)
Name: Olive oil
Manufacturer, lot number and grade
Manufacturer: Nikko Pharmaceutical Co., Ltd.
Lot number: 242509
Grade: Japanese pharmacopeia grade

Reason for vehicle selection
Olive oil was used as the vehicle in “28-day repeated oral dose toxicity study of SH-0850 in the rats” (study number B11-1025). Therefore, olive oil was also selected as a vehicle in this study. As a result of the solubility check, the test substance formed a homogeneous suspension at the concentration of 200 mg/mL in olive oil. Change of color tone, heat generation and bubbling were not observed for the 200 mg/mL test substance suspension prepared with olive oil up to 4 hours after preparation at a room temperature. Therefore, olive oil was determined to be stable and was selected as vehicle.

Details on exposure:

Preparation of the test substance solution
Preparation method: In the preliminary test, 2.00 g of weighed test substance was added to olive oil, suspended by stirring (with a tube mixer) and by ultrasonication (with an ultrasonic cleaner) to prepare a total volume of 10 mL of the test substance solution at 200 mg/mL. The solution was serially diluted with olive oil by stirring with a magnetic stirrer to prepare the test substance solutions at 100, 50.0, 25.0, 12.5 and 6.25 mg/mL. In the micronucleus test, 2.00 g of weighed test substance was added to olive oil, suspended by stirring (with a tube mixer) and by ultrasonication (with an ultrasonic cleaner) to prepare a total volume of 10 mL of the test substance solution at 200 mg/mL. The solution was serially diluted with olive oil by stirring with a magnetic stirrer to prepare the test substance solutions at 100 and 50.0 mg/mL. The solution was prepared in the safety cabinet.
Preparation timing and storage: The solution was prepared on each administration day and used within 4 hours after preparation. The solution was stored at room temperature until use.

Preparation of positive control solution
Preparation method: Water for injection (lot No.: K2J85, Otsuka Pharmaceutical Factory, Inc.,) was added to a bottle containing 2 mg of MCC. MMC was dissolved in water for injection and a total volume of 10 mL of the solution was prepared at 0.2 mg/mL. The positive control substance solution was handled in the safety cabinet.
Preparation timing and storage: The solution was prepared immediately before use and used within 4 hours after preparation. The solution was stored at a room temperature until use.

Duration of treatment / exposure:

24 hours

Frequency of treatment:

2 serial oral gavage administrations were given to the animals with an interval of 24 hours.

Post exposure period:

1 day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Preliminary test: 42 animals (21 male/21 female)
Micronucleus test: 30 males

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive control substance: Mitomycin C (MMC)
Reason for selection: MMC is stated as a positive control substance in the guideline listed in the section of Study method and its background data are available at Hita Laboratory.
Storage conditions: MMC was stored at a room temperature (allowable temperature range: 10-30 °C) in Cabinet 2 in the test material storage room.

Examinations

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

Preparation timing
At 24 hours after the final administration.

Method
After animals were euthanized by cervical dislocation, femur was removed and bone marrow cells were washed out to a centrifuge tube with approximately 0.8 mL of inactivated fetal bovine serum. It was centrifuged at 1000 rpm (210 xg) for 5 minutes and sediment of collected cells was smeared onto a slide glass. After air dried, it was fixed with methanol. Slide was stained with 3 vol% Giemsa solution (Sφerenzen buffer, prepared at pH 6.8) and then was stained differentially with 0.004 w/v% citric acid solution.
Two specimens were prepared from an animal which survived at the specimen preparation phase.

Observation of specimen
Specimens from negative control, positive control and 3 dose levels of test substance treated groups were observed. For each control and test substance groups, specimens from 5 animals in ascending order of the animal number were observed.
Specimens for observation were assigned with random slide numbers and were observed with blinded method using a biological microscope (x1000).

Frequency of micronucleus
For 1 animal, 2000 (1000 per slide) of polychromatic erythrocyte (PCE) was observed to determine the frequency of micronucleated polychromatic erythrocyte (MNPCE).

Growth inhibition for bone marrow cell
For 1 animal, 200 (100 per slide) of total erythrocyte (TE) was observed to determine ratio of PCE (PCE/TE).

Evaluation criteria:

Criteria for the valid test
Mean value of MNPCE/PCE of both negative and positive control should be within the background data (negative: mean +/- 3 S.D., positive: mean +/- 3 S.D.) from the latest 20 tests in Hita Laboratory.
At specimen observation, there should be 3 or more dose levels at which their PCE/TE is greater than 20% of the PCE/TE of the negative control.

Statistics:

Determination of MNPCE/PCE
For MNPCE/PCE of each test substance groups and of positive control groups, one-tailed 1% and 5% significant tests were conducted by the conditional binominal test (Kastenbaum and Bowman) compared to the negative control. Cochran-Armitage Trend Test was planned to check the dose-dependency if the test substance groups showed the significant differences, but no significant difference was noted and no trend test was conducted. The result was considered to be positive when significant increase of MNPCE/PCE was noted for the test substance group compared with the negative control and also dose-dependency was noted in the increase.

Determination of PCE/TE
Homogeneity of variances of each group (except the positive control) was examined by Bartlett Test. As a result, the homogeneity of variances was noted and therefore Williams test was conducted. If the result of Williams test did not show any significant difference, Dunnett test was planned to check the difference of mean values between the negative control and each test substance groups, but the significant difference was noted and Dunnett test was not conducted.
For comparison between the negative and positive control, homogeneity of variances between the 2 groups was examined by F test. As a result, variance was judged to be homogeneous and the difference of mean value was examined by Student’s t-test.
A statistical tool, StatLight, was used for the tests. The significance level of each test were 5% for Bartlett test, 5% (two-tailed) for Williams test, 5% for F test, and 5% and 1% (two-tailed) for other tests.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary test
Clinical signs
No change in clinical conditions and death were noted in the negative control and all test substance groups up to day 1 after second administration.

Body weight
No significant body weight change was noted in all test substance groups up to day 1 after second administration compared with the negative control group.

Micronucleus test
Clinical signs
No change in clinical conditions and death were noted in the negative control and all test substance groups up to day 1 after second administration and in the positive control up to day 1 after single administration.

Body weight
No significant body weight change was noted in all test substance groups up to day 1 after second administration and in the positive control up to day 1 after single administration compared with the negative control group.

MNPCE/PCE/PCE and PCE/TE
Mean values of MNPCE/PCE of the negative and positive control are 0.09 and 5.39%, respectively. The mean values are 0.06, 0.08 and 0.10% at 500, 1000 and 2000 mg/kg/day of the test substance groups, respectively, and no statistically significant difference was noted for all test substance groups compared with the negative control. For the positive control, significant increase was noted at 1% level.
Mean values of PCE/TE of the negative and positive control are 47.0 and 39.2%, respectively. The mean values are 51.1, 55.6 and 60.9% at 500, 1000 and 2000 mg/kg/day of the test substance groups, respectively. Statistically significant difference was noted at 5% level (two-tailed) for the test substance groups of 1000 and 2000 mg/kg/day compared with the negative control.

Background data of Hita Laboratory
MNPCE/PCE for the negative control is 0.10 +/- 0.021% (mean +/- S.D.) and 6.91 +/- 1.030% (mean +/- S.D.) for the positive control in the background data from the latest 20 tests at Hita Laboratory. Based on this, the following range was determined as appropriate.
Negative control: 0.04 – 0.16% (mean +/- 3 S.D.)
Positive control: 3.82 – 10.00% (mean +/- 3 S.D.)

Discussion
Mean values of MNPCE/PCE of both the negative and positive control were within the background data (from the latest 20 tests) of Hita Laboratory and PCE/TE of 3 dose levels of the test substance exceeded 20% of PCE/TE of the negative control for all test substance groups. Therefore, it was concluded that the criteria of the valid test was satisfied.
Statistically significant differences were not noted in values of MNPCE/PCE of all test substance groups by specimen observation compared with the negative control group.
Therefore, it was concluded that the test substance administration did not increase the frequency of micronucleated polychromatic erythrocyte.
Statistically significant differences were noted in values of PCE/TE at 1000 and 2000 mg/kg of the test substance groups at 5% level (two- tailed). Exposure of bone marrow to the test substance that is indicated by a decrease of PCE/TE could not be verified because these significant differences were due to their higher values compared with the negative control in PCE/TE. However, it was concluded that the result of this study was proper for evaluation of the potential of micronucleus induction because the dose levels were set up to 2000 mg/kg/day which is stipulated in the guideline as the upper limit when no toxicity is noted by continuous administration.

Any other information on results incl. tables

Clinical symptoms of males in the preliminary test

Test animal: Crlj:CD1(ICR) mouse, male, 7 weeks old

Number of administrations: 2 serial administrations with an interval of 24 hours

Route of administration: Oral gavage

Dose level: 10 mL/kg

Test group	Dose level (mg/kg/day)	Animal number	First administration	Second administration
			Up to 1 hour after administration	Before administration	Up to 1 hour after administration	At day 1 after administration
Negative control [olive oil]	0	1	No abnormality	No abnormality	No abnormality	No abnormality
		2	No abnormality	No abnormality	No abnormality	No abnormality
		3	No abnormality	No abnormality	No abnormality	No abnormality
Test substance [SH-0850]	62.5	4	No abnormality	No abnormality	No abnormality	No abnormality
		5	No abnormality	No abnormality	No abnormality	No abnormality
		6	No abnormality	No abnormality	No abnormality	No abnormality
	125	7	No abnormality	No abnormality	No abnormality	No abnormality
		8	No abnormality	No abnormality	No abnormality	No abnormality
		9	No abnormality	No abnormality	No abnormality	No abnormality
	250	10	No abnormality	No abnormality	No abnormality	No abnormality
		11	No abnormality	No abnormality	No abnormality	No abnormality
		12	No abnormality	No abnormality	No abnormality	No abnormality
	500	13	No abnormality	No abnormality	No abnormality	No abnormality
		14	No abnormality	No abnormality	No abnormality	No abnormality
		15	No abnormality	No abnormality	No abnormality	No abnormality
	1000	16	No abnormality	No abnormality	No abnormality	No abnormality
		17	No abnormality	No abnormality	No abnormality	No abnormality
		18	No abnormality	No abnormality	No abnormality	No abnormality
	2000	19	No abnormality	No abnormality	No abnormality	No abnormality
		20	No abnormality	No abnormality	No abnormality	No abnormality
		21	No abnormality	No abnormality	No abnormality	No abnormality

 

Clinical symptoms of females in the preliminary test

Test animal: Crlj:CD1(ICR) mouse, male, 7 weeks old

Number of administrations: 2 serial administrations with an interval of 24 hours

Route of administration: Oral gavage

Dose level: 10 mL/kg

Test group	Dose level (mg/kg/day)	Animal number	First administration	Second administration
			Up to 1 hour after administration	Before administration	Up to 1 hour after administration	At day 1 after administration
Negative control [olive oil]	0	22	No abnormality	No abnormality	No abnormality	No abnormality
		23	No abnormality	No abnormality	No abnormality	No abnormality
		24	No abnormality	No abnormality	No abnormality	No abnormality
Test substance [SH-0850]	62.5	25	No abnormality	No abnormality	No abnormality	No abnormality
		26	No abnormality	No abnormality	No abnormality	No abnormality
		27	No abnormality	No abnormality	No abnormality	No abnormality
	125	28	No abnormality	No abnormality	No abnormality	No abnormality
		29	No abnormality	No abnormality	No abnormality	No abnormality
		30	No abnormality	No abnormality	No abnormality	No abnormality
	250	31	No abnormality	No abnormality	No abnormality	No abnormality
		32	No abnormality	No abnormality	No abnormality	No abnormality
		33	No abnormality	No abnormality	No abnormality	No abnormality
	500	34	No abnormality	No abnormality	No abnormality	No abnormality
		35	No abnormality	No abnormality	No abnormality	No abnormality
		36	No abnormality	No abnormality	No abnormality	No abnormality
	1000	37	No abnormality	No abnormality	No abnormality	No abnormality
		38	No abnormality	No abnormality	No abnormality	No abnormality
		39	No abnormality	No abnormality	No abnormality	No abnormality
	2000	40	No abnormality	No abnormality	No abnormality	No abnormality
		41	No abnormality	No abnormality	No abnormality	No abnormality
		42	No abnormality	No abnormality	No abnormality	No abnormality

 

Body weight of males in the preliminary test

Test animal: Crlj:CD1(ICR) mouse, male, 7 weeks old

Number of administrations: 2 serial administrations with an interval of 24 hours

Route of administration: Oral gavage

Dose level: 10 mL/kg

Test group	Dose level (mg/kg/day)	Animal number	Body weight (g)
			Before the first administration	Before second administration	At 1 day after second administration
Negative control [olive oil]	0	1 2 3	31.3 32.2 33.0	31.6 32.6 33.4	32.0 33.0 33.9
	Mean +/- S.D.		32.2+/-0.85	32.5+/-0.90	33.0+/-0.95
Test substance [SH-0850]	62.5	4 5 6	30.7 30.5 32.5	30.3 31.0 32.4	30.7 31.5 32.8
	Mean +/- S.D.		31.2+/-1.10	31.2+/-1.07	31.7+/-1.06
	125	7 8 9	30.0 31.0 32.1	29.4 31.2 32.2	29.9 31.7 32.8
	Mean +/- S.D.		31.0+/-1.05	30.9+/-1.42	31.5+/-1.46
	250	10 11 12	29.3 27.8 31.2	29.2 28.3 31.3	29.2 28.5 32.2
	Mean +/- S.D.		29.4+/-1.70	29.6+/-1.54	30.0+/-1.97
	500	13 14 15	31.3 31.0 32.4	30.8 31.8 31.7	31.5 32.4 31.8
	Mean +/- S.D.		31.6+/-0.74	31.4+/-0.55	31.9+/-0.46
	1000	16 17 18	30.0 31.6 32.0	30.7 32.0 32.0	31.0 32.6 31.7
	Mean +/- S.D.		31.2+/-1.06	31.6+/-0.75	31.8+/-0.80
	2000	19 20 21	29.6 32.7 31.7	29.5 32.4 31.9	29.7 32.1 33.0
	Mean +/- S.D.		31.3+/-1.58	31.3+/-1.55	31.6+/-1.71

 

Body weight of females in the preliminary test

Test animal: Crlj:CD1(ICR) mouse, male, 7 weeks old

Number of administrations: 2 serial administrations with an interval of 24 hours

Route of administration: Oral gavage

Dose level: 10 mL/kg

Test group	Dose level (mg/kg/day)	Animal number	Body weight (g)
			Before the first administration	Before second administration	At 1 day after second administration
Negative control [olive oil]	0	22 23 24	23.1 24.9 27.0	22.4 25.9 26.5	23.7 25.3 26.3
	Mean +/- S.D.		25.0+/-1.95	24.9+/-2.21	25.1+/-1.31
Test substance [SH-0850]	62.5	25 26 27	23.5 25.8 27.5	23.4 25.6 27.6	23.9 25.4 27.5
	Mean +/- S.D.		25.6+/-2.01	25.5+/-2.10	25.6+/-1.81
	125	28 29 30	24.9 25.9 27.8	25.0 25.0 28.0	24.3 25.2 27.9
	Mean +/- S.D.		26.2+/-1.47	26.0+/-1.73	25.8+/-1.87
	250	31 32 33	24.5 25.3 26.2	24.3 25.4 25.9	24.2 24.7 26.3
	Mean +/- S.D.		25.3+/-0.85	25.2+/-0.82	25.1+/-1.10
	500	34 35 36	25.5 25.3 26.5	24.9 25.3 26.1	24.8 25.3 26.1
	Mean +/- S.D.		25.8+/-0.64	25.4+/-0.61	25.4+/-0.66
	1000	37 38 39	23.7 26.6 28.7	24.2 25.9 28.9	24.0 25.6 29.7
	Mean +/- S.D.		26.3+/-2.51	26.3+/-2.38	26.4+/-2.94
	2000	40 41 42	25.1 25.7 26.4	25.3 26.1 27.0	25.4 26.3 27.5
	Mean +/- S.D.		25.7+/-0.65	26.1+/-0.85	26.4+/-1.05

 

Clinical symptoms in the micronucleus test

Test animal: Crlj:CD1(ICR) mouse, male, 7 weeks old

Number of administrations: 2 serial administrations with an interval of 24 hours (single

administration for the positive control)

Route of administration: Oral gavage (intraperitoneal administration for the positive control)

Dose level: 10 mL/kg

Test group	Dose level (mg/kg/day)	Animal number	First administration	Second administration
			Up to 1 hour after administration	Before administration	Up to 1 hour after administration	At day 1 after administration
Negative control [olive oil]	0	101	No abnormality	No abnormality	No abnormality	No abnormality
		102	No abnormality	No abnormality	No abnormality	No abnormality
		103	No abnormality	No abnormality	No abnormality	No abnormality
		104	No abnormality	No abnormality	No abnormality	No abnormality
		105	No abnormality	No abnormality	No abnormality	No abnormality
		106	No abnormality	No abnormality	No abnormality	No abnormality
Test substance [SH-0850]	500	107	No abnormality	No abnormality	No abnormality	No abnormality
		108	No abnormality	No abnormality	No abnormality	No abnormality
		109	No abnormality	No abnormality	No abnormality	No abnormality
		110	No abnormality	No abnormality	No abnormality	No abnormality
		111	No abnormality	No abnormality	No abnormality	No abnormality
		112	No abnormality	No abnormality	No abnormality	No abnormality
	1000	113	No abnormality	No abnormality	No abnormality	No abnormality
		114	No abnormality	No abnormality	No abnormality	No abnormality
		115	No abnormality	No abnormality	No abnormality	No abnormality
		116	No abnormality	No abnormality	No abnormality	No abnormality
		117	No abnormality	No abnormality	No abnormality	No abnormality
		118	No abnormality	No abnormality	No abnormality	No abnormality
	2000	119	No abnormality	No abnormality	No abnormality	No abnormality
		120	No abnormality	No abnormality	No abnormality	No abnormality
		121	No abnormality	No abnormality	No abnormality	No abnormality
		122	No abnormality	No abnormality	No abnormality	No abnormality
		123	No abnormality	No abnormality	No abnormality	No abnormality
		124	No abnormality	No abnormality	No abnormality	No abnormality
Positive control [MMC]	2	125		No abnormality	No abnormality	No abnormality
		126		No abnormality	No abnormality	No abnormality
		127		No abnormality	No abnormality	No abnormality
		128		No abnormality	No abnormality	No abnormality
		129		No abnormality	No abnormality	No abnormality
		130		No abnormality	No abnormality	No abnormality

MMC: Mitomycin C

 

Body weight in the micronucleus test

Test animal: Crlj:CD1(ICR) mouse, male, 7 weeks old

Number of administrations: 2 serial administrations with an interval of 24 hours (single

administration for the positive control)

Route of administration: Oral gavage (intraperitoneal administration for the positive control)

Dose level: 10 mL/kg

Test group	Dose level (mg/kg/day)	Animal number	Body weight (g)
			Before the first administration	Before second administration	At 1 day after second administration
Negative control [olive oil]	0	101 102 103 104 105 106	34.6 35.1 35.0 35.9 36.4 36.5	34.7 35.2 35.3 36.3 36.7 36.9	35.3 36.1 35.2 36.9 37.3 37.0
	Mean +/- S.D.		35.6+/-0.79	35.9+/-0.90	36.3+/-0.91
Test substance [SH-0850]	500	107 108 109 110 111 112	33.1 35.5 34.8 35.4 35.9 37.4	33.7 35.5 35.2 35.6 35.7 37.3	33.6 35.9 35.4 35.9 36.2 37.9
	Mean +/- S.D.		35.4+/-1.41	35.5+/-1.15	35.8+/-1.38
	1000	113 114 115 116 117 118	32.0 34.7 35.4 36.7 35.6 36.8	32.4 35.4 36.3 36.3 36.0 36.8	32.5 35.6 36.5 37.1 36.3 37.1
	Mean +/- S.D.		35.2+/-1.76	35.5+/-1.60	35.9+/-1.73
	2000	119 120 121 122 123 124	33.1 34.4 34.3 36.1 36.2 35.7	33.2 34.3 35.1 36.7 36.1 35.6	33.6 34.7 35.4 36.0 36.6 36.0
	Mean +/- S.D.		35.0+/-1.23	35.2+/-1.27	35.4+/-1.09
Positive control [MMC]	2	125 126 127 128 129 130		32.6 35.0 35.8 33.4 36.0 37.9	32.2 35.3 35.9 34.1 35.8 38.8
	Mean +/- S.D.			35.1+/-1.91	35.4+/-2.19

MMC: Mitomycin C

 

Specimen observation in the micronucleus test

Test animal: Crlj:CD1(ICR) mouse, male, 7 weeks old

Number of administrations: 2 serial administrations with an interval of 24 hours (single

administration for the positive control)

Route of administration: Oral gavage (intraperitoneal administration for the positive control)

Dose level: 10 mL/kg

Test group	Dose level (mg/kg/day)	Specimen preparation period after final administration (hours)	Animal number	Ratio of polychromatic erythrocyte to total erythrocyte^a) (%)	Frequency of micronucleated polychromatic erythrocyte^b) (%)
Negative control [olive oil]	0	24	101 102 103 104 105	50.0 52.0 37.0 43.0 53.0	0.05 0.10 0.10 0.10 0.10
	Mean +/- S.D.			47.0+/-6.82	0.09+/-0.022
	Maximum/Minimum			53.0/37.0	0.10/0.05
Test substance [SH-0850]	500	24	107 108 109 110 111	47.0 43.5 52.5 62.5 50.0	0.05 0.05 0.00 0.05 0.15
	Mean +/- S.D.			51.1+/-7.21	0.06+/-0.055
	Maximum/Minimum			62.5/43.5	0.15/0.00
	1000	24	113 114 115 116 117	52.0 53.0 56.0 60.5 56.5	0.15 0.05 0.10 0.10 0.00
	Mean +/- S.D.			55.6+/-3.334#	0.08+/-0.057
	Maximum/Minimum			60.5/52.0	0.15/0.00
	2000	24	119 120 121 122 123	61.0 56.0 58.5 64.0 65.0	0.05 0.10 0.10 0.15 0.10
	Mean +/- S.D.			60.9+/-3.75#	0.10+/-0.035
	Maximum/Minimum			65.0/56.0	0.15/0.05
Positive control [MMC]	2	24	125 126 127 128 129	36.0 38.5 37.0 38.5 46.0	4.75 3.95 7.50 3.85 6.90
	Mean +/- S.D.			39.2+/-3.95	5.39+/-1.702**
	Maximum/Minimum			46.0/36.0	7.50/3.85

MMC: Mitomycin C

a) 200 RBC/animal were observed

b) 2000 PCEs/animal were observed

#: Significant difference was noted compared with the negative control (p<0.05, Williams test)

**: Significant difference was noted compared with the negative control (p<0.01, conditioned binominal test (Kastenbaum and Bowmann))

 

Back group data of the micronucleus test in the mouse

Type on control	Ratio of polychromatic erythrocyte to total erythrocyte	Frequency of micronucleated polychromatic erythrocyte		Number of experiment
	Mean +/- S.D. (%)	Mean +/- S.D. (%)	Range (%)
Negative	51.5+/-5.48	0.10+/-0.021	0.04 – 0.16 (mean+/-3 S.D.)	20
Positive	41.6+/-4.77	6.91+/-1.030	3.82 – 10.00 (mean+/-3 S.D.)	20

Data from the latest 20 test completed before July 19, 2013.

For positive control, MMC was administered by single intraperitoneal administration at 2 mg/kg/day.

Applicant's summary and conclusion

Conclusions:

It was concluded that SH-0850 does not induce micronuclei under the condition of this study.

Executive summary:

Potential of micronucleus induction of SH-0850 was assessed using male Crlj:CD1(ICR) mouse of 7-weeks of age. 2 serial oral gavage administrations were given to the animals with an interval of 24 hours.

 

In the preliminary test at the dose levels of 62.5-2000 mg/kg/day, maximum tolerated dose was estimated to be 2000 mg/kg/day or greater for both male and female when death of animal was served as an index. Therefore, 3 dose levels of 2000 mg/kg/day that is the highest dose level, 1000 and 500 mg/kg/day by dilution with the common ratio of 2 were selected for the micronucleus test.

Male animals only were used because it was concluded that the toxicity level was not different between male and female. A negative and a positive control groups were set as control groups and 2 serial oral gavage administrations of vehicle (olive oil) was given to the negative control group at the dose level of 10 mL/kg. For the positive control, Mitomycin C was administered by single intraperitoneal administration at 2 mg/kg/day.

 

No death was observed in any groups treated with the test substance at any dose levels up to the highest dose level of 2000 mg/kg/day at the time of specimen preparation which was at 24 hours after the second administration. Therefore, 3 dose levels were supplied for specimen observation and the frequency of micronucleated polychromatic erythrocyte (MNPCE/PCE) and ratio between polychromatic and total erythrocyte (PCE/TE) were examined.

 

No statistically significant difference was noted in MNPCE/PCE compared to the negative control at any dose levels in test substance groups in specimen observation. It was concluded that the test substance administration did not increase the frequency of micronucleated polychromatic erythrocyte.

 

Based on above, it was concluded that SH-0850 does not induce micronuclei under the condition of this study.