Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
No historical negative or positive control data were reported, dose volume was 20 mL/kg bw instead of max. 10 mL/kg bw, 2000 immature erythrocytes/animal were scored instead of 4000.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted Sep 2014
Deviations:
yes
Remarks:
No historical negative or positive control data were reported, dose volume 20 mL/kg bw, 2000 immature erythrocytes/ animal were scored
Qualifier:
according to
Guideline:
other: The Guidelines for the Testing of Chemicals (State Environmental Protection Administration of China) 2004.5
Deviations:
not specified
Qualifier:
according to
Guideline:
other: The Guidelines for the Hazard Evaluation of New Chemical Substances (HJ/T 154-2004)
Deviations:
no
GLP compliance:
not specified
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Test animals

Species:
mouse
Strain:
other: Kunming (SPF grade)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Vital River Laboratory Animal Technology Co. Ltd., Beijing, China
- Weight at study initiation: 25 - 28 g (females, main study), 26 - 28 g (males, main study); 18.1 - 20.4 (females, preliminary study), 18.7 - 20.1 (males, preliminary study)
- Fasting period before study: from 12 h before dosing until 3 h after (preliminary study only)
- Housing: the animals were housed in clear plastic cages with stainless steel tops
- Diet: feed (SCXK (JIN) 2012-0001, Tianjin Huarong Laboratory Animal Science and Technology Co., Ltd., Tianijin, China), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 25
- Humidity (%): 50 ± 20
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: vegetable oil
- Justification for choice of solvent/vehicle: the test substance formed a dosable and apparently miscible suspension in vegetable oil
- Concentration of test material in vehicle: approximately 100 mg/mL; administered as 2 mL/100 g bw
Duration of treatment / exposure:
2 days
Frequency of treatment:
daily (2 doses in total)
Post exposure period:
24 h
Doses / concentrations
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: intraperitoneal injection
- Doses / concentrations: 40 mg/kg bw

Examinations

Tissues and cell types examined:
Tissue: bone marrow
Cell type: bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: No mortality or signs of toxicity were noted in mice administered a single dose of 5000 mg/kg bw in the range-finding study. The recommended limit dose according the OECD guideline 474 is 2000 mg/kg bw for administration periods of less than 14 days. Therefore the limit dose of 2000 mg/kg bw/day was selected.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): the bone marrow cells were harvested 24 h after the last test substance administration.

DETAILS OF SLIDE PREPARATION: Slides were fixed with methanol and stained with Giemsa solution.

METHOD OF ANALYSIS: 2000 polychromatic erythrocytes (PCE) were scored per animal to determine the frequency of micronucleated polychromatic erythrocytes (MNPCE). The ratio of polychromatic to monochromatic erythrocytes (PCE/NCE) was determined.
Statistics:
The statistical significance between groups was analysed using ANOVA. All results were expressed as mean ± standard deviation; values were considered significant at p < 0.05.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 5000 mg/kg bw
- Clinical signs of toxicity in test animals: none
- Rationale for exposure: the range-finding study was performed to establish the highest dose level at which no or low systemic toxicity was observed.
- Other: 5 mice/sex were administered 5000 mg/kg bw by gavage as a single dose. There was no mortality during the 14-day observation period. No adverse clinical signs were observed and the body weight gain was within the expected range for this species and strain. The histopathological examination did not show any abnormal results.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): the induction of micronuclei was not significantly increased in the treatment group, compared with the control group (see Table 1 - 3 under 'Any other information on results incl. tables').
- Ratio of PCE/NCE (for Micronucleus assay): For females, the ratio of PCE/NCE was 1.06, 1.06 and 1.05 for the vehicle, treatment and positive control group, respectively. For males, the ratio of PCE/NCE was 1.05, 1.03 and 1.02 for the vehicle, treatment and positive control group, respectively.
- Appropriateness of dose levels and route: the route and dose level was suitable under the conditions of the study.
- Statistical evaluation: the difference between the vehicle control and test substance group was not significant.

OTHER:
No effects on body weight were observed in any of the control or treatment groups.

Any other information on results incl. tables

Table 1: Results of the in vivo micronucleus assay in male animals

 

 

 

Total micronuclei

per 2000

PCEs at

sampling time

Exp group

Number

of animals/sex

Dose [mg/kg bw]

24 h

Vehicle control

(vegetable oil)

5

0

7

Test substance

 

5

2000

11

Positive control

(cyclophosphamide)

5

40

192*

*statistically significant (p<0.05);

  Table 2: Results of the in vivo micronucleus assay in female animals.

 

 

 

Total micronuclei

per 2000

PCEs at

sampling time

Exp group

Number

of animals/sex

Dose [mg/kg bw]

24 h

Vehicle control

(vegetable oil)

5

0

10

Test substance

 

5

2000

8

Positive control

(cyclophosphamide)

5

40

205*

*statistically significant (p<0.05);

 

Table 3: Results of the in vivo micronucleus assay

Treatment group

Dose

[mg/kg bw]

Sampling time

[h]

Mean frequency of PCE with MN

 

PCE/NCE ratio

Females

Vehicle control (vegetable oil)

0

24

0.01

1.06 ± 0.04

Test substance

2000

24

0.01

1.06 ± 0.04

Positive control (cyclophosphamide)

40

24

2.05

1.05 ± 0.02

Males

Vehicle control (vegetable oil)

0

24

0.07

1.05 ± 0.03

Test substance

2000

24

0.08

1.03 ± 0.03

Positive control (cyclophosphamide)

40

24

1.92

1.02 ± 0.04

 

Applicant's summary and conclusion