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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
No analytical purity is given.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
THE DEPARTEMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM.
Type of assay:
other: micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
other: Hsd: ICR (CD-1®)
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan UK
- Age at study initiation: 5-8 weeks old
- Weight at study initiation: 23-30 g
- Assigned to test groups randomly: yes; selected at random and given a number unique within the study by tail marking and a number written on a colour coded cage card
- Housing: in groups of up to 7, by sex, in solid-floor polypropylene cages with wood-flake bedding
- Diet: Harlan Teklad 2014 Rodent Pelleted Diet; ad libitum
- Water: drinking water; ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Arachis Oil BP
- Amount of vehicle: 10 mL/kg
- Lot/batch no.: W72
Duration of treatment / exposure:
24 h for all dose groups
48 h for the high dose group
Frequency of treatment:
single treatment
Post exposure period:
Animals were scarificed by cervical dislocation 24 or 48 h following treatment.
Doses / concentrationsopen allclose all
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
7 (exception: in the positive control group only 5 animals were used)
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: oral (via gavage)
- Doses: 50 mg/kg

Examinations

Tissues and cell types examined:
Femur; bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Based on the outcome of the range-finding study (see table 1), the maximum tolerated dose (MTD) of the test substance is 600 mg/kg. In addition, 150 and 300 mg/kg of the test substance were selected as lower dose levels for the use in the main test.

DETAILS OF SLIDE PREPARATION: Immediately following termination (24 h or 48 h following dosing), both femurs were dissected from each animal, aspirated with foetal calf serum and bone morrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Grünwald/Giemsa, allowed to air-dry and a cover slip applied using mounting medium.
Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei.

METHOD OF ANALYSIS:
Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei.
The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.
Evaluation criteria:
A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test material groups and the number occurring in the corresponding vehicle control group.
A positive mutagenic response was demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour kill times when compared to their corresponding control group.
If these criteria were not fulfilled, then the test material was considered to be non-genotoxic under the conditions of the test.
A positive response for bone marrow toxicity was demonstrated when the dose groupan polychromatic to normochromatic ratio was shown to be statistically significantly lower than the concurrent vehicle control group.
Statistics:
All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed following a  √(X + 1) transformation using Student's t-test (two tailed) and any significant results were confird using the one way analysis of variance.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Clinical signs were observed in animals dosed with the test material at 600 mg/kg in both the 24 and 48‑hour groups, these were as follows: Hunched posture, lethargy, ataxia and ptosis.
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 600, 800, 1000 and 2000 mg/kg bw
- Clinical signs of toxicity in test animals: In animals dosed with the test material via the oral route premature deaths occurred at 800 and 2000 mg/kg (see table 1). Clinical signs were observed at and above 600 mg/kg as follows: Hunched posture, ptosis, ataxia, lethargy, pilo-erection, splayed gait, decreased respiratory rate, laboured respiration, elevated tail and tonic convulsions. The test material showed no marked difference in its toxicity to male or female mice, it was therefore considered to be acceptable to use males only for the main test. 
- Other: Adequate evidence of test material toxicity was demonstrated via the oral route of administration, therefore, this route was selected for use in the main test. The maximum tolerated dose (MTD) of the test material, 600 mg/kg, was selected for use in the main test, with 300 and 150 mg/kg as the lower dose levels.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test substance when compared to the concurrent vehicle control groups (see table 2).
- Ratio of PCE/NCE: There were no statistically significant decreases in the PCE/NCE ratio in the 24- or 48-hour test material groups when compared to their concurrent vehicle control groups (see table 3). However, the observation of clinical signs was taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved.
- Other: There were no premature deaths seen in any of the dose groups. Clinical signs were observed in animals dosed with the test material at 600 mg/kg in both the 24 and 48‑hour groups, these were as follows: Hunched posture, lethargy, ataxia and ptosis. The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the test system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

Any other information on results incl. tables

Table 1: Range-finding toxicity test

Dose Level

(mg/kg)

Sex

Number of Animals Treated

Route

Deaths on Day

Total Deaths

0

1

2

2000

Male

1

oral

1*

-

-

2/2

Female

1

1

-

-

1000

Male

1

oral

0

0

0

0/2

Female

1

0

0

0

800

Male

1

oral

0

0

0

1/2

Female

1

1

-

-

600

Male

1

oral

0

0

0

0/2

Female

1

0

0

0

600

Male

2

oral

0

0

0

0/2

Female

0

-

-

-

*= Killed in extremis

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

 

Total PCE + MN

at sampling time

Total NCE + MN at sampling time

Treatment group

Number

of animals

Dose [mg/kg]

24 h

48 h

24 h

48 h

Number scored

Vehicle control

(Arachis oil BP, 10 mL/kg)

7

0

5

3

3

0

566 ± 46 (24 h)

500 ± 44 (48 h)

Test substance

7

600

4

5

0

1

510 ± 70 (24 h)

504 ± 31 (48 h)

Test substance

7

300

11

n.d.

0

n.d.

555 ± 45

Test substance

7

150

7

n.d.

0

n.d.

570 ± 87

Positive control

(cyclophosphamide)

5

50

150

n.d.

0

n.d.

615 ± 55

n.d.= not determined; *The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE) per animal was scored; **The number of normochromatic erythrocytes (NCE) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei.

 

Table 3: Summary of the results of the in-vivo micronucleus assay.

Treatment group

Dose

[mg/kg]

Sampling time [h]

Mean frequency of PCE with MN per 2000 PCE

 

PCE/NCE ratio

Vehicle control (Arachis oil BP, 10 mL/kg)

0

48

0.4 ± 0.5

1.02 ± 0.18

 

0

24

0.7 ± 0.8

0.78 ± 0.16

Test substance

600

48

0.7 ± 0.8

0.99 ± 0.12

 

600

24

0.6 ± 0.8

1.0 ± 0.32

 

300

24

1.6 ± 1.5

0.81 ± 0.15

 

150

24

1.0 ± 1.0

0.79 ± 0.30

Positive control (Cyclophosphamide)

50

24

30.0 ± 4.7***

0.64 ± 0.14

***=P<0.001

 

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of the in vivo micronucleus test the test substance did not induce micronuclei in erythrocytes of mice treated with the test substance.