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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read-across from supporting substance. Guideline study to GLP.
Justification for type of information:
See attached justification document
Cross-reference
Reason / purpose:
read-across: supporting information
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Justification for type of information:
See attached document
Reason / purpose:
read-across source
Genotoxicity:
negative

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): ITC 826 Concentrate

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Margate, UK
- Age at study initiation: Phase I: 4-13 weeks; Phase II: 9-10 weeks
- Weight at study initiation: not available
- Fasting period before study: not available
- Housing: 5 per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12:12

Administration / exposure

Route of administration:
oral: unspecified
Vehicle:
Sterilised deionised water.
Duration of treatment / exposure:
Single exposure
Frequency of treatment:
Once
Post exposure period:
24 and 48 hours
No. of animals per sex per dose:
Male: 200 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 200 mg/kg; No. of animals: 5; Sacrifice time: 48 hours
Female: 320 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 320 mg/kg; No. of animals: 5; Sacrifice times: 48 hours
Positive control(s):
Cyclophosphamide
- Justification for choice of positive control(s): Standard positive control substance
- Route of administration: oral
- Doses / concentrations: 65 mg/kg

Examinations

Tissues and cell types examined:
Bone marrow erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: MTD in preliminary (Phase I) test

TREATMENT AND SAMPLING TIMES : examination of bone marrow from femur at 24 and 48 hours post dosing

DETAILS OF SLIDE PREPARATION: four smears of marrow per slide. Slides were allowed to air dry and stained with polychrome methylene blue and eosin using an automatic staining machine

METHOD OF ANALYSIS:
Slides were coded and scored blind. for each animal, 2 x 1000 polychromatic erythrocytes were examined for the presence of micronuclei. Extended analysis of a further 2000 polychromatic erythrocytes was conducted for female vehicle control and female treated mice at the 24 hour sampling time.
OTHER:
Slides were examined for evidence of cytotoxicity (alterations in ratio of polychromatic to normochromatic erythrocytes in a sample of 1000 erythrocytes).
Evaluation criteria:
As described by Schmid (1976).
Statistics:
Analysis of variance at 24 and 48 hours, separately for males and females.

Results and discussion

Test resultsopen allclose all
Sex:
female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Doses producing toxicity: Males - 200mg/kg Females - 320mg/kg At 200 mg/kg, males showed clinical signs of toxicity including splayed gait and urinary incontinence. In females at 320 mg/kg hunched posture was observed.
Vehicle controls validity:
valid
Positive controls validity:
valid
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
at 200 mg/kg splayed gait and signs of urinary incontinence were observed
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Observations:
No biologically significant increases in micronucleus
formation were seen in any animals at any timepoint.

Any other information on results incl. tables

Phase I: Determination of MTD

Group

Test substance

Dose (mg/kg)

Sex

No. deaths/No. dosed

1

ITC 826 Concentrate

500

Male

0/2

2

ITC 826 Concentrate

800

Male

2/2

3

ITC 826 Concentrate

500

Male

Female

1/2

2/5

4

ITC 826 Concentrate

320

Male

Female

3/5

0/5

5

ITC 826 Concentrate

200

Male

0/5

 Phase II: Incidence of micronucleated PEs:

Males:  

Group

Treatment

Dose

Mean incidence of MPE/1000 PE +/- SD

24 hours

48 hours

11

Vehicle control

20 ml/kg

1.0 +/- 0.9

1.5 +/- 0.9

12

Cyclophosphamide

65 mg/kg

16.3 +/- 5.7 **

 

13

ITC 826 Concentrate

200 mg/kg

0.8 +/- 0.5

0.8 +/- 0.3

PE:   polychromatic erythrocytes

MPE:micronucleated polychromatic erythrocytes

SD:   standard deviation

** statistically significant increase p<0.01

 Females (original count):

Group

Treatment

Dose

Mean incidence of MPE/1000 PE +/- SD

24 hours

48 hours

11

Vehicle control

20 ml/kg

0.2 +/- 0.5

1.1 +/- 0.7

12

Cyclophosphamide

65 mg/kg

15.9 +/- 8.0 **

 

14

ITC 826 Concentrate

320 mg/kg

1.6 +/- 0.7*

0.9 +/- 0.2

PE:   polychromatic erythrocytes

MPE:micronucleated polychromatic erythrocytes

SD:   standard deviation

** statistically significant increase p<0.01

* statistically significant increase p<0.05

A statistically significant increase in micronuclei in high dose females at 24 hours was considered to be due to
low control values and was therefore considered not to be of toxicological significance. An extended count of a further
2000 polychromatic erythrocytes conducted at the 24 hour timepoint in females did not show any increase in the incidence of micronucleated polychromatic erythrocytes.

Females (additional count) 

Group

Treatment

Dose

Mean incidence of MPE/1000 PE +/- SD

24 hours

48 hours

11

Vehicle control

20 ml/kg

0.8 +/- 0.8

 

14

ITC 826 Concentrate

320 mg/kg

0.7 +/- 0.5

 

 Combined original and additional counts

Group

Treatment

Dose

Mean incidence of MPE/1000 PE +/- SD

24 hours

48 hours

11

Vehicle control

20 ml/kg

0.5 +/- 0.5

 

14

ITC 826 Concentrate

320 mg/kg

1.2 +/- 0.3*

 

 All means based on 20 observations (4 counts of 1000 PE per animal)

There was no change in polychromatic/normochromatic ratio.

Percentage of polychromatic erythrocytes:

Males:

Group

Treatment

Dose

Mean percentage of PE +/- SD

24 hours

48 hours

11

Vehicle control

20 ml/kg

44.1 +/- 3.6

45.6 +/- 4.2

12

Cyclophosphamide

65 mg/kg

41.1 +/- 3.2

 

13

ITC 826 Concentrate

320 mg/kg

44.8 +/- 3.5

45.3 +/- 2.3

PE:   polychromatic erythrocytes

SD:   standard deviation

Females:

Group

Treatment

Dose

Mean percentage of PE +/- SD

24 hours

48 hours

11

Vehicle control

20 ml/kg

35.9 +/- 9.1

36.0 +/- 4.7

12

Cyclophosphamide

65 mg/kg

31.5 +/- 13.2

 

14

ITC 826 Concentrate

320 mg/kg

37.3 +/- 5.1

37.9 +/- 5.5

PE:   polychromatic erythrocytes

SD:   standard deviation

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of the test, ITC 826 Concentrate is not clastogenic in the mouse bone marrow micronucleus test.
Executive summary:

ITC 826 Concentrate was evaluated for its ability to induce micronucleated polychromatic erythrocytes in the bone marrow of CD-1 mice. A single oral dose was given to groups of 5 male mice at a dose level of 200 mg/kg and to groups of 5 female mice at a dose level of 320 mg/kg. In each case the dose level used represented the maximum tolerated dose (MTD) based on patterns of clinical signs and lethalities over a four day observation period . Bone marrow samples were taken 24 and 48 hours after dosing.

No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes, over the control values, were seen in males at the 24 hour sampling time or in either males or females at the 48 hour sampling time. Although a small but statistically significant increase in the incidence of micronucleated polychromatic erythrocytes, compared to the vehicle control, was observed at the 24 hour sampling time in females dosed with ITC Concentrate at 320 mg/kg, this was not reproduced in an extended analysis of a further 2000 polychromatic erythrocytes and is therefore considered of no biological significance.

Comparison of the percentage of polychromatic erythrocytes showed no statistically or biologically significant differences in either sex at either of the sampling times between the vehicle control animals and those treated with ITC 826 Concentrate.

The test system positive control, cyclophosphamide, induced statistically significant and biologically meaningful increase in the incidence of micronucleated polychromatic erythrocytes, compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known clastogen.

Under the conditions of the test, ITC 826 Concentrate is not clastogenic in the mouse bone marrow micronucleus test.