Registration Dossier

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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
21 July 1997
GLP compliance:
yes (incl. certificate)
Remarks:
Landesanstalt für Pflanzenbau und Pflanzenschutz, Essenheimer Str. 144, D-55128 Mainz
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF and 33-0988
- Expiration date of the lot/batch: 15 August 2002
- Purity test date: 15 August 2000

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerator (4-10 °C)
- Stability under test conditions: confirmed for two years
- Solubility and stability of the test substance in the solvent/vehicle: stability in propylene carbonate verified analytically

FORM AS APPLIED IN THE TEST solution in vehicle

Test animals

Species:
mouse
Strain:
NMRI
Remarks:
Crl:NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH
- Age at study initiation: 5 - 8 weeks
- Weight at study initiation: mean = 27 g
- Assigned to test groups randomly: no, based on a randomization plan (computer program)
- Housing: at least 5 days the animals were housed in Makrolon cages; before the start of study: Makrolon cages, type MI
- Diet: ad libitum, Kliba Haltungsdiät, Provimi Kliba SA, Kaiseraugst, Switzerland
- Water: ad libitum, drinking water from bottles
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12 hours (12 hours light from 6.00 - 18.00 hours and 12 hours darkness from 18.00 - 6.00 hours)

IN-LIFE DATES: From: 09 March 2001 To: 28 August 2001

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Due to the hydrolytical sensitivity of the test substance in water, propylene carbonate was selected as the vehicle as requested by the sponsor.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: substance to be administered per kg body weight was dissolved in propylene carbonate

- Volume administered: 10 mL/kg bw
Duration of treatment / exposure:
single oral administration
Frequency of treatment:
once
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day
Remarks:
1st experiment
Dose / conc.:
100 mg/kg bw/day
Remarks:
1st experiment
Dose / conc.:
200 mg/kg bw/day
Remarks:
1st experiment
Dose / conc.:
100 mg/kg bw/day
Remarks:
2nd experiment
Dose / conc.:
150 mg/kg bw/day
Remarks:
2nd experiment
Dose / conc.:
200 mg/kg bw/day
Remarks:
2nd experiment
No. of animals per sex per dose:
5
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Positive control(s):
Once orally each in a volume of 10 mI/kg body weight: 20 mg cyclophosphamide and 0.15 mg vincristine sulphate.
The stability of both chemicals is welI-defined under the selected conditions, since both positive control articles are welI-defined clastogens and aneugens respectively.

Examinations

Tissues and cell types examined:
Bone marrow cells
Details of tissue and slide preparation:
In a pretest for the determination of the acute oral toxicity, deaths were observed down to a dose of 200 mg/kg body weight (male and femnale). At this dose, at which one animal died 48 hours after administration of the test substance, evident signs of toxicity were observed, such as gasping respiration, abdominal position and squatting posture, and the general state of the animais was poor. However, there were no symptomatic differences between the male and female animais. Thus, only male animals were used for the cytogenetic investigations.
Therefore, a dose of 200 mg/kg body weight was selected as the highest dose in the 1st cytogenetic study. 100 mg/kg and 50 mg/kg body weight were administered as further doses. In the 2nd experiment, doses of 200 mg/kg, 150 mg/kg and 100 mg/kg body weight were evaluated.
Evaluation criteria:
- test chemical considered positive:
- dose-related and significant increase in the number of micronucleated polychromatic erythrocytes at any of the intervals
- proportion of cells containing micronuclei exceeded both the values of the concurrent negative control range and the negative historical control range
- test chemical considered negative:
- no significant increase in the number of micronucleated polychromatic erythrocytes at any dose above concurrent control frequencies and at any time
- frequencies of cells containing micronuclei were within the historical control range
Statistics:
- Wilcoxon hypothesis of equal medians: test for the number of micronuclei in polychromatic erythrocytes, comparison of dose group with the vehicle control
- Labels of significance: * for p 0.05, ** for p 0.01

Results and discussion

Test results
Sex:
male
Genotoxicity:
positive
Remarks:
1st experiment: statistically significant increase in number of micronuclei at 200 mg/kg bw; 2nd experiment: slightly pronounced dose-dependent and statistically significant increase of small micronuclei at 100 mg/kg up to 200 mg/kg body
Toxicity:
yes
Remarks:
test substance led to signs of toxicity; no signs after administration of cyclophosphamide or vincristine
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY (acute oral toxicity study)
- Dose range:
- Solubility:
- Clinical signs of toxicity in test animals: evident signs of toxicity observed, such as gasping respiration, abdominal position and squatting posture and general poor state of animals;
- Evidence of cytotoxicity in tissue analyzed:
- Rationale for exposure: 200 mg/kg bw selected as high dose in 1st cytogenetic study
- Other: no symptomatic differences between male and female; therefore, only male animais were used for the cytogenetic investigations

Any other information on results incl. tables

For table 1-4:

PCE: polychromatic erythrocytes, NCE: normochromatic erythrocytes, CPP: Cyclophosphamide, VCR: Vincristine Sulphate

Wilcoxon test (one-sided): *: p<= 0.05, **: p<=0.01; a pairwise comparison of each dose group with the vehicle control group

Table 1: Summary table of the results of the 1st experiment for polychromatic and normochromatic erythrocytes

 

Interval: 24 hours

Interval: 48 hours

 

Total No. of

MN (o/oo) in

Total No. of

MN (o/oo) in

 

PCE’s

NCE’s

PCE’s

NCE’s

PCE’s

NCE’s

PCE’s

NCE’s

untreated control:

10000

3227

1.1

0.6

10000

2862

1.7

0.0

propylene carbonate:

10000

3583

1.9

0.8

10000

2661

1.2

0.0

 

 

 

 

 

 

 

 

 

50 mg/kg

10000

2756

2.1

1.1

 

 

 

 

100 mg/kg

10000

3985

2.3

0.8

 

 

 

 

200 mg/kg

10000

3678

3.9*

0.5

10000

4617

3.9

0.9

CPP 20 mg/kg

10000

3710

21.1**

1.6

 

 

 

 

VCR 0.15 mg/kg

10000

4680

49.6**

1.1

 

 

 

 

Table 2: Summary table of the results of the 1st experiment for polychromatic erythrocytes, differentiation between small and large micronuclei

 

Interval: 24 hours

Interval: 48 hours

 

Total No. of

Cells (o/oo) with

Total No. of

Cells (o/oo) with

 

PCE’s

MN. d<D/4

MN. d>=D/4

PCE’s

MN. d<D/4

MN. d>=D/4

untreated control:

10000

1.1

0.0

10000

1.7

0.0

propylene carbonate:

10000

1.9

0.0

10000

1.2

0.0

 

 

 

 

 

 

 

50 mg/kg

10000

2.1

0.0

 

 

 

100 mg/kg

10000

2.1

0.2

 

 

 

200 mg/kg

10000

3.9*

0.0

10000

3.9

0.0

CPP 20 mg/kg

10000

21.0**

0.1

 

 

 

VCR 0.15 mg/kg

10000

42.3**

7.3**

 

 

 

Table 3: Summary table (summary of 1st, 2nd and 3rd evaluation) of the results of the 2nd experiment for polychromatic and normochromatic erythrocytes

 

Interval: 24 hours

 

Total No. of

MN (o/oo) in

 

PCE’s

NCE’s

PCE’s

NCE’s

propylene carbonate:

30000

9207

1.9

1.2

 

 

 

 

 

100 mg/kg

30000

12231

4.5**

1.4

150 mg/kg

30000

13246

4.5**

1.0

200 mg/kg

30000

15903

5.0**

1.1

Table 4: Summary table (summary of 1st, 2nd and 3rd evaluation) of the results of the 2nd experiment for polychromatic erythrocytes, differentiation between small and large micronuclei

 

Interval: 24 hours

 

Total No. of

Cells (o/oo) with

 

PCE’s

MN. d<D/4

MN. d>=D/4

propylene carbonate:

30000

1.9

0.0

 

 

 

 

100 mg/kg

30000

4.4**

0.1

150 mg/kg

30000

4.5**

0.0

200 mg/kg

30000

4.9**

0.1

Applicant's summary and conclusion

Conclusions:
According to the results of the present study, the single oral administration of the test substance led to a slight but relevant and statistically significant increase in the number of polychromatic erythrocytes containing small micronuclei in two experiments carried out independently of each other.
A slight and dose-dependent inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected only in the 2nd experiment.

Under the experimental conditions chosen here, the test substance considered to be a chromosome-damaging (clastogenic) agent in bone marrow cells in vivo.