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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
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:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Purity: 99.932 area %

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 26.9 g
- Housing: individual housing in appropriately labeled cages, Makrolon cages, type M I
- Bedding: Type Lignocel FS 14 fibres, dustfree bedding, supplied by SSNIFF, Soest, Germany
- Diet: standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water: drinking water from bottles, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70%
- Air changes (per hr): fully air-conditioned rooms with central air conditioning
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
purified water
Details on exposure:
The substance to be administered per kg body weight was dissolved in purified water. To achieve a solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly. All test substance formulations were prepared immediately before administration.
Duration of treatment / exposure:
once intraperitoneally
Frequency of treatment:
once intraperitoneally
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
vincristine sulfate

Examinations

Tissues and cell types examined:
Bone marrow cells
Details of tissue and slide preparation:
The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration from the animals of the highest dose group of 2000 mg/kg bw and those of the vehicle control group. In the test groups of 1000 mg/kg and 500 mg/kg bw and in the positive control groups, the 24-hour sacrifice interval was investigated only. After staining of the preparations, 2000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 2000 polychromatic erythrocytes were also recorded.
As vehicle control, male mice were administered merely the vehicle purified water by the same route and in the same volume as the animals of the dose groups, which gave frequencies of micronucleated polychromatic erythrocytes within the historical vehicle control data range.
Evaluation criteria:
A finding is considered positive if the following criteria are met:
• Statistically significant and dose-related increase in the number of PCEs containing micronuclei.
• The number of PCEs containing micronuclei has to exceed both the concurrent vehicle control value and the range of the historical vehicle control data.
A test substance is considered negative if the following criteria are met:
• The number of cells containing micronuclei in the dose groups is not statistically significant increased above the concurrent vehicle control value and is within the range of the historical vehicle control data.
Statistics:
The statistical evaluation of the data was carried out using the program system MUKERN (BASF SE). The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there are statistically significant differences between the untreated control group and the treated dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal were used as a criterion for the rank determination for the U test. Statistical significances were identified as follows: * p ≤ 0.05 ** p ≤ 0.01. However, both biological relevance and statistical significance were considered together.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Both positive control substances, cyclophosphamide for clastogenicity and vincristine sulfate for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei.
A slight inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected at 2 000 mg/kg body weight at 48-hour sacrifice interval.
According to the results of the present study, the single intraperitoneal administration of Diethylene glycol did not lead to a relevant increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was close to the range of the concurrent vehicle control in all dose groups and at all sacrifice intervals and within the range of the historical vehicle control data.

Any other information on results incl. tables

Summary table - Induction of Micronuclei in bone marrow cells - single intraperitoneal administration

Test group

Sacrifice

Animal

Micronuclei in PCE

Number

 

interval [hrs]

No.

totala
[
]

large MNb
[
]

of NCEc

Vehicle control
purified water

24

5

1.3

0.0

3 820

Test substance
500 mg/kg bw.

24

5

0.6

0.0

4 389

Test substance
1 000 mg/kg bw.

24

5

0.6

0.0

4 227

Test substance
2 000 mg/kg bw.

24

5

0.6

0.0

3 941

Positive control
cyclophosphamide 20 mg/kg bw.

24

5

20.8**

0.1

6 045

Positive control
vincristine sulfate
0.15 mg/kg bw.

24

5

54.3**

17.1**

5 979

Vehicle control
purified water

48

5

0.9

0.1

3 138

Test substance
2 000 mg/kg bw.

48

5

1.9

0.0

5 331

PCE

= polychromatic erythrocytes

NCE

= normochromatic erythrocytes

bw.

= body weight

 

 

a

= sum of small and large micronuclei

b

= large micronuclei (indication for spindle poison effect)

c

= number of NCEs observed when scoring 10 000 PCEs
   (indication for target organ toxicity)

 

 

*

= p ≤ 0.05

**

= p ≤ 0.01

Applicant's summary and conclusion