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
Study period:
1995-11-07 to 1995-12-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1992
Deviations:
yes
Remarks:
1600 PCE scored instead of 2000 (due to shift towards NCE)
GLP compliance:
yes (incl. certificate)
Remarks:
Ministerium für Umwelt, Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winklemann, Borchen, Germany
- Age at study initiation: young adult
- Weight at study initiation: 35.8 ± 7.2 grams (male) / 31.8 ± 6.4 grams (female)
- Assigned to test groups randomly: yes, under following basis: colour code applied to the back of each animal; cage cards
- Fasting period before study: 16 - 18 hours prior to dosing procedure
- Housing: conventional, 5 animals/sex/Makrolon cage type III
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: ≥ 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ˚c
- Humidity (%): 30 - 70 %
- Air changes (per hr): 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
Route of exposure: intraperitoneal
Duration of treatment / exposure:
24 to 48 hours
Frequency of treatment:
Test compound and controls were administered once by intraperitoneal injection of 10 mL/kg bw.
Post exposure period:
The positive control group was sacrificed 24 hours after administration of the test substance. The vehicle control and test substance groups were sacrificed 24 - 48 hours after administration of the test substance.
Doses / concentrations
Remarks:
Doses / Concentrations:
1600 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
- cyclophosphamide

- Justification for choice of positive control(s): standard guideline positive control

- Route of administration: interperitoneal

- Doses / concentrations: 10 mL/kg bw of 0.9 % solution in physiological saline

Examinations

Tissues and cell types examined:
Bone marrow and erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: limit dose

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): no further information

DETAILS OF SLIDE PREPARATION: pure erythrocyte suspension was used to prepare flat cells on glass by means of a Shandon Cytospin 3 (2 slides per animal). Slides were air dried and stained with May-Grundwald/Giemsa

METHOD OF ANALYSIS: A MIAMED image analyser equipped with the "Micronucleus Test V 4.00" software was used for fully automated slide scoring. Where possible, a total of approximately 2000 PCE (i.e. 10,000 PCE per treatment group) were analysed for micronuclei. The corresponding NCE were also scored for micronuclei. As an indicator for chemical-induced bone marrow toxicity, for each animal the PCE/NCE ratio was determined on the basis of 2000 PCE scored.
Evaluation criteria:
A test compound is considered an inducer of micronuclei in PCE, if at least one group of mice treated with the test compound reveals a statistically and biologically relevant increase in micronucleated PCE compared with the negative control.
Statistics:
Means and standard deviations of the following perameters were calculated for each treatment group:

-the number of PCE with micronuclei

-the number of NCE with micronucei

-the PCE/NCE ratio.

"Statgraphics" statistical software was used to analyse data. Test compound groups were compared with negative control groups of the same sampling time. heterogeneity between animals of the same dose group was tested for by calculating the heterogeneity χ2 for the differences between the proportions of micronuclei for each animal. Pearson's contingency χ2 with one degree of freedom including a Yates correcting factor used to make comparisons in homogenous groups. In the case of inhomogeneity between groups, after transformation of the data, a one-sided two-sample t-test is performed, and can also be used to compare the micronucleus frequencies of the positive control with the negative control groups for comparison of PCE/NCE ratios.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
PCE/NCE ratio reduced
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
- Clinical signs of toxicity in test animals: all animals exhibited clinical syptoms - trembling, staggering, prone/lateral position, sedation, hypothermia. On necroscopy, blood was observed in the small intestine and discoloured livers (particularly in female mice).

Any other information on results incl. tables

Table 3: Results of in vivo micronucleus test with Wacker BS 1701 

 

Vehicle

Control

Vehicle

Control

Positive Control

1600 mg/kg

bw

1600 mg/kg

 bw

Number of cells evaluated

2000

approx.

2000

approx.

2000

approx.

2000

approx.

2000

approx.

Sampling time (h)

24

48

24

24

48

Number of erythro-cytes

normo­chromatic

NR

NR

NR

NR

NR

poly­chromatic

2000

approx.

2000

approx.

2000

approx.

2000

approx.

2000

approx.

polychromatic with micronuclei (%)

Male

0.16 %

Female

0.13 %

Male

0.20 %

Female

0.11 %

Male

4.1 %

Female

2.2 %

Male

0.15 %

Female

0.13 %

Male

0.2 %

Female

0.18 %

Ratio of erythro­cytes

polychromatic / normochromatic

(ratio)

Male

0.7

Female

0.81

Male

0.48

Female

1.38

Male

0.57

Female

0.83

Male

0.1

Female

0.14

Male

0.07

Female

0.09

polychromatic with micro­nuclei / normochromatic

NR

NR

NR

NR

NR

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The substance was tested in an in vivo mouse micronucleus assay according to EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test) and under GLP. It was not genotoxic under the conditions of the test. It is noted that the PCE / NCE ratio was reduced in the test substance indicating toxicity and demonstrating that the test substance had reached the target tissue.