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:
10 Sept 2002 - 25 Oct 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according to Guideline OECD 474

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003

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

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: Male 8 - 10 weeks.


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:
intraperitoneal
Vehicle:
1% carboxymethyl cellulose in water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
An individual stock suspension of the test substance was prepared for each group of animals as close to the time of dosing as possible. The dosing volume was 10 ml/kg bodyweight
Duration of treatment / exposure:
24 or 48 hours
Frequency of treatment:
Single dose
Post exposure period:
Not relevant
Doses / concentrations
Remarks:
Doses / Concentrations:
2000 mg/kg; 1000 mg/kg; 500 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide given as a single oral dose, 65 mg/kg, in 1% carboxy methyl cellulose in water

Examinations

Tissues and cell types examined:
Bone marrow erythrocytes
Details of tissue and slide preparation:
Femurs were removed and stripped clean of muscle. The iliac end of the femur was removed and a fine paint brush was rinsed in saline, and wetted with a solution of albumin (6% w/v in physiological saline). This was then dipped into the marrow canal and two smears were painted on a labelled clean, dry microscope slide.
This procedure wasrepeated to give four smears of marrow per slide.
The slides were allowed to ar dry and were stained with polychrome methylene blue and eosin using an automatic staining machine.
Evaluation criteria:
Criteria for identificaiton of micronuclei are as described by Schmid (1976):
i)Spherical (or rounded) with well-defined edges
ii)Diameters of not less than approx 1/20 of a polychromatic erythrocyte diameter
iii)Dark purple/dark blue staining
iv)Lie in the same plane as the polychromatic erythrocyte in which it is contained

To be considered positive there must be a statistically and biological significant increase in the incidence of micronucleated polychromatic erythrocytes which is in excess of a three-fold increase when compared with both historical and concurrent vehicle control incidences.
Statistics:
Analyses were carried out using the MIXED procedure in SAS (1999). Each treatment group mean was compared with the control group mean at the corresponding sampling time using a one-sided Student's test, based on the error mean square in the analysis.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In the dose finding study, both male and female mice were used. No deaths were seen up to 2000mg/kg (i.p.) but some clinical signs were seen at the highest dose. There was no difference between the sexes so only males were used for the main study.

Any other information on results incl. tables

Group

Treatment

Dose

Mean incidence of MPE/1000 PE +- SD

24 hours

48 hours

 

11

Vehicle Control

10 ml/kg

0.9 ± 0.42

0.7 ± 0.27

12

Cyclophsphamide

65 mg/kg

20.9 ±10.43

 

13

Lactam 204,636

2000 mg/kg

0.6 ±0.65

0.5 ±0.35

14

Lactam 204,636

1000 mg/kg

0.8 ±0.57

 

15

Lactam 204,636

500 mg/kg

1.1 ±0.22

 

MPE = micronucleated polychromatic erythrocytes

PE =    polychromatic erythrocytes

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of the test, Lactam 204,636 is not clastogenic in the mouse bone marrow micronucleous test