Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP-Guideline study, tested with the source substance 68424-31-7. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): only trade name given
- Physical state: pale yellow liquid
- Analytical purity: >98%
- Lot/batch No.: RMEST240
- Storage condition of test material: at room temperature in the dark

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories (UK Limited, UK)
- Age at study initiation: 5-9 weeks
- Assigned to test groups randomly: yes
- Housing: animals were housed in groups of 5 per sex per cage in mobile mouse cage racks
- Diet: Porton Combined Diet, ad libitum
- Water: filtered tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Air changes (per hr): 25
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Amount of vehicle: 10 mL/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dosing solutions of the test material were prepared in corn oil. A solution of cyclophosphamide was prepared in physiological saline.
Duration of treatment / exposure:
not applicable
Frequency of treatment:
single treatment
Post exposure period:
24 and 48 h after treatment
Doses / concentrations
Remarks:
Doses / Concentrations:
5000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: intraperitoneal injection
- Doses / concentrations: 65 mg/kg bw in physiological saline

Examinations

Tissues and cell types examined:
Monochromatic and polychromatic erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
No deaths or severe adverse effects occurred in Phase I (range finding study) of the study with doses up to 5000 mg/kg bw. This dose was selected as maximum tolerated dose.

TREATMENT AND SAMPLING TIMES: 24 h and 48 h after dosing

DETAILS OF SLIDE PREPARATION: Bone Marrow smears were stained with polychrome methylene blue and eosin.

METHOD OF ANALYSIS: 1000 polychromatic erythrocytes were evaluated for micronuclei per slide. In addition, 1000 erythrocytes were counted to determine the percentage of polychromatic erythrocytes in the total erythrocyte population.

Evaluation criteria:
Increase in the incidence of micronucleated polychromatic erythrocytes in any sex or at any time point.
Percentage of polychromatic erythrocytes.
Statistics:
The incidence of micronucleated polychromatic erythrocytes and percentage of polychromatic erythrocytes in the erythrocyte sample were considered by analysis of variance regarding each combination of sampling time, dose level and sex as a separate group. Results were examined to determine whether any differences between vehicle control and test substance treated groups were consistent between sexes and across sampling times.
Each group mean was compared with the vehicle control group mean at the corresponding sampling time using a one-sided Student´s t-test based on the error mean square in the analysis.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 50-5000 mg/kg bw
- Clinical signs of toxicity in test animals: no deaths or severe adverse reactions were recorded over the 4-day observation period at the high dose.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes over the vehicle control values were seen in either sex at either of the sampling times.
Comparison of the percentage of polychromatic erythrocytes showed no significant differences between the female animals treated with the vehicle control or with the test material. A small, but significant decrease was, however, noted in male mice treated with the test material at 5000 mg/kg bw. This small decrease is, however, considered not to be biologically significant compared to the concurrent control values.
The positive control induced statistically significant and biologically meaningful increases in micronucleated polychromatic erythrocytes, compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known clastogen.

Any other information on results incl. tables

Table 1. Results male animals.

Treatment group

Dose

Sampling time

Mean incidence of MPE/1000 PE ± SD

Mean [%]

PE ± SD

[mg/kg]

[h]

Vehicle control

0

24

0.8 ± 0.8

48.0 ± 5.6

 

0

48

1.0 ± 1,2

44.3 ± 7.5

Test substance

5000

24

0.6 ± 0.6

42.2 ± 7.0*

 

5000

48

0.4 ± 0.6

43.3 ± 1.9

Positive control (Cyclophosphamide)

 

65

 

24

 

24.4 ± 6.0**

 

41.4 ± 4.4*

PE = polychromatic erythrocytes

MPE = micronucleated polychromatic erythrocytes

SD = standard deviation

*: statistically significant decrease in percentage of polychromatic erythrocytes at p<0.05 in the Students t´test (one-sided).

Table 2. Results female animals.

Treatment group

Dose

Sampling time

Mean incidence of MPE/1000 PE ± SD

Mean [%]

PCE ± SD

[mg/kg]

[h]

Vehicle control

0

24

0.2 ± 0.5

41.9 ± 4.8

 

 

0

48

1.4 ± 1.1

41.9 ± 1,7

 

Test substance

5000

24

0.4 ± 0.9

46.5 ± 5.8

 

 

5000

48

0.4 ± 0.9

48.0 ± 5.2

Positive control (Cyclophosphamide)

 

65

 

24

 

18.4 ± 7.3**

 

45.9 ± 3.49

PE = polychromatic erythrocytes

MPE = micronucleated polychromatic erythrocytes

SD = standard deviation

**: statistically significant decrease in percentage of polychromatic erythrocytes at p<0.01 in the Students t´test (one-sided).

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative