Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: other:
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 September to 16 October 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline and GLP-compliant study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
: Relative humidity ranged between 30 - 80% (and not between 30 - 70%). 5 animals per sex and group were used in the current study instead of 5 animals per group.
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): BEPD
- Physical state: white solid
- Analytical purity: >99%
- Batch/Lot number: production May 2003
- Stability in solvent: stable for duration of the study in water, saline, PEG and CMC
- Storage conditions: at room temperature in closed container
- Expiry date: 31 December 2003

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
Mice obtained from RCC Ltd., Biotechnology and Animal Breeding Division, CH-4414 Füllinsdorf. They were 8-10 weeks old with a mean bodyweight of 33.9 g (SD ±2.7 g) males and 27.3 g (SD ±2.3 g) females at the start of treatment. The animals were under quarantine for a minimum of five days after arrival. The animal room temperature was at 22 ± 3°C with a relative humidity of 30 - 80%. Artificial light was controlled to be switched on between 6:00 - 18:00.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
PEG 400
Details on exposure:
A preliminary study on acute toxicity was performed with two animals/sex under identical conditions as in the mutagenicity study. The animals were treated orally with the test material and examined for acute toxicity symptoms at intervals of 1, 2-4, 6, 24, 30 and 48 hours after administration.
Single dose at 312.5, 625 and 1250 mg BEPD/kg bw formulated in PEG 400. The volume administered was 10 mL/kg bw.
Duration of treatment / exposure:
The mutagenicity study was conducted on six male and six female mice per dose group and sampling time, but only 5/group were evaluated.
Frequency of treatment:
Once
Post exposure period:
Toxicity symptoms were examined at 1, 2-4, 6 and 24 hours after administration of the test material.
Sampling of bone marrow was done 24 and 48 hours after treatment.
Doses / concentrations
Remarks:
Doses / Concentrations:
312.5, 625 and 1250 mg/kg
Basis:
other: achieved doses
No. of animals per sex per dose:
6 males and 6 females per dose. The high dose group was conducted twice, also with 6 males and 6 females.
Control animals:
yes, concurrent vehicle
Positive control(s):
CPA (cyclophosphamide)

Examinations

Tissues and cell types examined:
Bone marrow smears were prepared from the shafts of both femurs. After staining, slides of 5 animals/sex/dose were scored (2000 polychromatic erythrocytes per animal).
Details of tissue and slide preparation:
Bone marrow smears were prepared from the shafts of both femurs. After staining, slides of 5 animals/sex/dose were scored (2000 polychromatic erythrocytes per animal).
Evaluation criteria:
Negative controls in the range of 0.01 - 0.15% (mean = 0.066 ± 0.32 PCEs with micronuclei).
Positive controls in the range of 0.91 - 2.975% (mean = 1.644 ± 0.446 PCEs with micronuclei).
At least 80% of animals are evaluable.
PCE to erythrocyte ratio should not be less than 20% of the negative control.
Statistics:
Nonparametric Mann-Whitney test

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000, 1250, 1500 and 2000 mg/kg bw
- Clinical signs of toxicity in test animals: 2 males and 2 females showed signs of reduced spontaneous activity, abdominal position and apathy for up to 6 hours when dosed at 1000 mg/kg bw.
All 4 animals (2 males, 2 females) dosed at 1250 mg/kg bw showed reduced spontaneous activity up to 6 hours after dosing. All male mice had ruffled fur during the 48-hour period of observation. Both female mice had ruffled fur up to 6 hours after dosing, but this was not observed at 24 hours. One male and one female had closed eyelids one hour after dosing but this was not observed at 2-4 hours. One male mouse showed apathy one hour after dosing.
Death of one male dosed at 1500 mg/kg bw after 4 hours and one female after 6 hours. Reduced spontaneous activity and ruffled fur were observed in all 4 mice up to 4 hours after dosing. This observation was reduced to one male and one female mouse between 6 and 30 hours after dosing. Abdominal position was observed in one male and one female mouse up to 4 hours after dosing. One male and one female mousee had their eyelids closed up to one hour after dosing. One male mouse showed apathy one hour after dosing and one female mouse up to 4 hours after dosing.
Death of two females dosed at 2000 mg/kg bw after 4 hours and one male after 6 hours. All four mice showed a reduction of spontaneous activity, ruffled fur and apathy one hour after dosing.
On the basis of these data, 1250 mg/mg bw was chosen to be the highest dose level.

RESULTS OF DEFINITIVE STUDY
- Dose range: 312.5, 625 and 1250 mg/kg bw
- Clinical signs of toxicity in test animals:
No deaths were observed at 312.5 mg/kg bw. All signs of reduced spontaneous activity, eyelid closure and ruffled fur had disappeared by 24 hours after dosing.
No deaths were observed at 625 mg/kg bw. 3 males and 3 females had ruffled fur one hour after dosing but this was not observed by the end of the experiment at 24 hours. Eyelid closure was observed in 4 females at 4 hours after dosing but it was gone by hour 6. Reduction of spontaneous activity was observed in 3 males and 2 females one hour after dosing, 2 males and 4 females 4 hours after dosing and 2 males and 3 females 6 hours after dosing. No reduction in spontaneous activity was observed at 24 hours after dosing.
2/12 males died 4 hours after being dosed at 1250 mg/kg bw. No deaths were observed in the females. All 24 animals (12 males, 12 females) had ruffled hair one hour after dosing and this dropped to being only observed in 2 female mice by 24 hours after dosing. 12 females and 11 males had reduced spontaneous activity one hour after dosing, which dropped to being only observed in 3 females 24 hours after dosing.

Any other information on results incl. tables

To describe a cytotoxic effect due to the treatment with the test material, the ratio between polychromatic and total erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.

Test group

Dose (mg/kg bw)

Sampling time (h)

PCEs with micronuclei (%)

Range

PCE per 2000 erythocytes

Significance

p

Vehicle

0

24

0.055

0-3

1125

 

 

Test item (BEPD)

312.5

24

0.075

0-3

1046

-

0.2610

Test item (BEPD)

625

24

0.090

0-3

1051

-

0.1224

Test item (BEPD)

1250

24

0.100

0-4

1063

-

0.0820

Positive control

40

24

1.860

21-64

1052

+

< 0.0001

Test item (BEPD)

1250

48

0.060

0-4

980

-

0.5000

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Preliminary tests showed 1250 mg BEPD /kg bw was suitable as the highest dose level. In the main study, 2 males died after treatment with 1250 mg/kg bw 4 hours after dosing. The mean number of polychromatic erythrocytes did not increase after treatment with the test material when compared to the mean number of PCEs of the vehicle control, indicating that BEPD had no cytotoxic properties in the bone marrow. There were no statistically significant or biologically relevent enhancement on the frequency of the detected micronuclei at any preparation intercal and dose level after administration of the test material when compared with the corresponding vehicle controls. The mean values of micronuclei observed after treatment with BEPD were below or near the value of the vehicle control group. 40 mg cyclophosphamide/kg bw (positive control) was administered orally and showed a statistically significant increase of induced micronucleus frequency. Under the experimental conditions of this test, BEPD did not induce micronuclei.
Executive summary:

In a standard mouse in vivo micronucleus test, dose levels of 312.5, 625 and 1250 mg/kg bw were orally administered to NMRI mice (5/sex/group). Bone marrow smears were prepared from each femur. After staining the smear slides from 5 mice/sex/dose were scored for 2000 polychromatic erythrocytes per animal. The number of micronuclei was assessed. Under the conditions of this test, there was no evidence of genotoxicity. Appropriate responses to the positive control substance (cyclophosphamide) confirmed the sensitivity of the assay.