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:
between 19 June 2006 and 25 July 2006.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: Clear colourless viscous liquid
Details on test material:
-Batch number: 60401946
-Storage conditions: Room temperature in the dark

Test animals

Species:
mouse
Strain:
other: Crl:CD-1™(ICR)BR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: five to eight weeks old
- Weight at study initiation: 23 to 30 g
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: in groups of up to seven in solid-floor polypropylene cages with wood-flake bedding
- Diet (e.g. ad libitum): ad libitum,Certified Rat and Mouse Diet Code 5LF2, IPS Limited, London, UK
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30 to 70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
Arachis oil
Duration of treatment / exposure:
1 single administration
Frequency of treatment:
1 single administration
Post exposure period:
One group of mice from each dose level was killed by cervical dislocation 24 hours following treatment and a second group (seven male mice) dosed with test material at 30 mg/kg was killed after 48 hours.
Doses / concentrations
Remarks:
Doses / Concentrations:
30, 15 or 7.5 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
Group of 7 mice per dose and per sampling.
Group of 5 mice for positive control (only one sampling).
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide is a positive control material known to produce micronuclei under the conditions of the test.

Examinations

Tissues and cell types examined:
Bone marrow of femur.
Details of tissue and slide preparation:
Immediately following termination (i.e. 24 or 48 hours following dosing), both femurs were dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Grünwald/Giemsa, allowed to air-dry and cover-slipped using mounting medium.

Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE­ blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes were counted; these cells were also scored for incidence of micronuclei.
The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.
Evaluation criteria:
A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test material groups and the number occurring in the corresponding vehicle control group.
A positive mutagenic response would be demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour kill times when compared to their corresponding control group.
If these criteria were not fulfilled, then the test material was considered to be non-genotoxic under the conditions of the test.
A positive response for bone marrow toxicity would be demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the concurrent vehicle control group.
Statistics:
All data were statistically analysed using appropriate statistical methods as recommended by the
UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed following a (x+1) transformation using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Results of the range-finding study :
In animals dosed with the test material via the intraperitoneal route premature deaths occurred at and above 50 mg/kg, and clinical signs were observed at and above 30 mg/kg as follows: Hunched posture, ptosis, distended abdomen, pile-erection, lethargy, ataxia, decreased respiratory rate, laboured respiration, comatose and hypothermia. These adequately demonstrate that systemic absorption of the test material was being achieved.
The test material showed no marked difference in its toxicity to male or female mice; it was therefore considered to be acceptable to use males only for the main test. Adequate evidence of test material toxicity was demonstrated via the intraperitoneal route of administration; therefore, this was selected for use in the main test. Clinical signs (hunched posture and ptosis) were observed at 30 mg/kg and this was therefore selected as the maximum tolerated dose (MTD) for use in the main test, with 15 and 7.5 mg/kg as the lower dose levels.

Results of the main study:
There were no premature deaths seen in any of the dose groups. Clinical signs were observed in animals dosed with the test material at 30 mg/kg in both the 24 and 48-hour groups, these were as follows: Hunched posture and ptosis.
There were no statistically significant decreases in the PCE/NCE ratio in the 24 or 48-hour test material groups when compared to their concurrent vehicle control groups. However, the observation of clinical signs was taken to indicate that systemic absorption had occurred.
There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test material dose groups when compared to their concurrent vehicle control groups.
The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.
The test material was found not to produce a significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test.


Any other information on results incl. tables

Table : Micronucleus Test-Summary of Group Mean Data

 

 

 

Treatment Group

Number of PCE with

Micronuclei per 2000 PCE

 

PCE/NCE Ratio

 

 

 

 

GroupMean

SD

GroupMean

SD

1.   Vehicle Control (arachis oil)

10 ml/kg

48-hour Sampling Time

 

1.1

 

1.2

 

1.16

 

0.39

2.   Vehicle Control (arachis oil)

10 ml/kg

24-hour Sampling Time

 

0.7

 

1.0

 

0.65

 

0.25

3.   Positive Control (cyclophosphamide)

50 mg/kg

24-hour Sampling Time

 

61.6***

 

20.8

 

1.10

 

0.38

4.   test item

30 mg/kg

48 -hour Sampling Time

 

1.1

 

1.5

 

0.92

 

0.30

5.   test item

30 mg/kg

24-hour Sampling Time

 

1.3

 

1.9

 

0.83

 

0.20

6.  test item

15 mg/kg

24-hour Sampling Time

 

1.6

 

1.5

 

0.81

 

0.12

7.  test item

7.5 mg/kg

24-hour Sampling Time

 

1.7

 

1.0

 

0.76

 

0.18

 

 

PCE   =Polychromatic erythrocytes

NCE  =Normochromatic erythrocytes

SD     = Standard deviation

***    =p<0.001

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to the concurrent vehicle control groups. The test material was considered to be non-genotoxic under the conditions of the test.
Executive summary:

The study was performed to assess the potential of the test material to produce damage to chromosomes or aneuploidy when administered to mice.

A range-finding test was performed to find suitable dose levels of the test material, route of administration and to investigate to see if there was a marked difference in toxic response between the sexes. There was no marked difference in toxicity of the test material between the sexes; therefore the main test was performed using only male mice. The micronucleus test was conducted using the intraperitoneal route in groups of seven mice (males) at the maximum tolerated dose (MTD) 30 mg/kg and with 15 and 7.5 mg/kg as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow extracted, and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei.

Further groups of mice were given a single intraperitoneal dose of arachis oil (two groups each of 7 mice) or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls respectively. Vehicle control animals were killed 24 or 48 hours later, and positive control animals were killed after 24 hours There were no premature deaths seen in any of the dose groups. Clinical signs were observed in animals dosed with the test material at 30 mg/kg in both the 24 and 48-hour groups, these were as follows: Hunched posture and ptosis.

No statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48-hour test material dose groups when compared to their concurrent control groups. However, the observation of clinical signs was taken to indicate that systemic absorption had occurred.

There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to the concurrent vehicle control groups.

The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

The test material was considered to be non-genotoxic under the conditions of the test.