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
Study period:
18 June 2007 and 27 July 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

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
Details on test material:
Sponsor's identification: AS305BD
Description: dark brown, viscous liquid
Lot number: TS07002
Analytical purity: 90%
Storage conditions: room temperature in the dark

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, Kent
- Age at study initiation: approximately 5-8 weeks old
- Weight at study initiation: 21-29 g
- Assigned to test groups randomly: yes
- Housing: steel mesh bottom polypropylene cages
- Diet (e.g. ad libitum): Certified Rate and Mouse Diet Code 5LF2, IPS LTd, London, UK ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: minimum of seven days

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

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: arachis oil
Details on exposure:
Groups, each of seven mice, were dosed once only via the intraperitoneal route with the test material at 100, 50 or 25 mg/kg. One group of mice from each dose level was killed by cervical dislocation 24 hours following treatment and another group dosed with test material at 100 mg/kg was killed after 48 hours. In addition, three further groups of mice were included in the test; two groups (each of seven mice) were dosed via the intraperitoneal route with the vehicle alone (arachis oil) and a third group (five mice) was dosed orally with cyclophosphamide. The vehicle controls were killed 24 or 48 hours following dosing and positive control group animals were killed 24 hours following dosing. The experimental design is summarised as follows:

Dose group Dose level (mg/kg) Concentration (mg/ml) Dose volume (ml/kg) Kill time (hours after dosing) Animal numbers

Vehicle control 0 0 10 48 1-7
Vehicole control 0 0 10 24 8-14
Positive control 50 5 10 24 15-19
AS305BD 100 10 10 48 20-26
AS305BD 100 10 10 24 27-33
AS305BD 50 5 10 24 34-40
AS305BD 25 2.5 10 24 41-47

All animals were observed for signs of overt toxicity and death one hour after dosing and then once daily as applicable and immediately prior to termination.
Duration of treatment / exposure:
Single dose
Frequency of treatment:
Single dose
Post exposure period:
24 and 48 hours
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
100, 150, 200, 300, 100*, 500*, 2000* mg/kg (*denotes dose level not corrected for 90% purity)
Basis:
other: intraperitoneal
Remarks:
Doses / Concentrations:
25, 50, 100 mg/kg
Basis:
other: intraperitoneal
No. of animals per sex per dose:
Range finding: 2 (1male/1 female) per dose
Micronucleaus test: 7 males per dose
Control animals:
yes
Positive control(s):
Further groups of mice were given a single intraperitoneal dose of arachis oil (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.

POSITIVE CONTROL
Supplier's identification: cyclophosphamide
Lot number: A0164185
Storage conditions: approx 4 ºC in the dark

VEHICLE CONTROL
Supplier's identification: arachis oil
Lot number: P72
Description: pale, straw-coloured, slightly viscous liquid
Storage conditions: room temperature

Examinations

Tissues and cell types examined:
Femur bone marrow
Details of tissue and slide preparation:
Immediately following termination (i.e. 24 or 48 hours following dosing), both femurs were dissected fiom 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-Grûnwald/Giemsa, allowed to air dry and cover-slipped using mounting.
medium.
Evaluation criteria:
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 was counted; these cells were also scored for incidence of micronuclei. The ratio of poiychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.
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 (1 989). 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
Remarks:
hunched posture was noted at 24 and 48 hrs in animals treated at the MTD. In addition, although not staitically significant, all treated groups had PCE/NCE ratio values lower than concurrent vehicle control groups.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 100-300 mg/kg (corrected for test material purity); 100-2000 mg/kg (not corrected for test material purity)
- Clinical signs of toxicity in test animals: premature deaths occurred at and above 300 mg/kg and clinical signs were observed at and above 100 mg/kg as follows: hunched posture, ptosis, distended abdomen, letharfy, ataxia, hypothermia, splayed gait, decreased respiratory rate, laboured respiration and pilo-erection.
- Evidence of cytotoxicity in tissue analyzed: not applicable.

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay):
- Ratio of PCE/NCE (for Micronucleus assay): no statistically significant decreases in test material groups when compatred to vehicle control groups.
- Statistical evaluation: no statistically significant increases in frquency of micronucleated PCEs in any test material groups when compared to vehicle controls.
- Clinican observations: one clinical sign was observed in animals dosed with test material at 100 mg/kg in both 24 and 48 hour groups and this was hunched posture (1/7 in 24 hour group and 2/7 in 48 hour group).

Any other information on results incl. tables

Table 1. Micronucleus test - Summary of group mean data.

Treatment Group Number of PCE with Micronuclei per 2000 PCE PCE/NCE Ratio
Group mean SD Group mean SD
Vehicle control (Arachis oil)
10ml/kg
48-hour sampling time
1 0.8 1.36 0.44
Vehicle control (Arachis oil)
10ml/kg
24-hour sampling time
1.3 1.1 1.38 0.62
Positive Control (Cyclophospamide)
50 mg/kg
24-hour sampling time
65.2* 12.4 1.17 0.07
AS305BD
100mg/kg
48-hour sampling time
0.1 0.4 1.08 0.62
AS305BD
100mg/kg
24-hour sampling time
2 1.6 0.85 0.3
AS305BD
50mg/kg
24-hour sampling time
1.4 1.1 0.99 0.27
AS305BD
25mg/kg
24-hour sampling time
0.9 1.1 0.86 0.24

PCE = Polychromatic erythrocytes

NCE = Normochromatic erythrocytes

SD = Standard deviation

* = P<0.001

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test material was considered to be non-genotoxic under the conditions of the test.
Executive summary:

The study was performed in line with OCED Guideline 474 to determine the potential of the test material to produce damage to chromosomes or aneuploidy when administered to mice.

The test was conducted using the intraperitoneal route in groups of seven male mice at 25, 50 and 100 mg/kg. Animals were killed 24 or 48 hours later, bone marrow extracted and smear preparations made and stained. Vehicle and positive controls were also investigated. For all surviving animals, 2000 polychromatic erythrocytes (PCE) and the number of normochromatic erythrocytes (NCE) associated with 1000 erythrocytes were scored for the presence of micronuclei.

There were no premature deaths in any of the dose groups; hunched posture was seen in animals dosed with test material at 100 mg/kg in both the 24 and 48 hour groups. There was no statistically significant decrease in the PCE/NCE ratio in the 24 and 48 hour test material groups when compared to controls. There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with test material when compared to the controls. The results from the positive control groups confirmed the validity of the study.

The test material was not considered to be genotoxic under the conditions of the test.