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:
30 June 2008 - 15 August 2008
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

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

Test material

Constituent 1
Reference substance name:
SPP100 P0
IUPAC Name:
SPP100 P0
Constituent 2
Reference substance name:
90719-32-7
EC Number:
618-632-6
Cas Number:
90719-32-7
IUPAC Name:
90719-32-7
Details on test material:
Molecular formula C10H11NO2
Molecular weight 177.20
CAS Number 90719-32-7
Description Beige crystalline powder (determined at NOTOX)
Batch CHAD 0710
Purity 99.9%
Test substance storage At room temperature in the dark
Stability under storage conditions Stable
Expiry date 01 April 2009 (retest date)

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 34.2 - 36.0 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: 5 animals per sex per cage, cages type MII height: 14 cm containing sterilised sawdust as bedding material (Litalabo; S.P.P.S., Argenteuil, France). Paper bedding was provided as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5 - 22.8°C
- Humidity (%): 39 - 93%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle. Due to cleaning procedures or performance of functional observations in the room, temporary deviations from the light/dark cycle (with a maximum of 1 hour) and the maximum level for humidity (with max. 20%) occurred

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used:corn oil (Roth, Karlsruhe, Germany)
- Justification for choice of solvent/vehicle: Test substance dissolved in corn oil. Corn oil is accepted by international guidelines
Duration of treatment / exposure:
Highest dose level: 24 and 48 hours
Solvent control, low and mid dose: 24 hours
Positive control: 48 hours
Frequency of treatment:
once
Post exposure period:
none
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
500 mg/kg body weight
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
250 mg/kg body weight
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
125 mg/kg body weight
Basis:
nominal conc.
No. of animals per sex per dose:
5 male mice were treated per sampling time in each treatment group
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide CP; CAS no. 50-18-0; Endoxan, Asta-Werke, Germany) dissolved in physiological saline (B. Braun, Melsungen AG, Germany)

- Justification for choice of positive control(s): Accepted and approved by authorities and international guidelines
- Route of administration: intraperitoneal injection
- Doses / concentrations: 40 mg/kg body weight

Examinations

Tissues and cell types examined:
bone marrow smears
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg.

DETAILS OF SLIDE PREPARATION:
The preparations were air-dried, fixed for 5 min in 100% methanol and air-dried overnight. Two slides were prepared per animal. The staining was based on Giemsa.

METHOD OF ANALYSIS: Slides were scored at a magnification of 1000 x. The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes.
Evaluation criteria:
A micronucleus test is considered acceptable if it meets the following criteria:
a) The positive control substance induced a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes.
b) The incidence of micronucleated polychromatic erythrocytes in the control animals should reasonably be within the laboratory historical control data range
Statistics:
A test substance is considered positive in the micronucleus test if:
- It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time).

A test substance is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 500 and 2000 mg/kg body weight
- Clinical signs of toxicity in test animals:

2000 mg SPP100 P0 per kilogram body weight: one male and one female died within 1 hour after dosing

500 mg/kg body weight (three males and three females):
within 1 hour after dosing: lethargy, no reaction to a stimulus and ventral recumbency
within 5 hours after dosing 2 males and 2 females were still lethargic, 1 male had ventral recumbency and the other male and two females showed ataxia
within 23 hours after dosing 2 males and 3 females recovered from the treatment, one male had a rough coat
within 45 hours after dosing all animals recovered from the treatment

RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals: The animals of the groups treated with 125 mg SPP100 P0/kg body weight and the animals of the negative and positive control groups showed no treatment related clinical signs or mortality.

The following clinical observations were made in the groups treated with 500 and 250 mg SPP100 P0/kg body weight:

During the first 30 minutes after dosing, animals of the group treated with 500 mg/kg body weight showed the following clinical signs: lethargic, ventral recumbency (9 animals), no reaction to a stimulus (9 animals) and ataxia (1 animal). In the group treated with 250 mg/kg body weight all animals were lethargic and showed ataxia.

Within 2 hours after dosing in the groups treated with 500 mg/kg body weight all animals were lethargic, 7 animals had no reaction to a stimulus, 3 animals had ataxia and 3 had ventral recumbency. In the group treated with 250 mg/kg body weight all animals showed ataxia and 1 animal was still lethargic.

Within 19 hours after dosing all animals had recovered from the treatment.

- Induction of micronuclei (for Micronucleus assay): No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of SPP100 P0 treated animals compared to the vehicle treated animals.
- Ratio of PCE/NCE (for Micronucleus assay): no decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a lack of toxic effects of this test substance on the erythropoiesis. The animals of the groups treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic effects on erythropoiesis.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with SPP100 P0.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, both criteria for an acceptable assay were met.

It is concluded that this test is valid and that SPP100 P0 is not clastogenic or aneugenic in the micronucleus test under the experimental conditions described.