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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 November 2008 to 12 November 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to internationally accepted guidelines and to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
GLP compliance:
yes (incl. certificate)
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): ADK STAB PEP-36

- Substance type:
- Physical state: White powder
- Analytical purity: >=99.0%
- Lot/batch No.: 11328
- Expiration date of the lot/batch: Not reported
- Stability under test conditions: Responsibility of the Sponsor
- Storage condition of test material: Room temperature in the dark

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan UK.
- Age at study initiation: five to eight weeks
- Weight at study initiation: 22 to 30 g
- Assigned to test groups randomly: yes
- Fasting period before study: No
- Housing: Solid-floor polypropylene cages with wood-flake bedding.
- Diet (e.g. ad libitum): Harlan Teklad 2014 Rodent Pelleted Diet ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 7 days

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

IN-LIFE DATES: From: 5 November To: 7 November 2008

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: arachis oil
- Justification for choice of solvent/vehicle: Not given
- Concentration of test material in vehicle: 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Not reported

DIET PREPARATION
Not applicable - substance dosed by intraperitoneal route
Duration of treatment / exposure:
24 hrs & 48 hrs
Frequency of treatment:
Once
Doses / concentrations
Remarks:
Doses / Concentrations:
2000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
2 groups of 7 males dosed
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): not reported
- Route of administration: oral gavage
- Doses / concentrations: 50 mg/kg

Examinations

Tissues and cell types examined:
Bone marrow, Erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: based on a range finding conducted over the dose range 1000 to 2000 mg/kg. The test material showed no marked difference in its toxicity to male or female mice; it was therefore considered 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. The maximum
recommended dose (MRD) of the test material, 2000 mg/kg, was selected for use in the main test.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): no additional information.

DETAILS OF 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-Grunwald/Giemsa, allowed to air-dry and
cover-slipped using mounting medium.

METHOD OF ANALYSIS: 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 was 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 was 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 square root (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 applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000 - 2000 mg/kg
- Solubility: not investigated
- Clinical signs of toxicity in test animals: Yes but only via intraperitoneal dose route, no evidence of toxicity in animals dosed via the oral route.
- Rationale for exposure: Adequate evidence of test material toxicity was demonstrated via the intraperitoneal route of administration, therefore this was selected for use in the main test.
- Harvest times: not applicable
- High dose with and without activation: 2000 mg/kg


RESULTS OF DEFINITIVE STUDY

- Induction of micronuclei (for Micronucleus assay): Yes

- Ratio of PCE/NCE (for Micronucleus assay): 0.61 for 24 hr sampling time, 0.68 for 24 hour sampling time (means from 7 animals at each time point)

- Statistical evaluation: There were no statistically significant increases in the frequency of micronucleated PCE in any of the test material dose groups when compared to their concurrent vehicle control groups.

Any other information on results incl. tables

There were no premature deaths or clinical signs observed in any of the dose groups.

Although not statistically significant, modest decreases in the PCE/NCE ratio were observed in both the 24 and 48-hour test material dose groups when compared to their concurrent control groups. This was taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved. There were no statistically significant increases in the frequency of micronucleated PCE 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 statistically significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test.

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.