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:
2 April 2012 - 31 May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to OECD guideline and GLP. There were no significant deviations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

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)
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
gas under pressure: refrigerated liquefied gas

Test animals

Species:
mouse
Strain:
other: Hsd: ICR (CD-1®)
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK
- Age at study initiation: 6-10 weeks
- Weight at study initiation: 25-30g
- Assigned to test groups randomly: yes, under following basis: no data
- Fasting period before study: No
- Housing: The animals were housed in groups of up to seven in solid-floor polypropylene cages with wood-flake bedding.
- Diet (e.g. ad libitum): Free access to food (Harlan Teklad 2014C Global Certified Rodent Diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study.
- Water (e.g. ad libitum): Free access to mains drinking water was allowed throughout the study.
- Acclimation period: minumum 5 days

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

IN-LIFE DATES: 2 April 2012 - 31 May 2012

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: PBS
- Justification for choice of solvent/vehicle: solubility
- Concentration of test material in vehicle: 10, 5, 2.5 mg/ml
- Amount of vehicle (if gavage or dermal): 10 ml/kg
Duration of treatment / exposure:
24/48 hours
Frequency of treatment:
single injection
Post exposure period:
24/48 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 50, 25
Basis:
nominal conc.
mg/kg
No. of animals per sex per dose:
7 males
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): standard from guideline
- Route of administration: oral
- Doses / concentrations: 50 mg/kg

Examinations

Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A range-finding toxicity test was performed to determine a suitable dose level and route of administration for the micronucleus test. 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. The range-finding toxicity test was also used to determine if the main test was to be performed using both sexes or males only. With no evidence of toxicity via the oral route it was considered necessary to investigate the intraperitoneal route of administration.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The vehicle and positive control group animals were killed 24 hours following dosing.

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 bovine 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 a cover slip applied using mounting medium.

METHOD OF ANALYSIS:
Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. Where possible, 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 polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.

OTHER: -
Evaluation criteria:
A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test item groups and the number occurring in the 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 the vehicle control group.
If these criteria were not fulfilled, then the test item would be considered non-mutagenic 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 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
Remarks:
Although not statistically significant, a modest decrease in the PCE/NCE ratio was observed in the 24-hour 100 mg/kg dose group.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
In animals dosed with the test item at 2000 mg/kg via the oral route, clinical signs were not observed. Therefore, with insufficient evidence of toxicity or absorption, the intraperitoneal route was investigated using a dose level of 1000 mg/kg initially. Both animals dosed with the test item at 1000 mg/kg were killed in extremis due to the severity of the following clinical signs; hunched posture, ptosis, splayed gait, ataxia, lethargy, dehydration, pilo-erection, and emaciation. In animals dosed with the test item at 200 and 300 mg/kg the following clinical signs were observed: hunched posture, pilo-erection, ptosis, splayed gait, emaciation, decreased respiration, laboured respiration, and hypothermia. In animals dosed with the test item at 100 mg/kg the clinical signs hunched posture, ptosis, and pilo-erection were observed. Therefore, due to the severity and duration of the clinical signs observed at and above 200 mg/kg, the maximum dose level in the main test was set at 100 mg/kg, the considered maximum tolerated dose level, with 50 and 25 mg/kg as the two lower dose levels.

The test item showed no marked difference in its toxicity to male or female mice; therefore the main test was performed using male mice only.

RESULTS OF DEFINITIVE STUDY
Although not statistically significant, a modest decrease in the PCE/NCE ratio was observed in the 24-hour 100 mg/kg dose group. The decrease, together with the observation of clinical signs, was taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved. There was no evidence of any statistically significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group.

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. One animal in the group (No. 10) showed no marked increase in the incidence of micronucleated polychromatic erythrocytes and was therefore excluded from the statistical analysis. The reason for this was not known. However, with marked increases in the four remaining animals in the group, the purpose and integrity of the study was considered unaffected.

The test item was found not to produce a toxicologically 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 item was considered to be non-mutagenic under the conditions of the test.
Executive summary:

Introduction.

The study was performed to assess the potential of the test item to produce damage to chromosomes or aneuploidy when administered to mice. The method was designed to be compatible with the 1997 OECD Guidelines for Testing of Chemicals No.474 "Mammalian Erythrocyte Micronucleus Test", Method B12 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA (TSCA) OPPTS 870.5395, EPA 712-C-98-226, August 1998 guidelines, and be acceptable to the Japanese METI/MHLW/MAFF guidelines for testing of new chemical substances.

Methods.

A range-finding test was performed to find suitable dose levels of the test item, route of administration, and to investigate if there was a marked difference in toxic response between the sexes. There was no marked difference in toxicity of the test item 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) of 100 mg/kg and with 50 and 25 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. Additional groups of mice were given a single intraperitoneal dose of phosphate buffered saline (PBS) (7 male mice) or dosed orally with cyclophosphamide (5 male mice), to serve as vehicle and positive controls respectively. Vehicle and positive control animals were killed after 24 hours.

Results.

There were no premature deaths seen in any of the dose groups in the main test. Clinical signs were observed in animals dosed with the test item at and above 25 mg/kg in both the 24 and 48-hour groups, where applicable, and included hunched posture and ptosis.

Although not statistically significant, a modest decrease in the PCE/NCE ratio was observed in the 24-hour 100 mg/kg dose group. The decrease, together with the observation of clinical signs, was taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved.

There was no evidence of any statistically significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group.

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. One animal in the group (No. 10) showed no marked increase in the incidence of micronucleated polychromatic erythrocytes and was therefore excluded from the statistical analysis. The reason for this was not known. However, with marked increases in the four remaining animals in the group, the purpose and integrity of the study was considered unaffected.

Conclusion.

The test item was considered to be non-mutagenic under the conditions of the test.