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:
07 December 2005 - 01 February 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
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
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: Amine Synergist - Physical state: Pale yellow liquid- Storage condition of test material: Stored at room temperature in the dark

Test animals

Species:
mouse
Strain:
other: CrI: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: 5-8 wk- Weight at study initiation: 21-29 g- Assigned to test groups randomly: Yes- Housing: Seven animals/cage housed in solid-floor polypropylene cages with wood-flake bedding- Diet (e.g. ad libitum): Food (Certified Rat and Mouse Diet Code 5LF2, IPS Ltd., London, UK)- Water (e.g. ad libitum): Drinking water, ad libitum- Acclimation period: 7 d, ad libitumENVIRONMENTAL CONDITIONS- Temperature (°C): 19-25 °C- Humidity (%): 30-70 %- Air changes (per h): 15/h- Photoperiod (h dark / h light): 12 h dark / 12 h light

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: Arachis oil- Concentration of test material in vehicle: 5, 25, 50 and 100 mg/mL- Amount of vehicle: 10 mL/kg- Lot/batch no. (if required): SN365
Duration of treatment / exposure:
24 or 48 hours
Frequency of treatment:
Single administration
Doses / concentrations
Remarks:
Doses / Concentrations:250, 500 and 1,000 mg/kg bwBasis:nominal conc.
No. of animals per sex per dose:
Test and vehicle (control) group: Seven male micePositive control: Five male mice
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control: Cyclophosphamide- Route of administration: Oral- Doses / concentrations: 50 mg/kg bw

Examinations

Tissues and cell types examined:
Bone marrow smears
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Doses were selected based on the results of range-finding toxicity test in which 1,000 mg/kg was observed to be the maximum tolerated dose.DETAILS OF SLIDE PREPARATION: Immediately following termination (i.e. 24 or 48 h 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-Griinwald/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. Incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal; number of nonnochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes and NCE/PCE were recorded.
Evaluation criteria:
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 hours 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 nonnochromatic 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 theUKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed following a (x + 1)^1/2 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
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
at 2,000 mg/kg in range-finding test
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY- Dose range: 1,000 and 2,000 mg/kg- Mortality: 2/2 animals died at 2,000 mg/kg, no mortality at 1,000 mg/kg- Clinical signs of toxicity in test animals: Hunched posture, ptosis, ataxia, lethargy, pilo-erection and decreased respiratory rate.RESULTS OF DEFINITIVE STUDY- Induction of micronuclei (for Micronucleus assay): Increases in the frequency of micronucleated PCEs was statistically non-significant.- Ratio of PCE/NCE (for Micronucleus assay): Decreases in the PCE/NCE ratio in the 24 or 48 hours test material groups was statistically non-significant.

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)
10 mL/kg
48-h Sampling Time
0.6 0.8 0.88 0.19
Vehicle Control (Arachis oil)
10 mL/kg
24-h Sampling Time
1 1 0.72 0.25
Positive Control (Cyclophosphamide)
50 mg/kg
24-h Sampling Time
49.0** 19.2 1.26 0.31
Amine Synergist
1000 mg/kg
48-h Sampling Time
0.6 1.1 0.8 0.32
Amine Synergist
1000 mg/kg
24-h Sampling Time
1.1 0.9 0.95 0.24
Amine Synergist
500 mg/kg
24-h Sampling Time
1.3 1.5 0.85 0.28
Amine Synergist
250 mg/kg
24-h Sampling Time
0.3 0.5 0.69 0.19

PCE = Polychromatic erythrocytes

NCE = Normochromatic erythrocytes

SD = Standard deviation

** = P < 0.01

Applicant's summary and conclusion

Conclusions:
Under the test conditions, the test substance was negative in the mouse micronucleus test.
Executive summary:

A study was conducted to assess the potential of the test substance to produce damage to chromosomes or aneuploidy when administered to mice according to OECD Guideline 474 and EU Method B.12.

The test substance (250, 500 and 1,000 mg/kg bw) was administered intraperitoneally to seven male mice. Animals were killed 24 or 48 h 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 (7 mice) or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls, respectively.

There were no premature deaths seen in any of the dose groups. Clinical signs (hunched posture and ptosis) were observed in animals dosed with the test substance at and above 500 mg/kg bw in both the 24 and 48 h test groups. No statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48 h 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 and exposure to the bone marrow achieved. 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.

Under the test conditions, the test substance was negative in the mouse micronucleus test.