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:
22 September 2003 to 28 October 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: No deviations. Study done under GLP standards and performed according to Safepharm Standard Method Number that was designed to comply with Method B12 of the EEC Commission Directive 2000/32/EC and OECD test method 474.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
2003

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 (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: colourless liquid
Details on test material:
Description: colourless liquid
Storage conditions: room temperature, in the dark, over silica gel, under nitrogen

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals and environmental conditions:
- Sufficient male and female albino CD-1 Strain mice were supplied by Charles River (UK) Limited, Manston, Kent.
- At the start of the main study the mice weighed 26 to 30g, and were approximately 5 to 8 weeks old.
- Minimum acclimatisation period of 7 days.
- The animals were housed in groups of up to 5 in solid-floor polypropylene cages with woodflake bedding.
- Free access to mains drinking water and food.
- The animal room was maintained at a temperature of 19 to 25°C and relative humidity of 30 to 70%.
- The rate of air exchange was approximately 15 changes per hour.
- Lighting was controlled by a time switch to give 12 hours light and 12 hours darkness.


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Supplier's identification : Arachis oil
Supplier's batch number : T71
Safepharm serial number : V-2818 (V-2932 after drying)
Date received : 28 May 2003
Description : straw coloured slightly viscous liquid
Storage conditions : room temperature
Details on exposure:
In a range-finding study, groups of 1 male and 1 female were dosed with 2000 or 1500 mg/kg. In the main study, groups, each of seven male mice, were dosed once only via the oral route with test material at 375, 750 or 1500 mg/kg. One group of mice from each dose group was killed by cervical dislocation 24 hours following treatment and a second at 48 hours (1500 mg/kg only). In addition three further groups of ten mice (five males and five females) were included in the study; two groups were given a single oral dose of the vehicle (arachis oil) and the third group was dosed orally with cyclophosphamide, a positive control material known to produce micronuclei under the conditions of the test. The vehicle control groups were killed 24 and 48 hours following dosing and positive control group animals were killed 24 hours following dosing.
Duration of treatment / exposure:
24 and 48 hours
Frequency of treatment:
once
Post exposure period:
No
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 375, 750, 1500
Basis:
nominal conc.
No. of animals per sex per dose:
7
Control animals:
yes, concurrent vehicle
Positive control(s):
Supplier's identification : Cyclophosphamide
Supplier's lot number : 91K1176
Safepharm serial number : R-2827
Date received : 10 June 2003
Description : white powder
Storage conditions : 4°C in the dark
The concentration, homogeneity and stability of the positive control material and its preparation were not determined by analysis.

Examinations

Tissues and cell types examined:
Bone marrow polychromatic erythrocytes were examined for the presence or absence of micronuclei.
Details of tissue and slide preparation:
Immediately following sacrifice (ie. 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 and stained in May-Grünwald/Giemsa, dried and coverslipped 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 polychromatic 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 for males and females
separately and combined.
Statistics:
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.
All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part I11 (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:
clinical signs were observed
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

There was a marked and statistically significant decrease in the PCE/NCE ratio in the 48-hour 1500 mgkg test material group when compared to their concurrent vehicle control group. This accompanied with the clinical observations was taken to confrm that systemic absorption had occurred, and exposure to the target tissue achieved. There were no statistically significant increases in the frequency of micronucleated PCEs in any ofthe test material dose groups when compared to their concurrent vehicle control groups. The positive control group showed a marked increase in the incidenceofmicronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity ofcyclophosphamide under the conditions ofthe test. The test material was found not to produce a significant increase in the frequency of micronuclei in polychromatic erythrocytes ofmice under the conditionsofthe test.

Applicant's summary and conclusion

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

Introduction

The study was performed to assess the potential of the test material to produce damage to chromosomes or aneuploidy when administered to mice. The method was designed to comply with the 1997 OECD Guidelines for Testing of Chemicals No.474 "Micronucleus Test", Method B12 of the EC Commission Directive 2000/32/EC, the USA EPA, TSCA and FIFRA guidelines and the Japanese METL'MHLW guidelines for testing of new chemical substances.


Methods

The range-finding test was performed to find suitable dose levels of the test material, route of administration and investigate to see if there was a marked difference in toxic response between the sexes. There was no marked difference in test material toxicity between the sexes; therefore the main study was performed using only male mice. The micronucleus test was conducted using the oral route in groups of seven mice (males) at the maximum tolerated dose (MTD) 1500 mgkg with 750 and 375 mgkg as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow was 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 oral dose of dried arachis oil (7mice) 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.


Results

A statistically significant decrease in the PCE/NCE ratio was observed in the 48-hour1500 mgkg test material dose group when compared to the concurrent control group. This and the presence of clinical signs were taken to indicate that systemic absorption had occurred and exposure ofthe target tissue 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 ofcyclophosphamide under the conditionsofthe test.
Conclusion

The test material was considered to be non-genotoxic under the conditions of the test.