Page 1 of 30 

Working for a better future

HFCPE: Mouse In Vivo Micronucleus Test 

HLS study number: VHJ0009 

Version ID: Final Report 

Issue date: 17 September 2010 
 Huntingdon Life Sciences 

 VHJ0009 

 2 

Sponsor and Test Facility Details 

Sponsor Zeon Corporation 

 

 Shin Marunouchi Center Building 

 1-6-2 Marunouchi 

 Chiyoda-ku 

 Tokyo 

 100-8246 

 JAPAN 

Test facility Genetic Toxicology

Huntingdon Life Sciences 

 Eye Research Centre 

 Eye 

 Suffolk 

 IP23 7PX 

 UK 

Test facility Statistics

Huntingdon Life Sciences 

 Huntingdon Research Centre 

 Woolley Road 

 Alconbury 

 Huntingdon 

 Cambridgeshire 

 PE28 4HS 

 UK 
 Huntingdon Life Sciences 

 VHJ0009 

 3 

Table of Contents 

Sponsor and Test Facility Details ........................................................................................... 2

Compliance with Good Laboratory Practice............................................................................ 5

Quality Assurance Statement ................................................................................................. 6

Contributing Scientists ............................................................................................................ 7

Summary................................................................................................................................. 8

1. Introduction .................................................................................................................... 9

1.1 Objective ............................................................................................................ 9

1.2 Regulatory compliance....................................................................................... 9

1.3 Test system ........................................................................................................ 9

1.4 Route of administration .................................................................................... 10

2. Experimental procedure............................................................................................... 11

2.1 Duration of treatment........................................................................................ 11

2.2 Time schedule.................................................................................................. 11

2.3 Animal management ........................................................................................ 11

2.3.1 Clinical observations............................................................................. 12

2.3.2 Bodyweight ........................................................................................... 12

2.3.3 Mortality ................................................................................................ 12

2.4 Test substance................................................................................................. 12

2.5 Test substance formulation and administration................................................ 13

2.6 Preliminary toxicity test..................................................................................... 13

2.7 Micronucleus test ............................................................................................. 14

2.8 Fixation and slide staining ................................................................................ 15

2.9 Microscopic examination .................................................................................. 15

2.10 Assessment of results ...................................................................................... 15

2.10.1 Acceptance criteria ............................................................................... 15

2.10.2 Analysis of data .................................................................................... 15

2.10.3 Criteria for assessing clastogenic/aneugenic potential......................... 16

2.11 Maintenance of records.................................................................................... 16

3. Results ......................................................................................................................... 17

3.1 Preliminary toxicity test..................................................................................... 17

3.1.1 Clinical signs......................................................................................... 17

3.2 Micronucleus test ............................................................................................. 18

3.2.1 Clinical signs......................................................................................... 18

3.2.2 Micronucleated polychromatic erythrocyte counts (MPCE).................. 18

3.2.3 Micronucleated normochromatic erythrocytes (MNCE)........................ 18

3.2.4 Proportion of polychromatic erythrocytes (%PCE) ............................... 18

4. Conclusion ................................................................................................................... 19

5. References................................................................................................................... 20
 Huntingdon Life Sciences 

 VHJ0009 

 4 

List of Tables 

Table 1 Summary of results and statistical analysis................................................... 21

Table 2 Individual animal data.................................................................................... 22

List of Appendices 

Appendix 1 Bodyweights .................................................................................................. 23

Appendix 2 Clinical signs ................................................................................................. 25

Appendix 3 Historical control data.................................................................................... 28

List of Annexes 

Annex 1 GLP compliance statements ......................................................................... 29


 Huntingdon Life Sciences 

 VHJ0009 

 7 

Contributing Scientists 

HFCPE: Mouse In Vivo Micronucleus Test 

Genetic Toxicology Study Management 

William Barfield, BSc (Hons) 

Study Director 

Genetic Toxicology 

David Brown 

Senior Animal Technician 

Statistics 

Gareth D. Thomas, BSc (Hons) 

Head of Statistics and Data Management 
 Huntingdon Life Sciences 

 VHJ0009 

 8 

Summary 

This study was designed to assess the potential induction of micronuclei by HFCPE in bone 

marrow cells of CD1 mice. Animals were treated with HFCPE orally by gavage on two 

occasions approximately 24 hours apart. 

On the basis of results from the preliminary toxicity test, dose levels of 62.5, 125 and 250 

mg/kg/day were selected for the micronucleus test. No substantial differences in toxicity 

were observed between the sexes, therefore, in line with current guidelines the micronucleus 

test was performed using male animals only. 

All animals in the vehicle control and test substance dose groups were dosed orally by gavage 

using a dose volume of 10 mL/kg. The positive control group animals were dosed orally by 

gavage using a dose volume of 20 mL/kg. 

The negative control group received the vehicle corn oil and the positive control group 

received Mitomycin C at 12 mg/kg. 

Bone marrow smears were obtained from animals in the vehicle control and in each of the 

test substance groups 24 hours after administration of the second dose. In addition, bone 

marrow smears were also obtained from animals in the positive control group 24 hours after a 

single dose. One smear from each animal was examined for the presence of micronuclei in 

2000 polychromatic erythrocytes. The proportion of polychromatic erythrocytes was 

assessed by examination of at least 1000 erythrocytes from each animal. A record of the 

incidence of micronucleated normochromatic erythrocytes was also kept. 

Results 

No statistically significant increases in the frequency of micronucleated polychromatic 

erythrocytes and no statistically significant decreases in the proportion of polychromatic 

erythrocytes were observed in CD1 mice treated with HFCPE at any treatment level, 

compared to vehicle control values. 

The positive control compound, Mitomycin C, produced a statistically significant increase in 

the frequency of micronucleated polychromatic erythrocytes (p<0.01). 

Conclusion 

It is concluded that HFCPE did not show any evidence of causing an increase in the induction 

of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in male CD1 

mice when administered orally by gavage in this in vivo test procedure. 
 Huntingdon Life Sciences 

 VHJ0009 

 9 

1. Introduction 

1.1 Objective 

The purpose of this study was to assess the potential of HFCPE to induce an increase in the 

induction of micronucleated polychromatic erythrocytes in CD1 mice following oral 

administration using an in vivo cytogenetic system (Boller and Schmid 1970, MacGregor et 

al 1987, Mavournin et al 1990). 

1.2 Regulatory compliance 

The performance of this study was in compliance with the following Guidelines: 

OECD Guideline for the Testing of Chemicals. (1997) Genetic Toxicology: Mammalian 

Erythrocyte Micronucleus Test, Guideline 474. 

EC Commission Regulation No. 440/2008. Method B.12: Mutagenicity - In vivo 

Mammalian Erythrocyte Micronucleus Test. OJ L 142/240. 

US EPA (1998) Health Effects Test Guidelines; OPPTS 870.5395 Mammalian 

Erythrocyte Micronucleus Test. EPA 712-C-98-226. 

Japanese Ministry of Agriculture, Forestry and Fisheries. Test Data for Registration of 

Agricultural Chemicals, 12 Nohsan No. 8147, Guideline 2-1-19-3, Agricultural 

Production Bureau, November 24, 2000. 

ICH (1996) Guideline S2A: Guidance on Specific Aspects of Regulatory Genotoxicity 

Tests for Pharmaceuticals. PAB/PCD Notification No. 444. 

ICH (1998) Guideline S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing 

of Pharmaceuticals. PMSB/ELD Notification No. 554. 

Official notice of J MHLW, METI and ME (21 November 2003): 

 YAKUSHOKUHATSU No.1121002 

 SEIKYOKU No.2 

 KANPOKIHATSU No. 031121002 

1.3 Test system 

The bone marrow micronucleus test is a short-term assay for identification of genotoxic 

effects associated with mutagens and carcinogens (Mavournin et al 1990). Young adult 

animals are chosen for use because of the high rate of cell division in the bone marrow, the 

wealth of background data on this species, and their general suitability for toxicological 

investigations. 

In mitotic cells in which chromosomal breakage has been caused by the test substance or its 

metabolites, acentric fragments of the chromosomes do not separate at the anaphase stage of 

cell division. After telophase these fragments may not be included in the nuclei of the 

daughter cells and hence will form single or multiple micronuclei in the cytoplasm of these 

cells. 
 Huntingdon Life Sciences 

 VHJ0009 

 10 

A few hours after the last mitosis is completed, erythroblasts expel their nucleus. Young 

erythrocytes less than 24 hours old are termed polychromatic erythrocytes. More mature 

erythrocytes are termed normochromatic erythrocytes. 

Substances which interfere with the mitotic spindle apparatus will cause non-disjunction or 

lagging chromosomes at anaphase which may not be incorporated into the daughter nuclei. 

These lagging chromosomes are not excluded from the erythroblast with the main nucleus 

and hence also give rise to micronuclei. 

Any toxic effects of the test substance on the nucleated cells may lead either to a reduction in 

cell division or to cell death. These effects in turn lead to a reduction in the number of 

nucleated cells and polychromatic erythrocytes. If the proportion of polychromatic 

erythrocytes is found to be significantly less than the control value, this is taken as being 

indicative of toxicity. 

1.4 Route of administration 

The oral route was chosen for this particular study as directed by the sponsor to maximise 

exposure to the test system. 
 Huntingdon Life Sciences 

 VHJ0009 

 11 

2. Experimental procedure 

2.1 Duration of treatment 

The test substance was administered over a period of two consecutive days. 

2.2 Time schedule 

Study initiation: 5 May 2010 

(Protocol signed by the Study Director) 

Experimental start date: 17 May 2010 

 

Animals allocated to study at Huntingdon Life Sciences: 

Preliminary Toxicity Test: 17 May 2010 

 27 May 2010 

 16 June 2010 

 

Micronucleus Test: 7 July 2010 

Study completion: 

(Statistical analysis)

 4 August 2010 

2.3 Animal management 

All animals used on this study were CD1 mice. 

On the day after arrival from Charles River UK Limited, Margate, Kent, England, animals 

used on the study weighed between: 

Preliminary Test: Males weighed between 26.9 g to 31.4 g. 

 Females weighed between 20.7 g to 24.1 g. 

Micronucleus Test: Males weighed between 30.9 g to 34.0 g. 

Animal age on despatch and on Day 1 of dosing was: 

Preliminary Test: On despatch Males and females ca 35 days old. 

 Day 1 Males and females ca 47 - 56 days old. 

Micronucleus Test: On despatch Males ca 35 days old. 

 Day 1 Males ca 42 days old. 
 Huntingdon Life Sciences 

 VHJ0009 

 12 

The day after arrival the weight of the animals was checked and found to be acceptable. The 

animals were randomly assigned to groups and tail marked. Each group was kept, with the 

sexes separated, in cages and maintained in a controlled environment, with the thermostat and 

relative humidity target ranges set at 19 to 23°C and 40 to 70% respectively. Temperature 

was within range throughout the study; however, the relative humidity fell to 38%. The data 

indicates that the deviation in humidity, was minor, of short duration, and was not considered 

to have influenced the health of the animals or the outcome of the study. The room was 

illuminated by artificial light for 12 hours per day. All animals were allowed free access to 

pelleted expanded rat and mouse No.1 maintenance diet (SQC grade obtained from Special 

Diets Services Ltd, Witham, Essex, UK) and tap water ad libitum. Food, Nestlets and tap 

water are routinely analysed for quality at source. All animals were given Nestlets and a red 

triangular mouse house for environmental enrichment, were acclimatised for a minimum of 5 

days, examined daily and weighed prior to dosing. 

2.3.1 Clinical observations 

Animals were inspected at least twice daily for evidence of ill-health or reaction to treatment. 

Cages were inspected for evidence of animal ill-health amongst the occupants and any 

deviations from the normal was recorded at the time in respect of nature and severity, date 

and time of onset. 

2.3.2 Bodyweight 

Animal weights were recorded for each animal on the day after arrival, on each day of dosing 

and on the day of termination. 

2.3.3 Mortality 

Debilitated animals were observed carefully and animals were killed to prevent unnecessary 

or prolonged suffering. 

2.4 Test substance 

Identity: HFCPE 

Chemical name: 1,3,3,4,4,5,5-Heptafluorocyclopentene 

Batch number: 20090601 

Expiry date: End of December 2010 

Appearance: Colourless liquid 

Storage conditions: Room temperature 

Purity: 99.3% 

Date received: 22 June 2009 

The above information with regard to the physical characterisation of the test substance is the 

responsibility of the Sponsor. 
 Huntingdon Life Sciences 

 VHJ0009 

 13 

2.5 Test substance formulation and administration 

Suspensions of the test substance were prepared in corn oil obtained from Sigma, batch 

number 058K0070. 

Stability and homogeneity of the test substance and of the test substance in the vehicle were 

not determined in this test and remain the responsibility of the Sponsor. Chemical analysis of 

dosing formulations for achieved concentration was not performed in this study. 

Mitomycin C obtained from Sigma, batch number 049K0788 was used as the positive control 

compound. A solution was prepared using purified water at a concentration of 0.6 mg/mL 

just prior to administration. 

In the micronucleus test all animals in the vehicle control and test substance dose groups 

were dosed orally by gavage using a dose volume of 10 mL/kg. The positive control group 

animals were dosed orally by gavage using a dose volume of 20 mL/kg. 

2.6 Preliminary toxicity test 

The purpose of this test was to determine a suitable dose level for use in the micronucleus 

test. The dosages employed were used to give an approximate indication of the maximum 

tolerated dose, i.e. the highest dosage which would be expected to elicit signs of toxicity 

without producing extreme clinical signs or having a significant effect on survival. The 

experimental design is shown below: 

Group Treatment Concentration Dosage Number of animals 

 (mg/mL) (mg/kg/day) Males Females

1 HFCPE 50.0 500 2 2 

2 HFCPE 25.0 250 2 2 

3 HFCPE 37.5 375 2 2 

4 HFCPE 30.0 300 2 2 

Following dosing, the animals were examined regularly during the working day for a period 

of 48 hours after the first dose and any mortalities or clinical signs of reaction during the 

experiment were recorded. At the end of this observation period, surviving animals were 

killed and discarded. 
 Huntingdon Life Sciences 

 VHJ0009 

 14 

2.7 Micronucleus test 

Following the preliminary toxicity test no substantial differences in toxicity were observed 

between sexes. Therefore in line with current guidelines, the micronucleus test was 

performed using males only. 

From the results obtained in the preliminary toxicity study (see RESULTS), dose levels of

62.5, 125 and 250 mg/kg/day were used for the micronucleus test. The experimental design 

is shown below:

Group Treatment Concentration Dosage Number of animals 

 (mg/mL) (mg/kg/day) Males

1 Vehicle - - 6 

2 HFCPE 6.25 62.5 6 

3 HFCPE 12.5 125 6 

4 HFCPE 25.0 250 6 

5 Mitomycin C a 0.6 12 5 

Vehicle Corn oil 

a Positive control dosed once only approximately 24 hours prior to termination 

at a dose volume of 20 mL/kg/day. 

Following dosing, the animals were examined regularly and any mortalities or clinical signs 

of reaction were recorded. Animals from the vehicle control and test substance groups were 

sacrificed 24 hours after administration of the second dose. In addition animals in the positive 

control group were sacrificed 24 hours after a single dose. 

The animals were killed by exposure to rising levels of carbon dioxide and both femurs 

dissected out from each animal. The femurs were cleaned of all excess tissue and blood and 

the proximal epiphysis removed from each bone. The bone marrow of both femurs from each 

animal was flushed out and pooled in a total volume of 3 mL of filtered foetal calf serum by 

aspiration. 

The resulting cell suspensions were centrifuged at 1000 rpm (150 × g) for 5 minutes and the 

supernatant discarded. The final cell pellet was resuspended in a small volume of foetal calf 

serum to facilitate smearing in the conventional manner on glass microscope slides (Schmid 

1976). 
 Huntingdon Life Sciences 

 VHJ0009 

 15 

2.8 Fixation and slide staining 

1 Fixed for a minimum of 10 minutes in methanol and allowed to air-dry 

2 Rinsed in purified water 

3 Stained in acridine orange solution (0.0125 mg/mL using purified water) for 4 

minutes 

4 Washed in purified water for 5 minutes 

5 Rinsed in cold tap water for 2 minutes 

6 Stored at room temperature until required 

7 Immediately prior to scoring, slides are wet mounted with glass coverslips using 

purified water 

2.9 Microscopic examination 

Coded slides were examined by fluorescence microscopy and 2000 polychromatic erythrocytes 

per animal were examined for the presence of micronuclei. One smear was examined per 

animal, any remaining smears being held temporarily in reserve in case of technical problems 

with the first smear. 

The proportion of polychromatic erythrocytes was assessed by examination of a total of at 

least 1000 erythrocytes per animal and the number of micronucleated normochromatic 

erythrocytes was recorded. 

2.10 Assessment of results 

2.10.1 Acceptance criteria 

The following criteria were applied for assessment of assay acceptability: 

1. Each treated and control group should include at least 5 analysable animals. 

2. Vehicle control values for micronucleated polychromatic erythrocytes must be consistent 

with the laboratory historical negative control data. 

3. Positive controls must show clear unequivocal positive responses. 

2.10.2 Analysis of data 

For the proportion of polychromatic erythrocytes at 24 hours, an asymptotic one-tailed 

Jonckheere’s test for trend (Jonckheere 1954) with “step-down” was used on Groups 1 to 4 

for a decrease from control. If significant, then the analysis was carried out on Groups 1 to 3, 

then on Groups 1 and 2. Exact one-tailed Wilcoxon pairwise tests (Wilcoxon 1945), for a 

decrease from control, were also carried out on Group 1 (control) versus Groups 2, 3, 4 and 5. 

For micronucleated polychromatic erythrocytes at 24 hours, an exact one-tailed Linear-byLinear association test (Cytel 1995) with “step-down” was used on Groups 1 to 4 for an 

increase from control. If significant, then the analysis was carried out on Groups 1 to 3. 

Also, exact one-tailed pairwise Permutation tests (Cytel 1995), for an increase from control, 

were carried out on Group 1 (control) versus Groups 2, 3, 4 and 5. 
 Huntingdon Life Sciences 

 VHJ0009 

 16 

Statistical significance was declared at the 1% level for all tests. 

The data were received in an Excel document and analysed using SAS 9.1.3 (SAS Institute 

Inc., 2002) (Jonckheere's and Wilcoxon tests) and StatXact 3 (Cytel 1995) (Linear-by-Linear 

and Permutation tests).

2.10.3 Criteria for assessing clastogenic/aneugenic potential 

A positive response is normally indicated by a statistically significant increase in the 

incidence of micronucleated polychromatic erythrocytes for the treatment group compared 

with the vehicle control group (p<0.01); individual and/or group mean values should exceed 

the laboratory historical control range (Morrison and Ashby 1995). 

A negative result is indicated where individual and group mean incidences of micronucleated 

polychromatic erythrocytes for the group treated with the test substance are not significantly 

greater than incidences for the concurrent vehicle control group and where these values fall 

within the historical control range. 

An equivocal response is obtained when the results do not meet the criteria specified for a 

positive or negative response. 

Bone marrow cell toxicity (or depression) is normally indicated by a substantial and 

statistically significant decrease in the proportion of polychromatic erythrocytes (p<0.01). 

2.11 Maintenance of records 

All raw data, samples and specimens arising from the performance of this study will remain 

the property of the Sponsor. 

Types of sample and specimen that are unsuitable, by reason of instability, for long term 

retention and archiving may be disposed of. 

All other samples and specimens and all raw data will be retained by Huntingdon Life 

Sciences in its archive for a period of one year from the date on which the Study Director 

signs the final report. After such time, the Sponsor will be contacted and advice sought on 

the return, disposal or further retention of the materials. If requested, Huntingdon Life 

Sciences will continue to retain the materials subject to a reasonable fee being agreed with 

the Sponsor. 

Huntingdon Life Sciences will retain the Quality Assurance records relevant to this study and 

a copy of the final report in its archive indefinitely. 

. 
 Huntingdon Life Sciences 

 VHJ0009 

 17 

3. Results 

3.1 Preliminary toxicity test 

Detailed bodyweights are presented in Appendix 1 and clinical observations in Appendix 2. 

3.1.1 Clinical signs 

To determine suitable dose levels for use in the micronucleus test, initially one group 

consisting of two male and two female animals were dosed with HFCPE at 500 mg/kg/day on 

two consecutive days approximately 24 hours apart. 

At 500 mg/kg/day, no clinical signs of toxicity were observed in male or female animals until 

day 3. On day 3 both male animals were found dead, the female animals displayed clinical 

signs of toxicity including underactive behaviour, hunched posture, coat piloerection, both 

eyelids partially closed, pallor skin colour and deep breathing. One female animal was killed 

in extremis due to the clinical signs observed and the other culled as the dose was considered 

unacceptable for use in the micronucleus test. 

On the basis of the severity of the clinical signs and the deaths observed, two male and two 

female animals were administered HFCPE at 250 mg/kg/day. 

At 250 mg/kg/day no clinical signs of toxicity were observed and thus the maximum tolerated 

dose had not been achieved. 

Consequently, a further two groups of two male and two female animals were administered 

HFCPE at 375 mg/kg/day and 300 mg/kg/day respectively. 

At 375 mg/kg/day clinical signs of toxicity observed included, underactive behaviour, coat 

piloerection, hunched posture, deep breathing, reduced body temperature, muscle tremors, 

elevated gait and slow breathing. One male and one female animal were found dead on day 2, 

the remaining male and female were killed in extremis due to the severity of the clinical signs 

observed and did not receive a second administration of HFCPE at 375 mg/kg. 

At 300 mg/kg/day clinical signs of toxicity observed included underactive behaviour, coat 

piloerection, hunched posture, deep breathing, reduced body temperature, unsteady gait, slow 

breathing, muscle tremors and both eyelids partially closed. All animals were killed in 

extremis approximately six and a half hours after receiving the second dose of HFCPE at 300 

mg/kg. 

On the basis of the severity of the clinical signs and deaths observed it was considered that 

the maximum tolerated dose was exceeded at 375 and 300 mg/kg/day. 

Some small incidences of bodyweight loss were observed throughout the preliminary testing. 

The animals which died during the preliminary test were subjected to a macroscopic post 

mortem examination, no signs of mis-dosing were apparent in any of the animals analysed. 

Consequently, 250 mg/kg/day was considered to be the maximum tolerated dose in both male 

and female mice. In line with current guidelines and as no substantial differences in toxicity 
 Huntingdon Life Sciences 

 VHJ0009 

 18 

were observed between the sexes, male animals only were used in the micronucleus test, dose 

levels selected were 62.5, 125 and 250 mg/kg/day.

3.2 Micronucleus test 

The micronucleus test was carried out in male animals only. 

Table 1 gives a summary of the results of the micronucleus test and the results of statistical 

analysis. The results for individual animals are presented in Table 2. 

No mortalities were observed throughout the duration of the micronucleus test. Detailed 

bodyweights are presented in Appendix 1. 

3.2.1 Clinical signs 

Animals were treated with HFCPE at dose levels of 62.5, 125 and 250 mg/kg/day. No clinical 

signs were noted for the vehicle control animals, positive control animals and animals 

administered HFCPE at 62.5 and 125 mg/kg/day over the duration of the test. 

At 250 mg/kg/day no clinical signs of toxicity were observed on day 1 or day 2. On day 3 

underactive behaviour and coat piloerection were observed in one male animal. 

 

Some small incidences of bodyweight loss were observed throughout the duration of the test. 

3.2.2 Micronucleated polychromatic erythrocyte counts (MPCE) 

The test substance did not cause any statistically significant increases in the number of 

micronucleated polychromatic erythrocytes in male animals. 

Mitomycin C caused a statistically significant increase in the frequency of micronucleated 

polychromatic erythrocytes (p<0.01) in male animals. 

3.2.3 Micronucleated normochromatic erythrocytes (MNCE) 

The test substance did not cause any statistically significant increases in the incidence of 

micronucleated normochromatic erythrocytes in male animals. 

3.2.4 Proportion of polychromatic erythrocytes (%PCE) 

The test substance did not cause any statistically significant decreases in the proportion of 

polychromatic erythrocytes in male animals. 

Mitomycin C did not cause a statistically significant decrease in the proportion of 

polychromatic erythrocytes in male animals. 

Appendix 3 summarises the historical control data. 
 Huntingdon Life Sciences 

 VHJ0009 

 19 

4. Conclusion 

It is concluded that HFCPE did not show any evidence of causing an increase in the induction 

of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in male CD1 

mice when administered orally by gavage in this in vivo test procedure. 
 Huntingdon Life Sciences 

 VHJ0009 

 20 

5. References 

BOLLER, K. and SCHMID, W. (1970) Chemical mutagenesis in mammals. The bone 

marrow of the Chinese hamster as an in vivo test system. Haematological findings after 

treatment with Trenimon (translation). Humangenetik, 11, 34. 

CYTEL (1995) StatXact 3 for Windows: Statistical Software for Exact Nonparametric 

Inference. Cytel Software Corporation, NC, USA. 

JONCKHEERE, A.R (1954) A distribution-free k-sample test against ordered alternatives. 

Biometrics, 41, 133-145. 

MacGREGOR, J.T. et al. (1987) Guidelines for the conduct of micronucleus assays in 

mammalian bone marrow erythrocytes. Mutation Research, 189, 103. 

MAVOURNIN, K.H., BLAKEY, D.H., CIMINO, M.C., SALAMONE, M.F. and HEDDLE, 

J.A. (1990) The in vivo micronucleus assay in mammalian bone marrow and peripheral 

blood. A report of the US Environmental Protection Agency Gene-tox Program. Mutation 

Research, 239, 29. 

MORRISON, V. and ASHBY, J. (1995) High resolution rodent bone marrow micronucleus 

assays of 1,2-dimethylhydrazine : implication of systemic toxicity and individual responders. 

Mutagenesis, 10, 129. 

SAS INSTITUTE (2002) SAS OnlineDoc® Version Nine. SAS Institute Inc., Cary, NC, 

USA. 

SCHMID, W. (1976) The micronucleus test for cytogenetic analysis. In: HOLLANDER, A. 

(ed.) Chemical Mutagens, Principles and Methods for their Detection, 4, 31. Published by 

Plenum Press, New York. 

WILCOXON, F. (1945). Individual comparisons by ranking methods. Biometrics Bulletin, 

1, 80-83. 
 Huntingdon Life Sciences 

 VHJ0009 

 21 

Table 1 Summary of results and statistical analysis 

Male Data 

Sampling time 

after 2nd dose Treatment Dose 

(mg/kg/day) 

Proportion of 

PCE (%) # 

Incidence 

MPCE (mean) #

24 Hours Vehicle - 46.4 0.7 

 HFCPE 62.5 48.8 0.3 

 HFCPE 125 47.7 0.8 

 HFCPE 250 49.3 0.7 

 Mitomycin C a

 12 39.0 90.0** 

 

Vehicle Corn oil 

PCE Polychromatic erythrocytes 

MPCE Number of micronucleated polychromatic erythrocytes observed per 2000 

polychromatic erythrocytes examined 

a Positive control dosed once only 24 hours prior to termination 

# Occasional apparent errors of ± 1% may occur due to rounding of values for presentation 

in the table 

Results of statistical analysis using the appropriate nonparametric method of analysis based 

on permutation (one-sided probabilities): 

 ** p < 0.01 (significant) 

 otherwise p > 0.01 (not significant) 
 Huntingdon Life Sciences 

 VHJ0009 

 22 

Table 2 Individual animal data 

Male Data 

Results for individual animals - 24 hour sampling time 

Treatment 

(mg/kg/day) 

Animal 

number 

Proportion 

PCE (%) MPCE PCE NCE MNCE 

Vehicle 501 49.0 1 577 601 0

- 502 45.2 1 475 575 0 

 503 56.2 1 591 460 1 

 504 39.6 0 402 612 0 

 505 44.8 1 482 594 0 

 506 43.4 0 436 568 0 

HFCPE 551 41.5 1 419 590 0

(62.5) 512 52.9 0 542 485 0 

 513 50.2 1 552 548 0 

 514 47.9 0 481 524 0 

 515 49.4 0 528 540 0 

 516 51.3 0 545 518 0 

HFCPE 521 58.0 0 585 423 0

(125) 522 44.9 2 461 565 0 

 523 49.0 0 493 513 0 

 524 48.6 1 516 546 0 

 525 46.0 0 502 589 0 

 526 39.9 2 402 605 1 

HFCPE 531 41.7 1 479 669 0

(250) 532 53.6 2 553 479 0 

 533 55.3 0 571 462 0 

 534 39.7 0 400 608 0 

 535 56.6 1 571 438 0 

 536 49.1 0 503 522 0 

Mitomycin C a 541 39.8 70 400 606 0

(12) 542 40.5 101 409 601 0 

 543 35.6 79 370 670 0 

 544 40.3 115 406 602 0 

 545 39.1 85 398 619 0 

Vehicle Corn oil 

PCE Number of polychromatic erythrocytes per 1000 erythrocytes 

MPCE Number of micronucleated cells observed per 2000 polychromatic 

erythrocytes examined 

NCE Total number of normochromatic erythrocytes examined for micronuclei 

MNCE Number of micronucleated normochromatic erythrocytes observed a Positive control dosed once only 24 hours prior to termination 
 Huntingdon Life Sciences 

 VHJ0009 

 23 

Appendix 1 Bodyweights 

Preliminary Toxicity Test 

 Bodyweights (g)

Treatment At Treatment At Termination 

(mg/kg/day)

Animal

Number Day after arrival Day 1 Day 2 Day 3

 Individual Mean Individual Mean Individual Mean Individual Mean 

HFCPE M 25 27.4 33.2 33.2 FD 

(500) M 26 29.5 28.5 35.4 34.3 35.1# 34.2 FD 

 F 27 20.7 24.9 24.5 KIE 

 F 28 22.4 21.6 25.8 25.4 25.0 24.8 a 

HFCPE M 29 29.1 35.2 35.3 35.0# 

(250) M 30 31.4 30.3 36.4 35.8 35.4# 35.4 35.6 35.3 

 F 31 21.6 23.0 22.8# 23.0 

 F 32 22.2 21.9 24.7 23.9 24.8 23.8 25.2 24.1 

HFCPE M 89 29.2 35.6 KIE 

(375) M 90 27.4 28.3 33.0 34.3 FD 

 F 91 21.5 25.4 KIE 

 F 92 24.1 22.8 29.5 27.5 FD 

HFCPE M 473 26.9 34.6 31.1# KIE 

(300) M 502 27.6 27.3 36.1 35.4 34.2# 32.7 KIE 

 F 503 21.0 24.8 23.6# KIE 

 F 504 22.1 21.6 25.2 25.0 26.5 25.1 KIE 

 

# Denotes weight loss from previous weighing 

M Male 

F Female 

FD Found dead 

KIE Killed in extremis 

a Animal culled due to the severity of the clinical signs displayed in 27F 
 Huntingdon Life Sciences 

 VHJ0009 

 24 

Appendix 1 Bodyweights - continued 

Micronucleus Test 

Male Data 

 Bodyweights (g)

At Treatment At Termination 

Day after arrival Day 1 Day 2 Day 3 Treatment 

(mg/kg/day)

Animal

Number Individual Mean Individual Mean Individual Mean Individual Mean 

Vehicle 501 32.8 36.5 36.3# 36.6 

 502 33.3 36.6 37.1 37.1 

 503 32.5 36.6 35.9# 36.2 

 504 33.4 36.3 35.8# 36.6 

 505 31.8 35.2 35.4 35.8 

 506 33.2 

32.8 

35.1 

36.1 

37.3 

36.3 

38.2 

36.8 

HFCPE 551 33.4 33.6 33.7 35.0 

(62.5) 512 31.4 35.4 34.3# 35.8 

 513 31.6 36.5 37.0 37.3 

 514 33.2 35.7 35.5# 36.0 

 515 33.7 36.7 36.9 37.0 

 516 31.2 

32.4 

34.1 

35.3 

33.7# 

35.2 

35.2 

36.1 

HFCPE 521 32.5 35.5 34.4# 35.6 

(125) 522 33.0 34.9 35.0 35.4 

 523 32.4 32.5 32.4# 32.8 

 524 31.7 34.5 33.2# 33.9 

 525 32.4 35.2 34.8# 35.2 

 526 32.8 

32.5 

35.0 

34.6 

33.8# 

33.9 

35.6 

34.8 

HFCPE 531 33.4 35.4 34.4# 34.8 

(250) 532 31.2 33.9 32.8# 33.0 

 533 33.8 36.9 36.4# 37.0 

 534 32.8 33.5 33.2# 31.6# 

 535 32.3 35.3 34.6# 34.8 

 536 34.0 

32.9 

36.7 

35.3 

35.3# 

34.5 

36.4 

34.6 

Mitomycin C a

 541 31.9 - 34.0 33.8# 

(12) 542 30.9 - 34.0 33.9# 

 543 33.2 - 34.9 35.1 

 544 32.8 - 34.6 35.0 

 545 31.6 

32.1 

- 

- 

33.6 

34.2 

34.0 

34.4 

 

Vehicle Corn oil 

# Denotes weight loss from previous weighing 

a Positive control dosed once only 24 hours prior to termination 

 
 Huntingdon Life Sciences 

 VHJ0009 

 25 

Appendix 2 Clinical signs 

Preliminary Toxicity Test 

No adverse clinical signs were observed for male or female animals dosed at 250 mg/kg/day. 

Male animals administered HFCPE at 500 mg/kg/day were found dead on Day 3. No clinical 

signs of toxicity were observed prior to death. 

Preliminary Toxicity Test

Treatment/dosage HFCPE (500 mg/kg/day) 

Group/sex 1 / Female 

Day number 1 Pre-dose 2 3a

No abnormalities detected 2 2 2 0 

Behaviour – Underactive 0 0 0 2 

Posture – Hunched 0 0 0 1 

Coat – Piloerection 0 0 0 2 

Eyelids – Partially closed (both) 0 0 0 1 

Skin colour – Pallor 0 0 0 1 

Breathing – Deep 0 0 0 1 

Mortalities 2/2 

 

a Animal 27F killed in extremis due to the severity of the clinical signs observed. Animal 

28F culled due to the severity of the clinical signs observed in 27F

The number of animals displaying the clinical sign is indicated within the table. 
 Huntingdon Life Sciences 

 VHJ0009 

 26 

Appendix 2 Clinical signs - continued 

Preliminary Toxicity Test

Treatment/dosage HFCPE (375 mg/kg/day) 

Group/sex 3 / Male 

Day number 1 Pre-dose a 2 3 

No abnormalities detected 0 0 

Behaviour – Underactive 2 1 

Coat – Piloerection 2 1 

Posture – Hunched 2 1 

Breathing – Deep 1 0 

Body temperature – Reduced 1 1 

Muscle reaction – Tremors 1 0 

Mortalities 2/2 

 

a Animal 89M killed in extremis due to the severity of the clinical signs observed. Animal 

90M found dead.

The number of animals displaying the clinical sign is indicated within the table. 

Preliminary Toxicity Test

Treatment/dosage HFCPE (375 mg/kg/day) 

Group/sex 3 / Female 

Day number 1 Pre-dose a 2 3 

No abnormalities detected 0 0 

Behaviour – Underactive 2 1 

Coat – Piloerection 2 1 

Posture – Hunched 2 1 

Breathing – Deep 0 1 

Body temperature – Reduced 0 1 

Abnormal gait – Elevated 1 0 

Breathing – Slow 0 1 

Mortalities 2/2 

 

a Animal 91F killed in extremis due to the severity of the clinical signs observed. Animal 

92F found dead.

The number of animals displaying the clinical sign is indicated within the table. 
 Huntingdon Life Sciences 

 VHJ0009 

 27 

Appendix 2 Clinical signs – continued 

Preliminary Toxicity Test

Treatment/dosage HFCPE (300 mg/kg/day) 

Group/sex 4 / Male 

Day number 1 Pre-dose 2 a 3 

No abnormalities detected 2 2 0 

Behaviour – Underactive 0 0 1 

Coat – Piloerection 0 0 2 

Posture – Hunched 0 0 2 

Breathing – Deep 0 0 1 

Body temperature – Reduced 0 0 1 

Abnormal gait – Unsteady 0 0 1 

Breathing – Slow 0 0 1 

Muscle reaction – Tremors 0 0 1 

Mortalities 2/2 

 

a Animals killed in extremis due to the severity of the clinical signs observed

The number of animals displaying the clinical sign is indicated within the table. 

Preliminary Toxicity Test

Treatment/dosage HFCPE (300 mg/kg/day) 

Group/sex 4 / Female 

Day number 1 Pre-dose 2 a

 3 

No abnormalities detected 2 2 0 

Behaviour – Underactive 0 0 1 

Coat – Piloerection 0 0 2 

Posture – Hunched 0 0 2 

Body temperature – Reduced 0 0 1 

Abnormal gait – Unsteady 0 0 1 

Eyelids – Partially closed (both) 0 0 1 

Mortalities 2/2 

 

a Animals killed in extremis due to the severity of the clinical signs observed

The number of animals displaying the clinical sign is indicated within the table. 
 Huntingdon Life Sciences 

 VHJ0009 

 28 

Appendix 3 Historical control data 

CD1 Mice 

Vehicle Control Values – April 2008 – March 2010 (15 studies) 

Proportion of Polychromatic Erythrocytes (% PCE)

 Individual Group 

Minimum 32 39.2 

Maximum 73 61.8 

Mean 46.4 46.7 

Standard Deviation 6.5 5.1 

Micronucleated Polychromatic Erythrocytes (MPCE) 

(per 2000 PCE cells) 

 Individual Group 

Minimum 0 0.4 

Maximum 5 2.5 

Mean 1.4 1.4 

Standard Deviation 1.1 0.4 

Positive Control Values – April 2008 – March 2010 (15 studies) 

Proportion of Polychromatic Erythrocytes (% PCE)

 Individual Group 

Minimum 25.0 36.8 

Maximum 60.0 49.8 

Mean 42.7 42.7 

Standard Deviation 6.5 4.1 

Micronucleated Polychromatic Erythrocytes (MPCE) 

(per 2000 PCE cells) 

 Individual Group 

Minimum 27 50.4 

Maximum 179 154.6 

Mean 84.4 84.4 

Standard Deviation 30 26.6 

 
 Huntingdon Life Sciences 

 VHJ0009 

 29 

Annex 1 GLP compliance statements 
 Huntingdon Life Sciences 

 VHJ0009 

 30 

Annex 1 GLP compliance statements - continued