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### HFCPE: Mouse In Vivo Micronucleus Test

HLS study number: VHJ0009

Version ID: Final Report

Issue date: 17 September 2010Huntingdon 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 UKHuntingdon Life Sciences VHJ0009

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## 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................................................................................................................... 20Huntingdon Life Sciences VHJ0009

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## 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 ......................................................................... 29Huntingdon 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 ManagementHuntingdon Life Sciences VHJ0009

8 

## Summary

This study was designed to assess the potenti al induction of micronuclei by HFCPE in bone marrow cells of CD1 mice. Animals were tr eated 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 te st. 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 subs tance dose groups were dosed orally by gavage using a dose volume of 10 mL/kg. The positiv e control group animals were dosed orally by gavage using a dose volume of 20 mL/kg.

The negative control group rece ived the vehicle corn oil a nd 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 administra tion of the second dose. In addition, bone marrow smears were also obtained from animal s in the positive cont rol group 24 hours after a single dose. One smear from each animal was examined for the presence of micronuclei in 2000 polychromatic erythrocytes. The proporti on of polychromatic erythrocytes was assessed by examination of at least 1000 erythr ocytes from each animal. A record of the incidence of micronucleated normochro matic erythrocytes was also kept.

Results

No statistically significant increases in the frequency of micronucleated polychromatic erythrocytes and no statistica lly significant decreases in th e proportion of polychromatic erythrocytes were observed in CD1 mice treat ed with HFCPE at any treatment level, compared to vehicle control values.

The positive control compound, Mitomycin C, produ ced a statistically si gnificant 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

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## 1. Introduction

1.1 Objective

The purpose of this study was to assess the pote ntial 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 co mpliance with the following Guidelines:

OECD Guideline for the Testing of Chemi cals. (1997) Genetic Toxicology: Mammalian Erythrocyte Micronucleus Test, Guideline 474.

EC Commission Regulation No. 440/2008. Me thod B.12: Mutagenicity - In vivo Mammalian Erythrocyte Micronuc leus 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 a nd 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 Speci fic Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. PAB/PCD Notification No. 444.

ICH (1998) Guideline S2B: Genotoxicity: A St andard 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 th is 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 chromosome s 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 mu ltiple micronuclei in the cytoplasm of these cells.Huntingdon Life Sciences VHJ0009

10 A few hours after the last mitosis is completed, erythroblasts expel th eir nucleus. Young erythrocytes less than 24 hours old are termed polychromatic erythrocytes. More mature erythrocytes are termed normo chromatic erythrocytes.

Substances which interfere with the mitotic spindle apparatu s will cause non-disjunction or lagging chromosomes at anaphase which may not be incorporated into the daughter nuclei. These lagging chromosomes are not excluded fro m the erythroblast with the main nucleus and hence also give rise to micronuclei.
Any toxic effects of the test s ubstance 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 erythroc ytes. If the proportion of polychromatic erythrocytes is found to be signi ficantly 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 th e 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 ove r 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 a nd 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 mino r, of short duration, and was not considered to have influenced the health of the animal s or the outcome of the study. The room was illuminated by artificial light fo r 12 hours per day. All animal s 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 enrich ment, 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-he alth 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 ti me 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 a nd 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 substa nce 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 respon sibility of the Sponsor. Chemical analysis of dosing formulations for achie ved concentration was not pe rformed in this study.

Mitomycin C obtained from Sigma, batch num ber 049K0788 was used as the positive control compound. A solution was prepared using purif ied 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 vol ume 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 gi ve an approximate indication of the maximum tolerated dose, i.e. the highest dosage which would be expe cted to elicit signs of toxicity without producing extreme clinical signs or ha ving 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 a nd any mortalities or clinical signs of reaction during the experiment were recorded. At the end of th is observation period, surviving animals were killed and discarded.Huntingdon Life Sciences VHJ0009

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### 2.7 Micronucleus test

Following the preliminary toxicity test no substa ntial differences in t oxicity were observed between sexes. Therefore in line with cu rrent 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. Th e 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 regul arly and any mortalitie s or clinical signs of reaction were recorded. Anim als from the vehicle control a nd 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 risi ng levels of carbon dioxide and both femurs dissected out from each animal. The femurs we re 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 vol ume of 3 mL of filtere d 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 wa s resuspended in a small volume of foetal calf serum to facilitate smearing in the conven tional manner on glass microscope slides ( Schmid 1976 ).Huntingdon Life Sciences VHJ0009

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### 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 ar e wet mounted with glass coverslips using purified water



### 2.9 Microscopic examination

Coded slides were examined by fluorescence mi croscopy and 2000 polychromatic erythrocytes per animal were examined for the presence of micronuclei. One smear was examined per animal, any remaining smears bein g 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 th e 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 incl ude at least 5 analysable animals. 2. Vehicle control values for micronucleated poly chromatic 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 erythroc ytes 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, th en the analysis was carri ed 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 th e analysis was carried out on Groups 1 to 3. Also, exact one-tailed pair wise 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 cl astogenic/aneugenic potential
A positive response is normally indicated by a statistically significant increase in the incidence of micronucleated pol ychromatic erythrocytes for the treatment group compared with the vehicle control group ( p <0.01); individual and/or group mean values should exceed the laboratory histor ical control range ( Morrison and Ashby 1995 ).

A negative result is indicated where individual and group mean inciden ces 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 resu lts 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 significan t decrease in the pr oportion 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 unsuitabl e, by reason of instability, for long term retention and archiving may be disposed of.

All other samples and specimens and all ra w data will be retained by Huntingdon Life Sciences in its archive for a period of one y ear 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 material s 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

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## 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 te st, initially one group consisting of two male and two female animal s 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 animal s displayed clinical signs of toxicity including underactive behaviour, hunched pos ture, 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 administer ed HFCPE at 250 mg/kg/day.
At 250 mg/kg/day no clinical signs of toxicity were observed a nd 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 include d 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 si gns 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 preliminar y 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 toxicityHuntingdon 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 micronuc leus test and the results of statistical analysis. The results for individual animals are presented in Table 2

. No mortalities were observed throughout the du ration 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 an imals, 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 polychromat ic erythrocyte counts (MPCE)
The test substance did not cause any statisti cally significant increases in the number of micronucleated polychromatic eryt hrocytes 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 statistica lly significant increases in the incidence of micronucleated normochromatic erythrocytes in male animals.

3.2.4 Proportion of polychrom atic erythrocytes (%PCE)
The test substance did not cause any statistica lly 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) Chemi cal 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: Statistic al Software for Exact Nonparametric Inference . Cytel Software Corporation, NC, USA.
JONCKHEERE, A.R (1954) A di stribution-free k-sample test against ordered alternatives. Biometrics
, 41 , 133-145.

MacGREGOR, J.T. et al. (1987) Guidelines for the conduc t 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 Environmenta l 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 fo r 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). Individua l 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 2 nd 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 polychr omatic erythrocytes observed per 2000 polychromatic erythrocytes examined

a Positive control dosed once only 24 hours prior to termination

# Occasional apparent errors of ± 1% may o ccur due to rounding of values for presentation in the table

Results of statistical analysis using the appr opriate 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 er ythrocytes 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 normo chromatic erythrocytes observed

a Positive control dosed once only 24 hours prior to terminationHuntingdon Life Sciences VHJ0009

23 Appendix 1 Bodyweights

Preliminary Toxicity Test

Bodyweights (g) At Treatment At Termination Treatment (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 27FHuntingdon 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 terminationHuntingdon 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 ob served prior to death.

Preliminary Toxicity Test Treatment/dosage HFCPE (500 mg/kg/day) Group/sex 1 / Female Day number 1 Pre-dose 2 3 a
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 seve rity 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 seve rity 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 seve rity 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 Polychroma tic 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 Polychroma tic 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 26.6Huntingdon Life Sciences VHJ0009

29 Annex 1 GLP compliance statementsHuntingdon Life Sciences VHJ0009

30 Annex 1 GLP compliance statements - continued