Dimethyl Adipate Rat Micronucleus Test

DIMETHYL GLUTARATE

RAT MICRONUCLEUS TEST CONFIDENTIAL SOA 002l004851

DIMETHYL GLUTARATE

.Sponsor Research Laboratory

SOCMA Huntingdon Life Sciences Ltd. 1850 M Street NW Woolley Road Suite 700 Alconbury Washington Huntingdon DC 20036 Cambridgeshire USA PE28 4HS ENGLAND

Report issued Page 1 of 49

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CONTENTS

Page

COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS ...... 4

QUALITY ASSURANCE STATEMENT ......

RESPONSIBLE PERSONNEL ......

SUMMARY......

INTRODUCTION ...... :......

TEST SUBSTANCE..;...... 10

EXPERIMENTAL PROCEDURE ...... 11

ASSESSMENT OF RESULTS...... 15

MAINTENANCE OF RECORDS...... : 16

RESULTS ...... 17

CONCLUSION ...... 18

REFERENCES ...... 19

TABLES

1. Summary of results and statistical analysis...... 20

2. Results for individual animals ...... 21

3. Animal bodyweights...... 23

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Page

APPENDICES

I. Micronucleus test - Clinical signs and mortalities ...... 25

2. Historical vehicle control values ...... 27 . . .. 3. Historical positive control values ...... 28

4. Administration of Dimethyl Glutarate by inhalation to rats ...... 29

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COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS

The study described in this report was conducted in compliance with the following Good Laboratory Practice standards and I consider the data generated to be valid.

The United Kingdom Good Laboratory Practice Regulations 1999 (Statutory Instrument No 3 106).

OECD Principles of Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM(98)17.

EC Commission Directive 199911I/EC of 8 March 1999 (Official Journal No L 77/8).

US EPA, (TSCA), Title 40 Code of Federal Regulations Part 792, 1989.

The expiry date of the test substance was the Sponsor's responsibility.

_I_-" M~,~ \ Christinee E. Mason, B.Sc. (Hons.), Date Study Director, Huntingdon Life Sciences Ltd.

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QUALITY ASSURANCE STATEMENT

The following inspections and audits have been carried out in relation to this study,

Study Phase Date of Inspection Date of Reporting

Protocol Audit 1 December 2000 1 December 2000

Study Based Inspections Terminal procedures 20 December 2000 2 1 December 2000 Preparation of bone marrow smears 20 December 2000 21 December 2000 Study documentation 2 1 December 2000 21 December 2000

Process Based Inspections Housing and environment 2 October 2000 13 October 2000 Test substance control 2 October 2000 13 October 2000 Exposure 2 October 2000 13 October 2000 Sampling 2 October 2000 I3 October 2000 Clinical signs 2 October 2000 13 October 2000 Bodyweights 6 October 2000 13 October 2000 Feeding 6 October 2000 13 October 2000 Records 6 October 2000 13 October 2000 Slide scoring 10 January 2001 10 January 200 1

Report Audit 14 May 2001 14 May 2001

Protocol Audit: An audit of the protocol for this study was conducted and reported to the Study Director and Company Management as indicated above.

Study Based Inspections: Inspections and audits of phases of this study were conducted and reported to the Study Director and Management as indicated above.

Process Based Inspections: At or about the time this study was in progress, inspections of routine and repetitive procedures employed on this type of study were carried out. These were conducted and reported to appropriate Company Management as indicated above.

Report Audit: This report has been audited by the Quality Assurance Department. This audit was conducted and reported to the Study Director and Company Management as indicated above.

The methods, procedures and observations were found to be accurately described and the reported results of this study to reflect the raw data.

4-c rS M ,9D 0 1 s, B.Sc., M.Sc., &,h. Date Group MaMer, Department of Quality Assurance, Huntingdon Life Sciences Ltd.

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RESPONSIBLE PERSONNEL

Christine E. Mason, B.Sc.(Hons.), Study Director, Department of Genetic Toxicology.

Lincoin Pritchard, B.Sc.(Hons.): Study Supervisor, Department of Genetic Toxicology.

Terry Kenny, B.Sc.(Hons.), Senior Study Manager, Department of Toxicologicai Sciences.

Stuart Cracknell, B.Sc. (Hons.), Aerosol Technologist, Department of Toxicological Sciences.

Graham F. Healey, B.Sc., M.Sc., A.R.C.S., Head of Department, Department of Statistics. SOA 002/00485 1

SUMMARY

This study was designed to assess the potential induction of micronuclei by Dimethyl glutarate in bone marrow cells of the Fischer 344 rat. Animals were treated for two six hour periods of whole body inhalation exposure, to the test substance at dose levels of 0.5, 1.0 and 2.0 mg/l expressed in terms of the weight of test substance per unit volume of chamber air.

The test substance and negative control were administered by whole body exposure. The negative control group received clean air only. A positive control group was dosed orally, by gastric intubation, with cyclophosphamide at 20 mg/kg bodyweight.

Incidences of weight loss were recorded throughout the test but these were small and not considered to be significant. Clinical signs for animals treated with Dimethyl glutarate included partially closed eyes, smacking mouth, lethargy, piloerection, oily fur and brown staining around snout and jaws. No adverse clinical signs were obtained for the negative control treated animals over the duration of the test.

Bone marrow smears were obtained from five male and five female animals in the negative control and each of the test substance groups 24 hours after completion of the second exposure period and from the positive control group 24 hours after dosing. One smear from each animal was examined for the presence of micronuclei in 2000 immature erythrocytes. The proportion of immature erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated mature erythrocytes was also kept.

No statistically significant increases in the frequency of micronucleated immature erythrocytes and no substantial decrease in the proportion of immature erythrocytes were observed in rats treated with Dimethyl glutarate and killed 24 hours after two six hour periods of whole body inhalation exposure, compared to vehicle control values (P>O.OI in each case).

The positive control compound, cyclophosphamide, produced large, highly significant increases in the frequency of micronucleated immature erythrocytes (P

It is concluded that Dimethyl glutarate did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered by whole body inhalation exposure in this in vivo test procedure.

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INTRODUCTION

The purpose of this study was to assess the potential of Dimethyl glutarate to induce mutagenic effects in rats following whole body exposure using an in vivo cytogenetic system (Boiler and Schmid 1970, MacGregor et a1 1987, Mavournin et al 1990). The inhalation route was selected for use in this test as the most likely route of human exposure.

The procedures used were based on the recommendations of the following guidelines:

OECD Guideline for the Testing of Chemicals. (1997) Genetic Toxicology: Mammalian Erythrocyte Micronucleus Test, Guideline 474. EC Commission Directive 2000/32/EC Annex 4C - B.12. Mutagenicity - In vivo mammalian erythrocyte micronucleus test. No. L 136/50. US EPA (1998) Health Effects Test Guidelines. OPPTS 870.5395 Mammalian erythrocyte micronucleus test. EPA 7 12-C-98-226.

The bone marrow micronucleus test, originally developed by Matter and Schmid (1971), is a widely employed and internationally accepted short-term assay for identification of genotoxic effects (chromosome damage and aneuploidy) associated with mutagens and carcinogens (Mavournin et al 1990). This in vivo system allows consideration of various factors including pharmacokinetics, metabolism and DNA repair which cannot be accurately modelled in an in vim system. Young adult rats are chosen for use because of the high rate of cell division in the bone marrow, because of the wealth of background data on this species, and because of their general suitability for toxicological investigations.

In mitotic cells in which chromosomai 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 (Howell-Jolly bodies) in the cytoplasm of these cells. Micronuclei are seen in a wide variety of cells, but erythrocytes are chosen for examination since micronuclei are not obscured by the main nucleus and are therefore easily detected in this cell type (Boller and Schmid 1970).

Micronucleated immature erythrocytes appear in the bone marrow approximately 24 hours after induction of chromosome damage. These immature erythrocytes can be differentiated by a variety of staining techniques which rely on their relatively high content of residua1 RNA. Using the Feulgen/H&E method, they stain slate-blue while mature erythrocytes (which contain little RNA) are counterstained orange. An increased incidence of micronucleated immature erythrocytes is indicative of recent exposure to a chromosome-damaging agent. A simultaneous marked increase in the incidence of micronucleated mature erythrocytes is not expected and may be indicative of micronucleus-like artifacts (Schmid 1976).

Substances which interfere with the mitotic spindle apparatus will cause non-disjunction (unequal separation of the chromosomes at anaphase resulting in aneuploidy) 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.

:8: SOA 002/00485 1 immature erythrocytes; to compensate for this, peripheral blood is shunted into the bone marrow (von Ledebur and Schmid 1973). If the proportion of immature erythrocytes is found to be significantly less than the controi value, this is taken as being indicative of toxicity.

The protocol was approved by Huntingdon Life Sciences Management on 25 February 2000, by the Sponsor on 18 May 2000 and by the Study Director on 22 November 2000.

The in-life phase of the study and statistical analysis were performed at the Department of Inhalation Toxicology and the Department of Statistics, Huntingdon Life Sciences Ltd., Huntingdon, Cambridgeshire, PE28 4HS, England. Slide scoring and reporting were performed at the Department of Genetic Toxicology, Huntingdon Life Sciences Ltd., Eye, Suffolk, IP23 7PX, England.

Experimental start date (analytical procedure): 6 December 2000

Experimental completion date (slides decoded): 30 January 200 1

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TEST SUBSTANCE

Identity: Dimethyl glutarate

Appearance: Clear liquid

Storage conditions: Room temperature, in the dark

Batch number: H93 1363-A

Expiry date: Sponsor's responsibility; sufficiently stable for the duration of the study

Purity: 99.61%

Date received: 16 October 2000

SuppI ier: Dupont

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

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EXPERIMENTAL PROCEDURE

ANIMALS

All animals in this study were Fischer 3.44 outbred albino rats between six and eight weeks old, males weighing between 152.1 and 173.6 grams and females weighing between 125.7 and 148.6 gams on despatch from Charles River UK Ltd, Margate, Kent, England,

On arrival the weight of the animals was checked and found to be acceptable. The animals were randomly assigned to groups by random selection from the container and uniquely tail marked. Each group was kept, with the sexes separated, in cages and maintained in a controlled environment, with the temperature set at 21k2'C and relative humidity set at 5M10%. The humidity range throughout the study was 37-73% which fell outside the protocol stated range of 50 *IO%, no adverse reactions were recorded for any animals and this deviation was not considered to have affected the integrity of the study.

The room was illuminated by artificial light for 12 hours per day. Animals were provided with 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 except diet and water were withheld during the period of inhalation exposure. Food and tap water are routinely analysed for quality at source. Dietary contaminants are not suspected of having any significant effect on parameters measured in this test in this laboratory at any time over the last ten years. All animals were acclimatised for a minimum of 12 days and examined daily.

POSITIVE CONTROL COMPOUND

Cyclophosphamide, obtained from Sigma Chemical Co Ltd, batch number 87'110207; was used as the positive control compound. It was prepared as a solution in purified water, batch number 00101B26, at a concentration of 2 mg/ml just prior to administration.

TREATMENT PROCEDURE AND ATMOSPHERE ANALYSIS

Full details of the treatment procedure and atmosphere analysis are presented in Appendix 4 (Administration of Dimethyl glutarate by inhalation to rats).

DATES OF DOSING

Micronucleus test: 1 st exposure: 18 December 2000 2nd exposure: 19 December 2000

MICRONUCLEUS TEST

All animals were weighed prior to the first exposure period and sacrifice. Animals in the negative and test substance groups were treated by inhalation exposure for two six hour periods on consecutive days. Animals in the positive control group were treated once orally by intragastric gavage using a dose volume of 10 mI/kg and at a time consistent with the end of the second exposure period for the negative control and test substance groups.

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The experimental design is shown below:

Group Treatment Exposure Number of rats (m$l> Male Female 1 Air 5 5

2 Dimethyl glutarate . 0.5 5 5 3 Dimethyl glutarate I .O 5 5 4 Dimethyl glutarate 2.0 5 5

5 Cyclophosphamide 20 (mgkg) 5 5

ROUTE OF ADMINISTRATION The inhalation route was selected as the possible route of accidental exposure in man.

Exposure levels: The exposure levels were selected in consultation with the Sponsor taking account of all available data, including the results of preliminary studies, any acute inhalation and repeat dose inhalation toxicity study in rats.

Groups and exposure levels: The allocation of animals to the experimental groups, group numbers and exposure levels are shown above.

Exposure: The test atmospheres for Groups 2-4 were administered by inhalation as described beIow.

Exposure duration: Two six-hour exposures on consecutive days were used for all animals in the negative control and treatment groups.

Exposure chambers: Animals were exposed in whole-body exposure chambers constructed from stainless steel and glass. The internal volume of each chamber was approximately 750 litres.

Air was introduced into each exposure chamber at a total rate of 150 litres per minute.

The flow through each chamber was approximately 12 air changes an hour; normally sufficient to maintain oxygen concentration above 19% vh, temperature approximately 22 (f 1) OC and relative humidity between 40-60%.

The exposure chamber was maintained 1-10 mm H,O below ambient pressure.

Animals were housed singly in stainless steel mesh compartments during exposure.

Generation of test atmosphere: The liquid test material was delivered to a concentric jet atomiser and generated as dropiets into a stream of dry air for administration to the rats by inhalation while held in whole body exposure chambers. The target chamber concentrations were achieved by metering the test substance from polypropylene syringes mounted on syringe drivers. The atmospheres produced by the atomisers were further diluted with air to give the finaI chamber concentrations.

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PROCEDURE

The animals were removed from their cages and placed into the chamber appropriate to the treatment

Lcroup. A separate chamber was used for each group. The air supplies to the generators and chambers were turned on.

With the animals in position in the chamber. exposure commenced from the moment when generation . commenced. Operation of the exposure system was monitored frequently during the period of exposure.

During exposure the chamber atmosphere was sampled to determine the concentration of test material on at least 3 occasions during each exposure.

On completion of the 6-hour exposure period, the generation system was switched off, and the air supplies disconnected. The system was allowed to clear for approximately 25 minutes before the animals were removed from the chamber and returned to their holding cages.

The nominal rate of aerosol production was calculated by recording the amount of test substance delivered to the generation system during the exposure. The usage over the six hours exposure was divided by the total airflow through the chamber. Any losses during the generation process were quantified and included in the calculation of the nominal concentration.

The control animals were exposed to clean air only in an identical exposure chamber to that used for the test groups.

MONITORING OF CHAMBER CONDITIONS Method of analysis of the test substance in air: By chemical analysis of samples of chamber air.

Airflow: The total airflow through the chamber was 150 litreshinUte and a11 airflows were monitored continuously using rotameters. The airflow was recorded at 30-minute intervals.

Chamber temperature and relative humidity: Chamber temperature and calculated relative humidity were monitored continuously using a wet/dry bulb hygrometer and recorded at 30-minute intervals.

PREPARATION OF BONE MARROW SMEARS Following dosing, the animals were examined regularly by visual assessment and any mortalities or clinical signs of reaction were recorded. Five males and five females were sacrificed from the negative control and each of the test substance groups 24 hours after completion of the second exposure period. The positive control group was sacrificed 24 hours after dosing.

The animals were killed by cervical dislocation following carbon dioxide inhalation and both femurs dissected out from each animal. The femurs were cleared of tissue 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 10 ml Hanks, balanced salts solution by aspiration through a 2 1 g needle fitted to a syringe. The resulting cell suspensions were centrifuged at 1000 rpm (1 50 x g) for 5 minutes and the supernatant discarded. Each resulting cell pellet was resuspended in 2 ml of filtered foetal calf serum before being sedimented by centrifugation. The supernatant was discarded and 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). Several smears were prepared from each femur.

Due to the presence of mast cell granules in rat bone smears, which appear identical to micronuclei when stained using the Romanowsky methods. a modified Feulgen staining method is employed for

13 : SOA 002/00485 1 the rat micronucleus test in this laboratory. This method specifically stains DNA-containing bodies deep purple while leaving mast cell granules unstained. The method also allows reasonable differentiation of mature and immature erythrocytes and produces permanent preparations.

The slides were fixed and stained as described in the following schedule:

1. Fixed for 10 minutes in SLR grade methanol. 2. Hydrolysed in Bouin's fluid at room temperature for 30 hours. 3. Washed three times in purified water (5 minutes per wash). 4. Stained in Schiff s reagent for one hour at room temperature. 5. Washed three times in purified water (5 minutes per wash). 6. Counter-stained for ten minutes in very dilute (approximately 0.06 g/l) aqueous Eosin yellowish. 7. Washed for five minutes in purified water. 8. Stained for 30 minutes in Mayer's Haemalum diluted 9 volumes: 1 volume with aqueous acridine orange solution in purified water (lmgml). 9. Rinsed in purified water. 10. Rinsed in running tap water. * 1 1. Washed for 5 minutes in purified water. 12. Air-dried. 13. Slides were mounted with coverslips using DPX mountant. 14. The mountant was allowed to air dry until hardened.

&Y? All stains and Bouin's fluid were filtered immediately prior to use to remove particulate material.

The stained smears were examined (under code) by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal. One smear per animal was examined. The remaining smears were held temporarily in reserve in case of technical problems with the first smear.

Micronuclei are identified by the following criteria:

Large enough to discern morphological characteristics Should possess a generally rounded shape with a clearly defined outline Should be deeply stained and similar in colour to the nuclei of other cells - not black Should lie in the same focal plane as the cell Lack internal structure, ie they are pyknotic There should be no micronucleus-like debris in the area surrounding the cell The proportion of immature erythrocytes for each animal was assessed by examination of at least 1000 erythrocytes. A record of the number of micronucleated mature erythrocytes observed during assessment of this proportion was also kept as recommended by Schmid (1 976).

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ASSESSMENT OF RESULTS

0 They are suited to analysis of data consisting of discretelinteger values with ties such as the incidence of micronucleated immature erythrocytes.

0 The methods make few assumptions about the underlying distribution of data and therefore the values do not require transformation to fit a theoretical distribution (where data can be approximately fitted to a normal distribution, the results of non parametric analysis and classical analysis of variance are very similar).

' 'Outliers' are frequently found in the proportion of immature erythrocytes for both control and treated animals; non-parametric analysis based on rank does not give these values an undue weighting. Unless there is a substantial difference in response between sexes (which occurs only rarely) results for the two sexes are combined to facilitate interpretation and maximise the power of statistical analysis.

For incidences of micronucleated immature erythrocytes, exact one-sided p-values are calculated by permutation (StatXact, CYTEL Software Corporation, Cambridge, Massachussens). Comparison of several dose lev& is made with the concurrent control using the Linear by Linear Association test for trend, in a step-down fashion if significance is detected (Agresti et al. 1990); for individual inter group comparisons (ie the positive control group) this procedure simplifies to a straightforward permutation test (Gibbons 1985). For assessment of effects on the proportion of immature erythrocytes, equivalent permutation tests based on rank scores are used, ie exact versions of Wilcoxon's sum of ranks test and Jonckheere's test for trend.

A positive response is normally indicated by a statistically significant dose-related increase in the incidence of micronucleated immature erythrocytes for the treatment group compared with the concurrent control group (PO.O 1) 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 dose-related decrease in the proportion of immature erythrocytes (P

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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 after the periods stated in Huntingdon Life Sciences, Standard Operating Procedures.

All other samples and specimens and all raw data will be retained by Huntingdon Life Sciences in its archive for a period of five years from the date on which the Study Director signs the final report. After such time, the Sponsor will be contacted and his 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.

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RESULTS

CHAMBER ATMOSPHERE GENERATION

Analysed concentrations were in general agreement with target concentrations although slightly greater variation was seen between individual sample values than is normally found. The exposure mean concentrations of Dimethyl glutarate were 0.545, 0.807 and 2.298 mg/l compared with target levels of 0.5, 1.0 and 2.0 mg/l respectively expressed in terms of the weight of test substance per unit volume of atmosphere. Full details of the results of analysis are presented in Appendix 4.

MICRONUCLEUS TEST The highest dose level selected for the test by the Sponsor was 2.0 mg/l and although the maximum tolerated dose as evidenced by clinical signs was not achieved, a higher concentration would have produced higher concentrations of droplets to vapour. In addition the respirability of the droplet aerosol, i.e. particles less than 7 pm, would decrease and the mass median aerodynamic diameter of the aerosol would decrease, resulting in the dosage administered to the animal being reduced.

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 and animal bodyweight data in Table 3. Appendix 2 summarises the vehicle control micronucleated polychromatic erythrocyte counts obtained in previous, unrelated experiments. Appendix 3 summarises the corresponding values obtained for the positive control in previous, unrelated experiments.

CLINICAL SIGNS AND MORTALITIES

No mortalities were obtained in the micronucleus test. Clinical signs for animals treated with the test substance are detailed in Appendix 1. Signs included, partially closed eyes, smacking mouth, lethargy, piloerection, oily fur and brown staining around snout and jaws. No adverse clinical signs were obtained for the negative control treated animals over the duration of the test. Incidences of weight loss were recorded throughout the test but these were small and not considered to be significant.

MICRONUCLEATED IMMATURE ERYTHROCYTE COUNTS (MIE)

The test substance did not cause any statistically significant increases in the number of micronucleated immature erythrocytes [P>O.Ol]. Cyclophosphamide caused large, highly significant increases in the frequency of micronucleated immature erythrocytes [P

MICRONUCLEATED MATURE ERYTHROCYTES (MME)

The test substance did not cause any substantial increases in the incidence of micronucleated mature erythrocytes.

PROPORTION OF IMMATURE ERYTHROCYTES ('10IEAE + ME)

The test substance failed to cause any significant decreases in the proportion of immature erythrocytes [P>O.O 13.

Cyclophosphamide did not caused any statistically significant decreases in the proportion [P

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CONCLUSION

No statistically significant increases in the frequency of micronucleated immature erythrocytes and no substantial decrease in the proportion of immature erythrocytes were observed in animals treated with Dimethyl glutarate for two six hour periods of whole body inhalation exposure, compared to negative control values (P>O.Ol in each case).

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REFERENCES

AGRESTI, A., MEHTA, C.R. and PATEL, N.R. (1990) Exact inference for contingency tables with ordered categories. Journal of the American Statistical Association, 85,453.

BOLLER, K. and SCHMID, W. (1 970) 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 3for Windows:Statistical Softwayefor Exact Nonparametric Inference. Cytel Software Corporation, NC, USA.

GIBBONS, J.D. (1 985) Nonparametric Statistical Inference, 2nd edition, Marcel Dekker, New York.

JONCKHEERE, A.R (1954) A distribution-free k-sample test against ordered alternatives. Biometrics, 41, 133-145.

KRUSKAL, W.H. and WALLIS, W.A (1952) Use of Ranks in One-Criterion Variance Analysis. Journal of the American StatisticaI Association, 47, 583-621.

KRUSKAL, W.H. and WALLIS, W.A (1953) Errata for Kruskal-Wallis (1952). Journal of the American Statistical Association, 47, 583-62 1.

MacGREGOR, J.T., HEDDLE, J.A., HITE, M., MARGOLIN, B.H., RAMEL, C., SALAMONE, M.F., TICE, R.R. and WILD, D. (1987) Guidelines for the conduct of micronucleus assays in mammalian bone marrow erythrocytes. Mutation Research. 189, 103.

MATTER, B. and SCHMID, W. (1971) Trenimon-induced chromosomal damage in bone marrow cells of six mammalian species, evaluated by the micronucleus test. Mutation Research, 12, 4 17. 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. (I 995) High resolution rodent bone marrow micronucleus assays of 12-dimethylhydrazine : implication of systemic toxicity and individual responders. Mutagenesis, 10, 129.

SAS INSTITUTE (1989) SASSTAT User's Guide, Version 6, Fourth Edition, Vol.2. SAS lnstitute Inc., Cary, NC, USA.

SCHMID, W. (1976) The micronucleus test for cytogenetic analysis. In: HOLLANDER, A. (ed.) Chemical Mutagens, Principles and Methods for their Derection, Vol. 4, p.3 I. Published by Plenum Press. New York. von LEDEBUR, M. and SCHMID. W. (197;) The micronucleus, test. Methodological aspects. ,klutation Research. 19, 109.

WILCOXON, F. (1945). Individual comparisons by ranking methods. Biometrics Bulletin, 1, 80-83.

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TABLE 1

Summary of results and statistical analysis

Sampling Treatment Exposure level % ie/(ie+me) t Incidence mie Incidence mme time (mm (mean) (group mean) 54 Hours a Negative control 31 0.1 0.6 Dimethyl glutarate 0.5 32 0.4 0.0 1.o 31 0.4 0.3 2.0 32 0.2 0.6 24 Hours Cyclophosphamide 20 mg/kg 27 5.9*** 0.0 a 24 hours after completion of the second exposure (48 hours from completion of first exposure) % ie/(ie+me) Proportion of immature erythrocytes mie Number of micronucleated cells observed per 2000 immature erythrocytes examined mme Number of micronucleated cells calculated per 2000 mature erythrocytes

Results of statistical analysis using the appropriate nonparametric method of analysis based on permutation (one-sided probabilities): *** P < 0.001 (highly significant) otherwise P > 0.01 (not significant)

5- Occasional apparent errors of i 1% may occur due to rounding of values for presentation in the table

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TABLE 2

Results for individua. animals

Treatment Exposure level Animal ie YOie/(ie+me) Incidence me Incidence (mg/l) number mie mme Jegative- control - M201 267 I' 25 0 818 0 M 202 399 40 0 60 1 0 M 203 453 43 0 594 0 M 204 387 37 0 672 0 M 205 387 38 0 622 0 F 206 225 22 0 80 1 1 F 207 279 27 0 772 0 F 208 31 1 30 1 714 0 F 209 248 24 0 787 1 F 210 26 1 25 0 764 0 Dimethyl 0.5 M 211 328 32 0 682 0 glutarate M 212 37 1 35 2 70 1 0 M 213 297 28 0 748 0 M 214 340 33 0 677 0 M 215 428 42 1 602 0 F 216 275 26 0 779 0 F 217 388 38 1 64 1 0 F 218 273 27 0 749 0 F 219 324 32 0 689 0 F 220 26 1 25 0 777 0 Dimethyl 1.o M 221 270 27 0 732 0 glutarate M 222 303 30 1 70 1 0 M 223 352 35 1 664 1 M 224 285 27 0 762 0 M 225 304 30 1 705 0 F 226 283 28 0 727 0 F 227 305 30 0 716 0 F 228 327 33 0 678 0 F 229 374 36 0 664 0 F 230 325 32 ' 1 68 I 0 Dimethyl 2.0 M 231 371 36 I 666 0 gi utarate M 232 33 1 32 0 692 0 M 233 357 35 0 662 0 M 234 29 1 29 0 708 0 M 235 394 38 0 655 0 F 236 292 26 0 826 0 F 237 276 28 0 727 0 F 238 313 30 0 72 1 1 F 239 354 34 0 690 0

F 240 292 29 ' 1 730 1 ie Number of immature erythrocytes observed per 1000 cells scored ?IO ie/(ie+me) Proportion of immature erythrocytes in ie Number of micronucleated cells observed per 2000 immature erythrocytes me Total number of mature erythrocytes recorded per 1000 cells scored mme Number of micronucleated mature erythrocytes observed

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TABLE 2 (continued)

Results for individual animals

mdkg number mie mme CP 20 M 241 262 26 9 763 0 M 242 392 37 7 669 0 M 243 293 29 5 722 0 M 244 378 37 4 636 0 M 245 227 22 6 826 0 F 246 200 20 3 800 0 F 247 26 1 25 6 802 0 F 248 234 22 4 815 0 F 249 267 27 6 740 0 F 250 243 24 9 775 0 CP Cyclophosphamide ie Number of immature erythrocytes observed per 1000 cells scored % ie/(ie+me) Proportion of immature erythrocytes mie Number of micronucleated cells observed per 2000 immature erythrocytes me Total number of mature erythrocytes recorded per 1000 cells scored mme Number of micronucleated mature erythrocytes observed

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TABLE 3 Animal bodyweights Animal Treatment Exposure Bodyweight (g) number (mg/l) Allocation to study At start of first At termination exposure period Individual Mean Individual Mean Individual Mean isd Isd t sd 20 1 M Negative control - 201 20357 218 22059 217* 221 I9 202 M 197 213 214 203 M 196 21 1 213 204 M 213 232 233 205 M 207 228 229 206 F 152 148 t 3 155 151 I2 153* 150 I3 207 F 150 151 152 208 F 147 149 147* 209 F 145 151 148* 210 F 145 150 150 21 1 M Dimethyl 0.5 216 207 I 7 237 226 3t 8 232* 222 I8 212 M glutarate 209 228 225* 213 M 205 22 1 213* 214 M 208 228 224* 215 M 197 217 214* 216 F 141 146 I4 152 152 I5 147* 148 i6 217 F 150 159 154* 218 F I43 146 139* 219 F 147 149 147* 220 F 149 156 151* 22 1 M Dimethyl 1.o 203 202 I5 219 221 14 214* 215 i3 222 M glutarate 204 223 218* 223 M 209 227 220* 224 M 200 22 1 213* 225 M 195 215 212* 226 F 141 147 2 5 148 152 2 5 145* 148 f 4 227 F 149 151 148* 22 8 F 142 149 143* 229 F 149 154 149* 230 F 153 I60 I54* 23 I M Dimethyl 2.0 20 1 199 I7 219 218 k 5 212* 208 I6 232 M glutarate I89 210 I99* 233 M 194 218 206* 234 M 206 220 212* 235 M 203 224 213* 336 F 151 142 i5 154 148 k 4 145* 139 I 3 237 F 141 148 138* 238 F 140 147 l38* 25 9 F 139 144 140* 240 -F 139 146 136* sd Standard deviation * Denotes weight loss from previous weighing

23 SOA 002/004851

TABLE 3 - continued Animal bodyweights

I Animal Treatment Exposure Bodyweight (g) number (mgkg) Allocation to study At treatment At termination day 1 Individual Mean Individual Mean Individual Mean 5 sd 2 sd 5 sd 241 M CP 20 20 1 20725 216 22455 216 225 5 6 242 M 206 227 228 243 M 206 222 220' 244 M 214 229 230 245 M 208 228 229

246 F 143 14654 148 153+4 146* 15214 247 F I43 153 152* 248 F 146 151 151 249 F 152 156 155* 250 F 148 157 157

CP Cyclophosphamide sd Standard deviation * Denotes weight loss from previous weighing

: 24 : SOA 002/004851

APPEM)M 1

Micronucleus test - Clinical signs and mortalities (1st exposure period)

Treatment Dimethyl glutarate Exposure 0.5 1 .o 2.0 (mg/l) Approx. M F M F M F time (hr : min) 0 :30 0 0 PC, SM PC, SM PC, SM PC, SM

1 :o 0 0 PC, SM PC, SM PC, SM, L PC, L, SM

2:o 0 0 PC, L PC, L PC, L PC, L

3 : 30 0 0 PC, L PC, L PC, P, L PC, L, P

5:0 PC PC PC, L PC, L PC, P, L PC, L, P

6:O PC PC PC, L PC, L PC, P, L PC, L, P

6 : 36 OF, BS OF, BS

6 : 39

6:41 0 0

6 : 52 0 0

6 : 53

6 : 54 OF,BS OF,BS

Mortalities 015 015 015 015 015 015

Approx. time: Calculated from start of commencement of generation for first exposure period.

NB No adverse clinical signs were noted for the vehicle control group throughout the experiment.

0 No reaction detected.

Type of reaction: BS Brown staining round snout. L Lethargic. OF Oily fur, P Piloerection, PC Partially closed eyes. SM Smacking mouth.

Clinical signs shown refer to all animals within that dose group and sex; except where x(...), x denoting the number of animals displaying the clinical sign(s) within the brackets

: 25 : SOA 002/00485 1

APPENDIX 1 (continued)

Micronucleus test - Clinical signs and mortalities (2nd exposure period)

Treatment I Dimethyl glutarate Exposure 0.5 1.o 2.0 (mg/l) Approx. M F M F M F time (hr : min) 0 : 30 0 0 PC PC PC PC

1 :30 0 0 PC PC PC, L PC, L

2:30 0 0 PC, L PC, L PC, L PC, L

3:o , 0 0 PC, L PC, L PC, L, P PC, L, P 5:O L L PC, L PC, L PC, L, P PC, L, P

6:O L L PC, L PC, L PC, L, P PC, L, P

6 : 38 0 0

6:39

6:41 OF,BS OF,BS Mortalities I 06 015 1 015 015 I 015 015

Approx. time Calculated from start of commencement of generation for second exposure period.

NB No adverse clinical signs were noted for the vehicle control group throughout the experiment.

0 No reaction detected.

Type of reaction: BS Brown staining around snout and jaws, OF Oily fur, L Lethargic, P Piloerection, PC Partially closed eyes

Clinical signs shown refer to all animals within that dose group and sex, except where x(...), x denoting the number of animals displaying the clinical sign(s) within the brackets

26 : SOA 002/004851

APPENDIX 2

Historical negative control values (1 February 1999 - 31 January 2001) Frequency of m icronucleated immature erythrocytes (individual animals)

0 1 2 3 4 5 6 7 8 Number of micronucleated immature erythrocytes (MIEper 2000) Individualmean 0.94

Historical negative control values (1 February 1999 - 31 January 2001) Frequency of m icronucleated immature erythrocytes (Group mean values)

‘Of35 30 25 (u 0 E 20 L.E 15 i; 10 5 0 0.0-0.2 0.3-0.5 0.6-0.8 0.9-1.1 1.2-1.4 1.51.7 1.8-2.0 2.1-2.3 2.4-2.6 Num ber of micronucleated immature erythrocytes (MIE per 2000) Group man 0.71

27 : SOA 002lOO4851

APPENDIX 3

Historical positive control vaiues (I February 1999 - 31 January 2001) Frequency of micronucleated immature erythrocytes (Individual animals) Cyclophosphamide at 20 qlkg

30 s 25 20 E 15 E3 V 5 10 5

0 0-20 21-40 41-60 61-80 81-100 101-120 121-140 Number of micronucleated immature erythrocytes (MIEper 2000 cells) Individual mean 48.03

Historical positive control values (1 February 1999 - 31 January 2001) Frequency of m icronucleated immature erythrocytes (Group mean values) Cyclophosphamide at 20 mglkg

40 35 30 1 E 25 0, V g 20 L 5 15 i; 10 5 0 0-10 11-20 21-30 31-40 41-50 51-60 61-70 71-80 81-90 91-100 Nurn ber of micronucleated immature erythrocytes (MIEper 2000) Group mean 49.2

: 28 : i SOA 002/00485 1

APPENDIX 4

ADMINISTRATION OF DIMETHYL GLUTARATE

BY INHALATION TO RATS

Author

S Cracknell

: 29 : SOA 002/003851

APPENDIX 4 (continued)

TEST SUBSTANCE AND ADMINISTRATION

TEST SUBSTANCE

The test substance, which was also identified as Dimethyl glutarate (batch number H93 1363-A), was supplied as a clear liquid in a giass container (net contents 3931 g) and was received at these laboratories on 16 October 2000. Information from the Sponsor indicated that the test substance was of 99.61% purity and was sufficiently stable for use in this study. The Dimethyl glutarate was administered to the rats as a liquid droplet aerosol.

ADMINISTRATION

The liquid test material was delivered to a concentric jet atomiser and generated as droplets into a stream of dry air for administration to the rats by inhalation while held in whole body exposure chambers. The target chamber concentrations were achieved by metering the test substance from polypropylene sdyringesmounted on syringe drivers. The atmospheres produced by the atomisers were further diluted with air to give the final chamber concentrations.

The flow of air to the atmosphere generation systems and the diluent air supply were caIibrated using a precision-made tapered glass tube flowmeter (generation air) or a dry type gas meter (diluent air) during the preliminary phase of the study. Airflow through the generation systems was monitored throughout each of the exposures using in-line tapered tube gas flowmeters.

The settings of the test substance metering system required to obtain the target chamber concentrations were determined during the preliminary phase of the study, based on the Gas Chromatographic analysis of atmosphere samples.

Animals assigned to the control group received an exposure to compressed air only, from the same source as used for the generation of the test atmospheres. All animals were exposed for 6 hours daily on two consecutive days.

The usage of Dimethyl glutarate was determined, for each day of treatment, for each of the three test groups.

EXF'QSURE SYSTEM

Each exposure system comprised a 0.75 m3 whole body inhalation exposure chamber, a stainless steel concentric jet atomiser, a syringe driver, a glass elutriator column, diluent air control valves, a compressed -air supply and control valves to each generator and in-line airflow monitoring flowmeters. During periods of atmosphere generation, the test material was dispensed from single- use disposable syringes.

Schematic diagrams of the atmosphere generation system and an exposure chamber system are presented in Figures A and B. The component parts of the systems are described in further detail below:

: 30 :

1_- SOA 002/004851

APPENDIX 4 (continued)

Atmosphere generation

Each test group inhalation chamber used on the study was connected to an atmosphere generation system supplying a flow of the appropriate dilution of Dimethyl glutarate droplet aerosol. Each exposure system comprised a whole body inhalation exposure chamber, a concentric jet atomiser, a glass elutriator and a diluent air control system.

The test material delivery system for each group comprised a polypropylene syringe located on a syringe driver (Precidor, Model 5003). The Liquid test material was delivered to the inlet of the concentric jet atomiser via a PolyTetraFluoroEthylene (PTFE) needle. The syringe driver settings required to achieve the target concentrations were established during the preliminary phase of the study. The atmosphere exiting each atomiser was delivered into a glass column with a volume of approximately 11 litres (elutriator) and further diluted with air that entered via a pair of inlet ports adjacent to the atomiser in the base of the unit. The test atmospheres exited the elutriator through a flexible duct located at the centre top of the column and were delivered directly to the top of the whole body exposure chambers.

A compressor supplied air to the atomisers and diluent lines. The air was filtered to remove any residual particulate and was dried (dew point -2T).

The syringes were refilled with Dimethyl glutarate as required during the exposure periods. On each day of exposure, the volume of test material dispensed to each exposure system was determined.

Inhalation chamber

The exposure chambers were of stainless steel and glass construction and consisted of a cuboidal body fitted with a pyramidal base and top. The internal volume of each chamber was approximately 0.75m3. At the apex of the upper pyramidal figure was the tangentially mounted air duct. Immediately below this was a perforated canister, which ensured equal distribution of the test atmosphere within the chamber.

Access to the chamber was through the front of the box section via a hinged door with a glass panel and stainless steel frame. The door was sealed using moulded'rubber sealing strip.

Exposure cages constructed of stainless steel mesh were suspended on a framework arranged on four levels. Each level held four cages, with each cage capable of housing 4 rats individually. This gave a total animal exposure capacity of 64 rats. In this investigation 10 animal compartments were used.

A wet and dry alcohol bulb thermohygrometer, used to monitor chamber teniperature and relative humidity, was suspended in the chamber, visible through the glass panelled door.

The pyramidal base of each chamber was fitted with a 2-inch drain. The drain connected with a common drainage system via a baIl valve.

A square tubular exhaust plenum, 3 inches in diameter and perforated along the ventral surface, was situated in the pyramidal base. This connected to the main extract system.

:;1 : SOA 002/00485 1

APPENDIX 4 (continued)

The total chamber airflow was 150 litredminute. Air entered the chamber through the inlet duct. Diluent air flow was measured using a tapered tube flow meter situated at the front of a purpose-built stainless steel trolley on which the elutriator was mounted. Generation air was measured on a similar flowmeter mounted on the vapour generation trolley.

A Magnehelic pressure gauge (0- 1OOmm water gauge) was connected with each chamber by a nylon tube. This was mounted on the elutriator trolley and was used to monitor the atmosphere pressure inside the chamber, relative to the exposure room.

Extraction of the chambers was accomplished by means of a single fan mounted on the outside wall of the building withdrawing air through a manifold to which all chambers were connected. The chamber air extract was vented to atmosphere via an exhaust stack.

Extract flow was adjusted using gate valves mounted in the extract ducting between the chamber and filters. The internal pressure within each chamber was maintained at approximately -2 to -4 mm H,O below ambient when operational.

The negative control animals were exposed using a similar system to that used for the test groups, but received compressed air only. PROCEDURE

A separate exposure chamber was used for each group. The negative control animals were exposed using an identical exposure chamber to that used for the test groups. The inhalation system was set up as described above and the atomiser, diiuent flows and chamber negative pressure were set. The syringes were filled with Dimethyl glutarate and attached to the syringe pumps.

The rats were removed from their cages and placed in ascending cage sequence from the front left hand side to back right hand side in the individual compartments of the exposure cages. AI1 of the animals were located on level 2 of the chamber with the males on the left side of the chamber.

The chamber doors were closed and secured and the diluent and generator air flows were switched on. The chamber sampling ports were sealed and the Magnehelic pressure gauges checked to ensure that operation of the chamber took place with internal pressure below that of the room.

The syringe pumps were switched on and the exposure start time recorded.

During the exposure (6 hours), samples were taken after approximately 1, 3 and 5 hours to determine the chamber concentration of Dimethyl glutarate. Each sample was removed through a modified blanking plug in a port on the level used to expose the animals.

At the end of 6 hours generation, the syringe drivers supplying test substance were switched off. The generator and diluent airflows were turned off and the chambers allowed to clear. Clearance air was allowed to enter each chamber through an open sampling port in the chamber wall. At the end of the clearance period, the rats were unloaded from the chambers into their respective holding cages. The chambers were then washed to remove animal waste.

: 32 : SOA 002/00485 1

APPENDIX 4 (continued)

TARGET CONCENTRATIONS

The target concentrations of Dimethyl glutarate were as follows:

Group Designation Concentration (mgN 2 (Low dose) 0.5 3 (Inter dose) 1.O 4 (High dose) 2.0

The target concentrations were selected in consultation with the Sponsor, following the review of available data.

EXPOSURE CHAMBER CONDITIONS

Analysis of chamber concentrations of Dimethyl gtutarate

The samples of chamber air were collected by drawing a previously selected volume of chamber atmosphere through a sintered glass bubbler containing 1,4-dioxane as a trapping agent. Sample collection wasperformed by inserting the inlet of the bubbler through a port located at the animal exposure level on the side wall of the chamber. The atmosphere samples were taken at a calibrated flow of 2 litres/minute using a laboratory pump. The air volume of each sample collected was measured using and in-line wet type gas meter. lnvestigations performed during the preliminary phase of the study confirmed satisfactory trapping of the test atmospheres with the equipment employed. Breakthrough to a second bubbler trap in series for a closely related material was found to be 0% (not detected) of the total collected and consequently the use of a single trap for the main study with this material was considered satisfactory.

Air samples were coIIected in sequence from each of the three exposure chambers containing the test atmospheres (chambers 2 to 4 sampled sequentially). The Aerosol Technology and Analysis Section of the lnhalation Studies Group developed the method of analysis and details of the analytical procedures used are given in Appendix A.

Analysis of droplet size distribution of Dimethyl glutarate

A single sample for the determination of the droplet size of Dimethyl glutarate in the chamber atmospheres was taken from each test chamber over the two days of treatment. The atmosphere samples were collected at a calibrated flow of 2 litres/minute using a laboratory pump. The air volume of each sample coilected was measured using an in-line wet type gas meter.

Air sample collection

The air samples were collected in sequence from chambers 2 to 4.

Airflow

The air flow into each chamber was monitored continuously using tapered tube rotameters and recorded at approximately 30-minute intervals throughout each expo-Sure. A documented check on the chamber negative pressure was also made at 30-minute intervals.

: 33 : SOA 002/00485 1

APPENDM 4 (continued)

Temperature

The wet and dry chamber temperatures in each exposure chamber were recorded at approximately 30-minute intervals during each exposure. The relative humidity (RH%) in each chamber was derived from these data.

CALCULATIONS

Chamber concentration

The mass of test material collection in each sample was determined by Gas Chromatography (GC) and the aerosol concentration derived from the mass of dimethyl glutarate found and the air volume sampled.

Droplet size distribution

The collection characteristics of the Maple sampler under the conditions of use in this study are as fo1lows:

Impactor stage 3 4 5 6 7 8 Filter Cut-point* (pm) 9.8 6.0 3.5 1.55 0.93 0.52 0 * Aerodynamic equivalent particle diameter for spherical particles of unit mass density in air at 25OC and 760 mm Hg

The material collected on each substrate was extracted and quantified by GC.

The particle size of the test aerosol was characterised by linear regression of the probit of cumulative percentage, by mass, of particles smaller than cut-point of each stage versus the logarithm of each stage cut-point. The mass median aerodynamic diameter (MMAD) and geometric standard deviation of the aerosol, together with the percentage of the atmosphere considered to be inhalable to the laboratory rat (< 7 pm equivalent aerodynamic diameter) was derived for each occasion of measurement.

In order to minimise the cumulative errors that result from repeated rounding of numbers, much of the data in this report has been calculated continuously using unrounded numbers and only rounded for printing. Consequently, these rounded numbers will include rounding errors in the last significant figure, possibly leading to small apparent discrepancies with other data in the report.

34 SOA 002100485 I

APPENDIX 4 (continued)

RESULTS

CHAMBER CONCENTPATION

Analysed concentration of Dimethyl glutarate

The data are presented as follows:

Table A Individual and daily mean concentrations

The target and study mean concentrations of Dimethyl glutarate are presented below:

Group Concentration (mg/l) Target Analysed 2 (Low dose) 0.5 0.545 3 (Inter dose) 1.o 0.807 4 (High dose) 2.0 2.298

Analysed concentrations were in general agreement with the target values, however, the achieved chamber concentration to the Intermediate exposure group was lower than expected on both days of treatment. The overall mean achieved concentrations were 109, 8 1 and 1 15% of the target values for Groups 2, 3 and 4, respectively, and the corresponding coefficients of variation for the individual samples, over the two days of treatment, were 19.5, 6.9 and 24.6 YO.It is possible that the presence of animals in the chamber through a combination of the additional surface area provided by the animal’s bodies and the water vapour contributed to the chamber air by the breathing action of the animals increased the degree of vapour condensation and therefore increased the relative proportion of aerosol in the test atmosphere. lmpaction of the aerosol on exposure system surfaces together with aggregation of droplets to a size which was no longer aerostable would reduce the generation efficiency as measured by the nominal to analysed concentration ratio.. In a dynamic system the degree of condensation, aerosol impaction and droplet aggregation will vary with time and this probably accounts for the variation in chamber concentration over the 6 hour exposure period. The reduction of generation efficiency resulted in achieved concentrations at the Intermediate and High level being slightly lower than target levels. However, the differences between analysed and target levels were small and are considered not to have affected the outcome of the study.

Nominal concentration of dimethyl glutarate

The data are presented in Table B and are summarised below:

Group Study mean nominal A/N Concentration (mgll) (Yo) 2 (Low dose) 1.016 53.5 3 (Inter. dose) 3.141 25.6 4 (High dose) 8.062 28.3

Analysed concentration) A/N= x 100 Nominal concentration I SOA 002/004851

The nominal concentrations were calculated from the mass of dimethyl glutarate used during the exposures together with the chamber calibrated airflow (150 l/min) and the period of atmosphere generation (360 min).

Examination of the derived values of analysed to nominal ratio showed that the lower than target achieved concentration for the Intermediate exposure group was associated with a lower than expected efficiency of delivery to this chamber. No explanation was found for this variation from the expected pattern of proportional reduction in efficiency with increasing chamber concentration other than the variation inherent in a dynamic exposure system as detailed above.

PARTICLE SIZE DISTRTBUTION

Results of the particle size determinations are presented in Table C.

Droplet size determination samples collected on one occasion from each test chamber during the study confirmed that there was a high proportion of inhalabie aerosol in the exposure atmospheres. The Mass Median Aerodynamic Diameter showed a trend of increasing droplet size with ascending concentration. This is a common finding in inhalation toxicity studies and is associated with reduced efficiency of generation as the rate of test fluid delivery to the atomiser is increased together with interactions between droplets in the aerosol, which are in closer proximity than occurs at lower concentrations.

CHAMBER TEMPERATURE AND RELATIVE HUMIDITY

The daily mean chamber temperatures and relative humidities are presented in Table D.

The chamber temperatures and humidities were similar for all groups throughout the duration of the study. The relatively low humidity recorded for all groups, on both occasions of exposure, was associated with the supply of dried air to the chambers. The low humidity in the chamber atmospheres had no observable effect on the animals and is not considered to have affected the outcome of the study.

: 36 : SOA 002lOO485 1

APPENDM 4 (continued)

FIGURE A

Schematic diagram of the aerosol generation system

Key a Dispensing syringe b Syringe driver C Test material delivery pipe d Aerosol generation air supply e Aerosol outlet to elutriator f Elutriator vessel g Diluent air supply h Diluent air inlet 1 Aerosol outlet to inhalation exposure chamber

: 37 : SOA 002t00485 1

APPENDIX 4 (continued)

FIGURE B

Schematic of the exposure system used to expose rats

Key A Glass eiutriator G Exhaust plenum B Air flow control and chamber monitoring H Drain C Dispersion device I Gate valve D Exposure chamber (0.75 m3) J Pre-filter E Animal exposure cages K Powered extract filter F Sampling port L Main exhaust SOA 002/00485I

APPENDIX 4 (continued)

TABLE A

Chamber comtrations of Dimethyl glutarate - individual and mean values

wure Sample Chamber concentration Daily mean sd I==-number wmkr identity (mg/l) (mm 2 1 SI 0.440 s2 0.510 0.499 (0.0543) S3 0.547

2 SI 0.476 s2 0.744 0.591 (0.1378) S3 0.554 Mean 0.545 sd 0.1065 cv (Oh) , 19.5 t-y-I I 0.820 s2 I 0.827 I 0.790 (0.0581) S3 0.723 2 I I 0.790 0.788 0.823 (0.0589) I 0.891 0.807

cv (Oh) 6.9 1 SI I .747 I s2 I 1.813 1 1.845 1 (0.1173) 1.975 I s3 I -3 3223 i 2.465 2.75 1 (0.4 11 8) 1 :;S3 2.565 Mean 2.298 sd 0.5653 cv (Yo) 24.6 I

CV Coefficient of variaxion (sd x 1OOimean)

: 39 : SOA 002/00485 1

APPENDIX 4 (continued)

TABLE B

Exposure nominal concentrations of Dimethyl glutarate

Exposure Vol used Wt used Concentration (mg/l) ANratio (ml> (8) Nominal’ Analysed (%) I 51 55.64 1.030 0.499 48.4 2 50 54.55 1.010 0.59 1 58.5 Mean 1.020 0.545 53.5

Exposure Vol used Wt used Concentration (m$l) AM ratio (mu (8) Nominal’ Analysed (”/) 1 159 173.47 3.212 0.790 24.6 2 153 166.92 3.091 0.823 26.6 Mean 3.152 0.807 25.6

Exposure Vol used Wt used Concentration (mg/l) ANratio (mI> (0) Nominal’ Analysed (”/.I 1 395 430.95 7.98 1 1.845 23.1 2 406 442.95 8.203 2.75 I 33.5 Mean 8.092 2.298 28.3

:40 :

_. SOA 002/00485I

APPENDIX 4 (continued)

TABLE C

Droplet size distribution of Dimethyl glutarate - individual sample and stage values

Group YOof total collected on each stage Particle size I number 3 4 5 6 7 8 Filter MMAD c~g YO . I (Pm) < 7pm -7 (Low dose) 0.0 9.1 19.7 31.9 9.1 0.0 30.2 1.4 2.39 96 I i (Interdose) 0.0 91.6 2.2 0.0 0.0 0.0 6.2 4.6 2.55 67 4 (Highdose) 13.3 24.2 45.7 14.9 0.4 0.0 1.5 5.3 2.32 62

:41 : SOA 002/004851

APPENDIX 4 (continued)

TABLE D

Chamber temperature and relative humidity - exposure mean values

Group 1 Group 2 Group 3 GrouDGroup 4 (Air control) (Low(LOW dose) (Inter. Dose) (High dose) Temp I RH Temp 1 RH Temp 1 RH Temp 1 FW 1 20.0 38 20.3 26 20.3 30 19.3 25 2 I 19.5 37 19.9 38 19.9 32 18.9 31 Mean I 19.8 38 20.1 52 20. I 31 19.1 28

42 : SOA 002/00485 1

APPENDIX 4 (continued)

APPENDIX A

COMPOUND SPECIFIC INHALATION ANALYTICAL PROCEDURE FOR DIMETHYL ADIPATE AND DIMETHYL GLUTARATE

The analysis of Dimethyl adipate and Dimethyl glutarate in air sample substrate

The method outlined in this document has been validated and is considered fit for the purpose of monitoring test atmospheres in an Inhalation Toxicology study.

This document details the basic procedures for the analysis of Dimethyl adipate (DMA) and Dimethyl glutarate (DMG) collected using solvent traps (bubblers) and Marple cascade impactors. 1.4-Dioxane is used as the trapping solvent for the analyte. The resulting solutions of approximate concentration 50 to 1000 pg/ml are quantified by GC. Study specific amendments and additions will be detailed within a supplementary document. NOTE Throughout this document, the symbol 5 indicates that the relevant information is included in a Study specific supplement.

1 EFFECTW DATE: 1 8 December 2000

Test substance

Dimethyl adipate, dimethyl hexanedioate, CgH1404, molecular weight 174.2, has the following structure:

Appearance Colourless liquid

Storage Room temperature, dark

Dimethyl glutatrate, dimethyl pentanedioate, C7H 1204, molecular weight 160.2, has the following structure:

Appearance Colourless liquid

Storage Room temperature, dark

: 43 : SOA 002/00485 1

APPI~ND~R4 [eahtihuedj

APPENDIX A - continued

Equipment

Balance and data printer Sartorius WOOD with YDP-02

Dispenser Bibby 2- 1Om1

Diluter Hamilton Microlab 500 Series

Ultrasonic bath Decon FS200b

General laboratory glassware

Consumables

Scintillation vials Packard ca 20 ml capacity Instruments BV

Autosampler vials, septa and Fisher Scientific 2 ml glass vials caps Ltd

Reagents

1,4-Dioxane Sigma Aldrich HPLC grade

Preparation of samples for analysis

Transfer the solution from a bubbler sample to a volumetric flask. Rinse the bubbler with further dioxane and add this to the volumetric flask.

Transfer the sample substrate from the cascade impactor to an appropriately labelled container. A volume of extraction solvent (dioxane) is added immediately using the dispenser, ensuring the substrate is submerged. The container is then sealed. The stainless steel substrates are gently swirled and left to stand for a minimum of IO mins. The filters are placed into an ultrasonic bath on full power for 10 mins. The volumes of extraction solvent added to each sample are as detailed in the study specific supplement.

All samples will be transferred to appropriately labelled scintillation vials prior to storage.

44 SOA 002/00485 I

APPENDIX 4 (continued)

APPENDM A - continued

Preparation of standard solutions '

Weigh approximately 100 mg of Dimethyl adipate and 1OOmg of Dimethyl glutarate (to 0.1 mg) into a votumetric flask (100 ml), dissolve in dioxane (10 ml), mix thoroughly and make up to volume with dioxane to provide standard SI. Using the diluter, prepare standards at the required concentrations, as detailed in the study specific supplement.

Storage of standards and samples

The maximum storage periods for the various sample types are detailed below:

Type Storage conditions Maximum storage period Samples sotutions ca 4OC 7 Days Standard solutions CQ 4% 7 Days

Calibration and quantification

Calibrate by injecting duplicates of each calibration standard solution, as detailed in the study specific supplement, at the beginning of each analytical sequence. Measure the peak area response in each injection of the calibration standard solutions and derive the line of best fit using a 1 /concentration2 weighted least squares method.

For each injection of the sample measure the peak area response and determine the amount present in the sample using the equation below:

(A -1) Amount(pg) = -X v S

Where A = Peak area response of Dimethyl adipate or Dimethyl glutarate in the sample chromatogram S = SIope of calibration iine derived from calibration data I = Intercept of calibration iine derived from calibration data V = Dilution volume of sample (ml); = extraction volume x dilution factor

: 45 : SOA 0021004851

APPENDIX 4 (continued)

APPENDIX A - continued

Chromatographic conditions

Analytical column PE-5 dimethyl silicone FSWCOT 0.25 dF, 5m x 0.25 mm Carrier gas He, 1 mI/min Septum purge 3 ml/min Air 450 ml/min Hydrogen 45 ml/min Injection volume 2 PJ Split vent 20 Split ratio 20: 1 Injector temperature 150°C Column temperature 120°C Detector FID 250"C, range 1, attenuation 1 Retention time Dimethyl giutarate and Dimethyl adipate approximately 0.6 and 0.9 minutes respectively Quality assurance measures

When the method is established on a chromatographic system six injections of a standard will be used to verify performance of the system. The parameters and acceptance criteria are set out below; Parameter Typical value Acceptance criteria DMA DMG Plate count (USP) 12510 8086 >SO% Tailing factor (USP) 1.20 1.25 * 20 Yo Measurement repeatability (n=6) 2.2% 2.5% <5% expressed as coefficient of variation

The highest calibration standard will be compared against a standard of similar concentration prepared independently. The ratio of response factors will be acceptable if within the range 0.95 to 1.05.

: 46 : SOA 002/00485 1

x. APPENDIX 4 (continued)

APPENDIX A - continued

A quality check standard must follow every 6 concentration samples (8 particle size sample stages) for the analysis to be regarded as valid. .The results of the.quality check standards must lie within the QC tolerance limits.

A quality check standard of low concentration will be run to verify the LOQ for the run. The LOQ for the run will be regarded as the concentration of the lowest acceptable quality check standard.

Summary of method validation

The raw data for the method validation is located in study SOA/OO 1.

Comparison of test blanks, standards and test samples showed that the analyte was well resolved from any potential interfering peak. Precision data showed coefficients of variation for Dimethyl adipate of less than 2.5% with solutions in the range of 1000 to 50 pg/ml increasing to 6% in the range 20 to 10 pg/ml.

Precision data showed coefficients of variation for Dimethyl glutarate of less than 3% with solutions in the range of 1000 to 100 pg/ml increasing to 6% in the range 50 to 10 yg/ml. Least squares regression analysis with a I/concentration’ weighting of the peak area response against concentration of Dimethyl adipate (50 to 1000 pg/ml) produced a correlation coefficient of 0.999924 and relative errors less than 4% in the range 1000 to 50 pg/ml.

Least squares regression analysis with a I/concentration2weighting of the peak area response against concentration of Dimethyl adipate (1 0 to 50 pg/ml) produced a correlation coefficient of 1.OOOOOO and relative errors less than 1% in the range 10 to 50 j~g/rnl.The Limit of Quantification (LOQ) for Dimethyl adipate will be set by the lowest acceptable check standard, however, the LOQ and Limit of Detection (LOD) are potentially as low as 6.01 and 1.98 pg/ml respectively (calculated statistically using the standard deviation obtained for a solution of concentration 10 pg/ml).

Least squares regression analysis with a 1/concentration2weighting of the peak area response against concentration of Dimethyl glutarate (50 to 1000 pg/ml) produced a correlation coefficient of 0.999946 and relative errors less than 4% in the range 1000 to 50 pg/ml. Least squares regression analysis with a 1/concentration2 weighting of the peak area response against concentration of , Dimethyl glutarate (10 to 50 pg/ml) produced a correlation coefficient of 0.999999 and relative errors less than 1% in the range 10 to 50 pg/ml. The Limit of Quantification (LOQ) for Dimethyl adipate will be set by the lowest acceptable check standard, however, the LOQ and Limit of Detection (LOD) are potentially as low as 6.06 and 2.00 pg/ml respectively (calculated statistically using the standard deviation obtained for a solution of concentration 10 pg/ml).

Standards of Dimethyl adipate and Dimethyl ,ahtarate in dioxane in the range 50 to 1000 pg/ml stored at approximately 4°C for 7 days and subsequently analysed against fresh standards showed concentrations within 5% of their nominal concentrations except at concentrations approaching 5Opg/ml, the Limit of Quantification, where concentrations within 10% of their nominal were observed.

: 47 : SOA 002/00485 1

APPENDIX 4 (continued)

APPENDIX A - continued

GAS CHROMATOGRAPHSIN INHALATION studies group at 4 October 2000 System Components of gas chromatography system NO. Manufacturer Model KO. Description 1 Hewlett Packard 5890A Chromatograph with capillary inlets, heated manual gas sampling valve, ECD and FID. Hewlett Packard G 1513A Autoinjector} Hewlett Packard 18596CX Controller)6890 Series Autosampler Hewlett Packard G15 12AX Turntable) Thermoquestl SP4500 A/D interface Thermoquest PC 1000 Integration software 2 Pye Unicam PU4550 Chromatograph with gas valve and FID. Pye Unicam PU4700 Autosampler Thermoquest SP4500 A/D interface Thermoquest PC 1000 Integration software Shimadzu GC-14A Chromatograph with FID. Shimadzu AOC- 1400 Autosampler Shimadzu AOC-14 Autoinjector S h imadzu Split injection system Thermoquest SP4500 A/D interface Thermoquest PC 1000 Integration software Shimadzu GC-14A Chromatograph with FID. Shimadzu MGS-4 Automated gas valve Shimadzu SPL-14A Split injection system Shimadzu CR4-A Integrator Shi madzu GC-14A Chromatograph with FID. Shimadzu MGS-4 Automated gas valve Shimadzu SPL-14A Split injection system Shimadzu CR4-A Integrator Hewlett Packard 5890A Chromatograph with capillary inlets, heated automatic gas sampling valve and FID. Hewlett Packard 18593B Autoinjector) Hewlett Packard 18596CX Controller) 7673 Autosampler Hewlett Packard G1512AX Turntabie} Thermoquest SP4500 A/D interface Thermoquest PC 1000 Integration software 9 Perkin Elmer Autosystem Automatic Chromatograph with programmable XL split'less capillary injector, heated automatic gas sampling valve and FID. ICD 1 tctron capture detector FID Flame ionisation detector

I Themoquest, formerly Thermo Separation Products and Spectra Physics

: 48 : SOA 002/00485 1

APPEMDIX 4 (continued)

SON001 - STUDY SPECIFIC SUPPLEMENT TO THE INHALATION ANALYTICAL PROCEDURE FOR DIMETHYL ADIPATE .4ND DIMETHYL GLUTARATE

This supplement details additions and amendments to the procedure to be used for the GC assay of Dimethyl adipate and Dimethyl glutarate obtained from air samples collected on the above study.

The assay, incorporating the additions and amendments, is suitable for the analysis of Dimethyl adipate and Dimethyl glutarate, in solution, at concentrations within the range of 50 to 1000 pg/ml.

Details given in this supplement supersede those in the compound specific IAP.

[ EFFECTIVE DATE : 1 8 December 2000

Analytical standard

Name Dimethyl adipate Batch number 5806339-A Purity 98.824% Expiry date Not known Supplier Sponsor

Name Dimethyl glutarate Batch number H93 1363-A Purity 99.61% Expiry date Not known Supplier Sponsor

Method of sample extraction

Volume (ml) of solvent required in sample extraction Sample Group 2 Group 3 Group 4 Bubbler 25 25 25 Cascade impactor stage 3 - 8 5 5 5 Cascade impactor stage F 5 5 5

Preparation of standard solutions

Prepare standard solutions in the nominal range 10 to 1000 pg/ml.

Calibration and Quantification

Calibration of the instrument is performed using standard solutions in the range 50 to 1000 pg/ml or in the range 10 to 50 pg/ml.

Chromatographs

The analysis is performed using chromatograph 9.

:49 :

Dibasic Esters Group 1850 IM Street. N.W.. Suite 700. Washington. D C 20036 DBE (202) 721-4160 * Fax (202) 296-8121)

May 25,2001

Vio Messenger/RETURN RECEIPT

Document Processing Center (7407) Room G99 East Tower Office of PoIlution Prevention and Toxics U.S. Environmental Protection Agency 401 M Street SW Washington, DC 20460

Dibasic Esters (DBEs) Testing Docket No: OPPTS-42190

Attention: Docunient Control Office /TSCA Section 4

In compliance with the Enforceable Consent Agreement (ECA) entered into with the United States EnvironmrntaI Protection Agency on August 5, 1999, the Dibasic Esters @BE) Group comprised of the following companies: Aceto Corporation, E.I. duPont de Nemours & Company, and Sclutia, Inc. submits the test protocols and final reports for the required Dimethyl Glutarate (DMG, CAS # 1119-40-0)and Dimethyl Adipate (DMA, CAS # 627-93 0) Rat Micronuclcus Test. Copies were also sent to our ECA Coordinator George Semeniuk.

Both male and fernale rats were treated by inhalation exposure for 6 hours at a time on two consecutive days with DMA and DMG to evaluate genotoxic effects (chromosome damage and aneuploidy) associated with mutagens and carcinogens. Groups of male and female rats were exposed via airflows containing 0.5, 1.O, and 2.0 mg/l of the test substance. It was concluded that bo!.hDimethyl Glutarate and Dimethyl Adipate showed no evidence of causing chromosome damage or bone marrow ceIl toxicity when administered by whole body inhalation exposure in this in vivo test procedure.

Please contact me at (202) 721-4145 if there are questions relating to this submission. Sincerely,

Edward W. Kordoski, MBA, Ph.D. Executive Director cc: DBE Group T. Hardy, Kirkland & Ellis, DBE Group Legal Counsel

Enclosure: 4 Copies ', Protocol reference: G~XICYVIMNWINHALISOCMA Enquiry number: 2165lA Huntinadon Lite Sciences

CONFI DENTIAL

PROTOCOL

DIMETHYL ADIPATE RAT MICRONUCLEUS TEST

Sponsor Research Laborat0ry

SOCMA Iuntingdon Life Sciences Limited 1850 M Street NW Yoolley Road Suite 700 qlconbury Washington !untingdon DC 20036 ----_ Jambridgesh ire USA PE28 4HS ENGLAND

Total number of pages: 15 Page i

Huntingdon Life Sciences Ltd., registered in England No: 1815730 Protocol reference: GTXICYVIMNRIINHALISOCMA Huntingdon. .e A . Enq~iirynumber: 21651A Lite Sciences

STUDY DETAILS PAGE

Study number: SOA/OO 1 Study title: Rat micronucleus test Test substance: Identity: Dimethyl adipate Lot number: J806339-A Expiry: Sponsor's responsibility; assumed stable for duration of study Appearance: Clear liquid Storage conditions: Room temperature PuritylAssay: 99.9% Route of administration: Inhalation exposure Stability of test substance formulation: Not assessed in this study Analysis of achieved concentration: To be assessed during exposure Sponsor's representative: Richard Opatick Study Director: Christine E Mason Study Supervisor: Lincoln Pritchard Monitoriqg of Inhalation exposure: T J Kenny I Location if study: In-life phase - Department of Inhalation Toxicology, Huntingdon Life Sciences Ltd., Huntingdon, Cambridgeshire, PE28 4HS Slide scoring and reporting - Department of Genetic Toxicology, Huntingdon Life Sciences Ltd., Eye, Suffolk, IP23 7PX

Proposed study dates Start: 30 November 2000 Completion: 19 January 200 1 Draft report: 16 February 200 I Modifications: 1. PURPOSE The EEC guideline has been updated to: EC Coinmission Directive 2000/32/EC Annex 4C - B.12. Mutagenicity - In vivo mammalian erythrocyte micronucleus test. No. L 136150. STUDY DIRECTOR APPROVAL OF PROTOCOL

EdMmW XLA ~~h a- ChristineC, E Mason Date Study Director Huntingdon Life Sciences Ltd

Final of 22 November 2000 Page ii I’rotocol refercnce: GTX/CYV/MNR/INHAL/SOCMA Huntinsdon Lite Sciences

PROTOCOL APPROVAL

DIMETHYL ADIPATE

RAT MICRONUCLEUS TEST

as Kevin Adams Date Management Huhtingdon Life Sciences Ltd

PIeuse sign both copies of this page, retain one for your records and return one to the Study Director ut Huntingdon Life Sciences.

3udy Director approval of the protocol is given on the study details page of the protocol once such detaiIs have been established and agreed. The completed page will be issued prior to the start of the study.

Page iii lJrotocol reference: GTX/CYV/MNR/INI iAL/SOCM A Huntingdon Life Sciences

TABLE OF CONTENTS

Page

1 . PURPOSE ...... 2

2 . BACKGROUND ...... 3

3 . EXPERIMENTAL PROCEDURE ...... 4

4 .ASSESSMENT OF RESULTS...... 10

5 . REPORTING ...... 11

6 .MAINTENANCE OF RECORDS ...... 11

7 . GOOD LABORATORY PRACTICE ...... 11

8 . QUALITY ASSURANCE ...... 11

9 . HEALTH & SAFETY ...... 12

10. REFERENCES ...... 12

Page I Protocol reference: GTX/CYV/MNK/INIIALISOCMA HuntingdonI .e A. Lite Sciences

1. PURPOSE

The object of this study is to assess the mutagenic potential of the test substance in an in vivo cytogenetic test system which coniplies with the following guidelines:

OECD (February 1997) Proposal for updating guideline No. 474. Genetic Toxicology Mammalian Erythrocyte Micronucleus Test. EEC Annex to Directive 92/69/EEC (1992) Part B : Methods for determination of toxicity, B.12. Mutagenicity (Micronucleus test). 0.J No. L 383 A, vol 35, pI54. US EPA (I 998) Health Effects Test Guidelines. OPPTS 870.5395 Mammalian erythrocyte micronucleus test. EPA 712-C-98-226.

Page 2 Study Number :soA/oo1 I’rotocol Amendment Number : 1 Huntingdon Life Sciences

2. BACKGROUND The bone marrow micronucleus test, originally developed by Matter and Schmid (1971), is a widely employed and internationally accepted short-term assay for identification of genotoxic effects (chromosome damage and aneuploidy) associated with mutagens and carcinogens (Mavournin et ul 1990). This in vivo system allows consideration of various factors including pharmacokinetics, metabolism and DNA repair which cannot be accurately modelled in an in vilro system. Young adult rats are chosen for use because of the high rate of cell division in the bone marrow, because of the wealth of background data on this species, and because of 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 (HoweI1-Jolly bodies) in the cytoplasm of these cells. Micronuclei are seen in a wide variety of cells, but erythrocytes are chosen for examination since micronuclei are not obscured by the main nucleus and are therefore easily detected in this cell type (Boller and Schmid 1970).

Micronucleated immature erythrocytes appear in the bone mmow approximately 24 hours after induction of chromosome damage. These immature erythrocytes can be differentiated by a variety of staining techniques which rely on their relatively high content of residual RNA. Using the Feulgen/H&E method, they stain slate-blue while mature erythrocytes (which contain little RNA) are counterstained orange. An increased incidence of micronucleated immature erythrocytes is indicative of recent exposure to a chromosome-damaging agent. A simultaneous marked increase in the incidence of micronucleated mature erythrocytes is not expected and may be indicative of micronucleus-like artifacts (Schmid 1976).

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 tun lead to a reduction in the number of nucleated cells and immature erythrocytes; to compensate for this peripheral blood is shunted into the bone marrow (von Ledebur and Schmid 1973). If the proportion of immature erythrocytes is found to be significantly less than the control value, this is taken as being indicative of toxicity. A very large decrease in the proportion would be indicative of a cytostatic or cytotoxic effect.

Page 3 Study Number : SOA/OOl Protocol Amendment Number : 1 Huntinqdon Lite Sciences

3. EXPERIMENTAL PROCEDURE

Animal management Species Rat Strain Fischer 344 Source Harlan Olac UK Ltd, Bicester, Oxon, England.

Age ca 6 - 8 weeks old Weight (male) 150 - 175 g Weight 125 - 150 g (female) Acclimatisation On receipt, animals will be randomly assigned to groups, identified by unique tailmark and weighed. Animals will be acclimatised for at least five days before the experiment is performed. Housing The animals will be housed and dosed within one room. Room temperature will be controlled within the limits 21 k 2°C and relative humidity will be controlled to 55 f 10% RH. These parameters will be continuously monitored using a Kent Clearspan chart recorder. Lighting will be controlled to provide 12 hours artificial light (0730 1930 hours) in each 24-hour period. Environmental controls provide at least 12 room-air changes per hour. An auxiliary power supply is available to ensure conditions are maintained in the event of main supply failure. Animals will be housed in groups, with the sexes separated, in suspended stainless steel cages (North Kent Plastic Cages Ltd) equipped with solid sides and wire grid front, back and floor. Each cage measures 53 cm long, 35 cm wide and 25 cm high. Plastic trays, lined with absorbent paper will be placed below each cage to collect excreta. The paper will be changed daily. Each cage will be identified by label (colour-coded according to group) which will display the study number, cage number, sex and individual animal numbers. The cages will be suspended on moveable batteries, a separate battery will be used for each dose group. Animals in the positive control group will be housed in the same conditions except that they will be held in disposable solid plastic cages with stainless steel lids with wood-chip bedding. The animals within each dosage group wili be kept in a separate ventilated cabinet within the dosing room to prevent cross-contamination between groups. Room air is exhausted through the holding cabinets, therefore, the cabinets draw their air supply fiom the main room. Environmental control sensors are mounted in the exhaust ducting ensuring that the main room and holding cabinet temperature and relative humidity are uniform. Diet Animals will be provided with pelleted expanded rat and mouse No. 1 maintenance diet (SQC grade obtained from Special Diets Services Ltd, Witham, Essex, UK) and tap water adlibifurnexcept diet and water will be withheld during the period of inhalation exposure. Food and water are routinely analysed for quality at source and a copy of the relevant certificates will be maintained in the study data file. Dietary contaminants, known or reasonably anticipated, are not expected to be present at levels that interfere with study objectives.

Page 4 !.

I Study Number : SOA/OOl Protocol Amendment Number : 1 Huntinadon Lite Sciences

Positive control Identity Cyclophosphamide Supplier Aldrich Chemical Co Ltd or other suitable supplier Appearance White crystalline powder Vehicle Purified water Dosage preparation Solution prepared just prior to use

Main test The study designs are shown below. Animals in negative and test substance groups will be treated by inhalation exposure for 6 hours at a time on two consecutive days. Negative control and test substance groups wili be sacrificed 24 hours after completion of the second exposure (48 hours from completion of first exposure). Animals in the positive control group will be treated once orally by intragastric gavage using a dosage volume of 10 mlkg. Positive control animals will be sacrificed 24 hours after dosing, concurrently with the negative control and treatment groups.

Animals will be weighed prior to the first exposure and regularly examined for adverse clinical signs until the time of sacrifice.

Study design

Group/ Material Exposure Sampling No. of Animal numbers colour code level time animals (hours) M F M F 1 :White Vehicle - 54* 5 5 201-205 206-210

2 :Yellow x14 54* 5 5 211-215 216-22.0

3 :Blue Dimethyl adipate XI2 54* 5 5 221-225 226-230

4 :Pink X 54* 5 5 231-235 236-240

5 :Orange Cyclophosphamide 20 mg/kg 24 5 5 241-245 246-250

where x = the estimated maximum tolerated exposure level

* 24 hours after completion of the second exposure (48 hours from completion of first exposure).

Page 5 Study Number : SOA/OOl Protocol Amendment Number : 1 Huntinadon Life SciFnces

Route of administration The inhalation route is selected since this is a possible route of accidental exposure in man.

Exposure levels: The exposure levels will be selected in consultation with the Sponsor taking account of all available data, including the results of preliminary studies, any acute inhalation and repeat dose-inhalation toxicity study in rats. ,

Groups and exposure levels: The allocation of animals to the experimental groups, group numbers and exposure levels are shown above.

Exposure: The test atmospheres for Groups 2-4 will be administered by inhalation as described below.

Exposure duration: Two six-hour exposures on consecutive days will be used for all animals in the negative control and treatment groups.

Exposure chambers: Animals will be exposed in whole-body exposure chambers constructed from stainless steel and glass. The internal volume of each chamber is approximately 750 litres.

Air will be introduced into each exposure chamber at a total rate of 150 litres per minute.

The flow through each chamber is approximately 12 air changes an hour; normally sufficient to maintain oxygen concentration above 19% v/v, temperature approximately 22 (& 1) "C and relative humidity between 40-60%.

The exposure chamber will be maintained 1-10 mm H,O below ambient pressure.

Animals will be housed singly in stainless steel mesh compartments during exposure.

Generation of test atmosphere: The test atmosphere will be produced using a using an all- glass vapouriser system to which the test substance is metered from gIass or polypropylene syringes mounted on an infusion pump. Adjustments may be made to the liquid test substance supply to each vapouriser during exposures in order to maintain the desired chamber concentrations. The airflow through each vapour generator will be determined during preliminary generation trials. Details will be presented in the final report.

Procedure

The animals are removed from their cages and placed into the chamber appropriate to the treatment group. A separate chamber is used for each group. The air supplies to the generators and chambers are turned on.

With the animals in position in the chamber, exposure commences from the moment when generation commences. Operation of the exposure system is monitored frequently during the period of exposure.

During exposure the chamber atmosphere is sampled to determine the concentration of test vapour on at least 3 occasions during each exposure. --

Page 6 Study Number : SOA/OUl Protocol Amendment Number : 1 Huntinsdon Life SciFnces

On completion of the 6-hour exposure period, the generation system is switched off, and the air supplies disconnected. The system is allowed to clear for approximately 25 minutes before the animals are removed from chamber and returned to their holding cages.

The nominal rate of vapour production is calculated by recording the amount of test substance delivered to the generation system during the exposure. The usage over the six hours exposure is divided by the total airflow through the chamber. Any losses during the generation process may be quantified and included in calculation of the nominal concentration.

The control animals will be exposed to clean air only in an identical exposure chamber to that used for the test groups.

Monitoring of chamber conditions Method of analysis of the test substance in air: By chemical analysis of samples of chamber air. When possible a method of analysis will be supplied by the Sponsor and this will be validated at Huntingdon Life Sciences. This will be followed in principle although specific conditions might be modified at the discretion of the Head of Section, Aerosol Technology and Analysis.

Airflow: The total airflow through the chamber will be 150 litres/minute and all airflows will be monitored continuously using rotameters. The airflow will be recorded at 30-minute intervals.

Chamber temperature and relative humidity: Chamber temperature and calcuIated relative humidity will be monitored continuously using a wetldry bulb hygrometer and recorded at 30 minute intervals.

Preparation of bone marrow smears At the appropriate time after completion of exposure (or administration of the positive control), the animals from each group will be weighed and killed by humane methods. The femurs will be dissected from each animal and the proximal heads removed. The distal heads will be kept intact and as much tissue as possible will be removed from the bones. The contents of both femurs from each animal will be eluted and pooled in 10 ml Hanks’ balanced salts solution by aspiration through a 21 g needle fitted to a plastic syringe. The resulting cell suspensions will be spun at 1000 rpm (150 x g) for 5 minutes using a centrifuge. Each resulting cell pellet will be resuspended in 2 ml of filtered foetal calf serum before being sedimented out using the centrifuge. The final cell pellet will be resuspended in a small volume of foetal calf serum to facilitate smearing in the conventional manner on glass microscope slides (Schmid 1976). Several smears will be prepared from each animal. At least one smear from each animal will be stained and examined, the remaining smears being held temporarily in reserve in case of technical problems with the first smear.

Due to the presence of mast cell granules in rat bone smears, which appear identical to micronuclei when stained using the Romanowsky methods, a modified Feulgen staining method is employed for the rat micronucleus test in this laboratory. This method specifically stains DNA-containing bodies deep purple while leaving mast cell granules unstained. The method also allows reasonable differentiation of mature and immature erythrocytes and produces permanent preparations.

Page 7 I Study Number : SOA/OOl Protocol Amendment Number : 1 Huntingdon Life Sciences

Fixation and staining of slides

1. Fixed for 10 minutes in SLR grade methanol. 2. Hydrolysed in Bouin's fluid at room temperature for 30 hours. 3. Washed three times in purified water (5 minutes per wash). 4. Stained in Schiffs reagent for one hour at room temperature. 5. Washed three times in purified water (5 minutes per wash). 6. Counter-stained for ten minutes in very dilute (approximately 0.06 g/l) aqueous Eosin yellowish. The dilution and staining time in Eosin may be adjusted to give optimum differentiation between mature and immature erythrocytes. 7. Washed for five minutes in purified water. 8. Stained for 30 minutes in Mayer's Haemalum diluted 9 volumes: 1 volume with 1 mg/m1 Acridine Orange solution in purified water. 9. Rinsed in purified water briefly. 10. Briefly rinsed in running tap water. 11. Washed for 5 minutes in purified water. 12. Air-dried. 13. Slides wiIl be mounted with coverslips using DPX mountant. 14. The mountant will be allowed to harden at approximately 37°C. NB All stains and Bouin's fluid will be filtered immediately prior to use to remove particulate material.

Microscopic examination Coded slides will be examined by light microscopy and 2000 immature erythrocytes per animal will be examined for the presence of micronuclei. Usually only one smear is examined per animal, any remaining smears being held temporarily in reserve in case of technical problems with the first smear.

Micronuclei are identified by the following criteria: Large enough to discern morphological characteristics Should possess a generally rounded shape with a clearly defined outline Should be deeply stained and similar in colour to the nuclei of other cells - not black Should lie in the same focal plane as the cell Lack internal structure, ie they are pyknotic There should be no micronucleus-like debris in the area surrounding the cell

The proportion of immature erythrocytes will be assessed by examination of a total of at least 1000 erythrocytes per animal.

Page 8 Study Number : soAfoo1 Protocol Amendment Number : 1 Huntingdon Life Sciences

4. ASSESSMENT OF RESULTS The results obtained for each treatment group will be compared with the results obtained for the vehicle control group using non-parametric statistical methods. Non-parametric statistical methods are chosen for analysis of results because: They are suited to analysis of data consisting of discrete/integer values with ties such as the incidence of micronucleated immature erythrocytes. The methods make few assumptions about the underlying distribution of data and therefore the values do not require transformation to fit a theoretical distribution (where data can be approximately fitted to a normal distribution, the results of non-parametric analysis and classical analysis of variance are very similar). “Outliers” are frequently found in the proportion of immature erythrocytes for both control and treated animals; non-parametric analysis based on rank does not give these values an undue weighting.

For incidences of micronucleated immature erythrocytes, exact one-sided p-values are calculated by permutation (StatXact, CYTEL Software Corporation, Cambridge, Massachussetts). Comparison of several dose levels is made with the concurrent control using the Linear by Linear Association test for trend in a step-down fashion if significance is detected (Agresti et al. 1990); for individual inter-group comparisons (eg the positive control group) this procedure simplifies to a straightforward permutation test (Gibbons 1985). For assessment of effects on the proportion of immature erythrocytes, equivalent permutation tests based on rank scores are used, ie exact versions of Wilcoxon‘s sum of ranks test and Jonckheere’s test for trend.

A positive response is normally indicated by a statistically significant dose-related increase in the incidence of micronucleated immature erythrocytes for the treatment group compared with the concurrent control group (PcO.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 immature erythrocytes for the group treated with the test substance are not significantly greater than incidences for the concurrent control group (P>O.OI) 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 dose-related decrease in the proportion of immature erythrocytes (PcO.0 1).

Additional slide reading or animal testing may very occasionally be required to clarify unclear or equivocal results.

Page 9 Study Number : SOA/OOl Protocol Amendment Number : 1 Huntinqdon ..C A .- Lite Sciences

5. REPORTING The report will contain details of the test substance, methodology, results and the interpretation of the data. Tabulated results will show individual animal results, and group mean results. Good Laboratory Practice and Quaiity Assurance statements will be included.

6. MAINTENANCE OF RECORDS

All raw data, samples and specimens (if appropriate) arising from the performance of this study will remain the property of the Sponsor.

Types of sample and specimen which are unsuitable, by reason of instability, for long term retention and archiving may ,be disposed of after the periods stated in Huntingdon Life Sciences Standard Operating Procedures.

Huntingdon Life Sciences will retain the Quality Assurance records relevant to this study and a copy of the final report in its archive indefinitely.

7. GOOD LABORATORYPRACTICE

The study will be conducted in compliance with the principles of Good Laboratory Practice Standards as set forth in: The UK Good Laboratory Practice Regulations 1999 (Statutory Instrument No 3 106). OECD Principles of Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM(98)17. EC Commission Directive 1999/1I/EC of 8 March 1999 (Official Journal No L 77/8).

8. QUALITY ASSURANCE

The following will be inspected or audited in relation to this study. Protocol Audit : Standard protocol and study specific pages. Process based inspections : Routine and repetitive procedures will be inspected on representative studies, not necessarily on this study. Report Audit : The draft report and study data will be audited before issue of the draft report to the Sponsor.

Page 10 Study Number : SON001 Protocol Amendment Number : 1 Huntingdon LifeSciences

QA findings will be reported to the Study Director and Company Management promptly on completion of each action, except for process based inspections which will be reported to appropriate Company Management only.

9. HEALTH & SAFETY

In order for Huntingdon Life Sciences to comply with the Health and Safety at Work etc. Act 1974, and the Control of Substances Hazardous to Health Regulations 1994, it is a condition of undertaking the study that the Sponsor shall provide Huntingdon Life Sciences with all information available to it regarding known or potential hazards associated with the handring and use of any substance supplied by the Sponsor to Huntingdon Life Sciences. The Sponsor shall also comply with all current legislation and regulations concerning shipment of substances by road, rail, sea or air.

Such information in the form of a completed Huntingdon Life Sciences or Sponsor test substance data sheet must be received by Safety Management Services at Huntingdon Life Sciences before the test substance can be handled in the laboratory.

Page 11

- Study Number : SOA/OOl Protocol Amendment Number : 1 Huntingdon Life Sciences

10. REFERENCES

AGRESTI, A., MEHTA, C.R. and PATEL, N.R. (1 990) Exact inference for contingency tables with ordered categories. Journal of the American Statistical Association, 85,453.

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). Humungenetik, 11, 34.

GIBBONS, J.D. (1985) Nonparametric Statistical Inference, 2nd edition, Marcel Dekker, New York.

MATTER, B. and SCHMID,W. (1971) Trenimon-induced chromosomal damage in bone marrow cells of six mammalian species, evaluated by the micronucleus test. Mutation Research, 12,417.

MAVOURNIN, K.H., BLAKEY, D.H., CIMTNO, 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.

SCHMID,W. (1976) The micronucleus test for cytogenetic analysis. In: Hollander, A. (ed.) Chemical mutagens, principles and methodsfor their detection, VoI. 4. Plenum Press, New York. von LEDEBUR, and SCHMID,W. (1973) The micronucleus test. Methodological aspects. Mutarion Research, 19, 109.

Page 12 Study Number : SOA/OOl Protocol Amendment Number : 2 Huntingdon Life Sciences

PROTOCOL AMENDMENT

DIMETHYL ADIPATE

RAT MICRONUCLEUS TEST

Total number of pages: 2

Study Director : Christine Mason

The signature of the Study Director authorises the implementation of this amendment to protocol. Any changes to the study design after the date of this authorising signature will be documented in a further formal amendment.

AMENDMENT APPROVAL

For Huntingdon Life Sciences Ltd

Authorised by: .E.%-)* Date: bkL& a (Study Director)

For the Sponsor

Approved by: Date: Study Number : SOAIOOI Protocol Amendment Number : 2 Huntinadon Life ScieYnces

PROTOCOL AMENDMENT

DIMETHYL ADIPATE

RAT MICRONUCLEUS TEST

Reasons for amendments

Due to difficulties obtaining the strain of rat required from Harlan Olac Limited, an alternative animal supplier (Charles River UK Limited, Margate, England) was selected.

Start date of study delayed by one week due to animal supply difficulties. All other timings remain unchanged.

Amendments

3. EXPERIMENTAL PROCEDURE

Animal management Species Rat Strain Fischer 344 Source Charles River UK Ltd., Margate, England.

Age ca 6 - 8 weeks old Weight (male) I50 - I75 g Weight 125 - 150g (female)

Proposed study dates:

Start: 6 December 2000 Completion: 19 January 2001 Draft report: 16 February 2001 C O ."4.- ~ ~

Page 2 Huntingdon.C A e Lite bcrences

FILE NOTE No. 1

Huntingdon Life Sciences Study Number SON001 ......

Study Director: Christine Mason

Effective date: 19 December 2000

Phase of study: Experimental phase

EXPERIMENTALPROCEDURE Animal Management: Housing

Lighting will be controlled to provide 12 hours artificial 1igL (06.30-18.30 hours) in each hour period and not (07.30-19.30 hours) as stated in the study protocol.

This minor deviation has'no impact on the integrity of the study.

Signature...... c.:.Ek.k...... Date:..j%%.b.%.&.h Study Director Huntinadon Lite 3ciences

FILE NOTE No. 2

Huntingdon Life Sciences Study Number SON001 ......

Study Director: Christine Mason

Effective date: 19 December 2000

Phase of study: Experimental phase

Slides were dated for use on 14 December 2000 but due to a delay to the start of the treatment the bone marrow smears were prepared on 20 December 2000.

This minor deviation has no impact on the integrity of the study.

Signature...... c?.i.%.:.b~ ...... Date:..&..b%&?d..m

Study Director Huntingdon Study Number: SOMOO1 Life Sciences

FILE NOTE NUMBER 4

Study Director : Christine E. Mason

Effective Date : 28 February 2001

Phase of Study : Reporting

Dose justification In consultation with the Study Sponsor and based on information supplied 2000 ppm was selected as the highest dose concentration for the study. Dose levels selected for the study were 500,1000 and 2000 pprn.

Issued and Authorised by: (Study Director)

I Form No.25-COM 1 Version~o.:I I Page~o.:I of1 I Huntingdon Study Number: SOA/OOl Life Sciences

FILE NOTE NUMBER 3

Study Director : Christine Mason

Effective Date : 15 February'2001

Phase of Study : Reporting

Fixation and staining of slides

The Protocol states that the mountant will be allowed to harden at approximately 37°C. However, mounted slides were left to air dry until mountant had hardened.

This minor deviation has no impact on the integrity of the study.

Issued by: ...... Zahid Wekw-d Date: ...... \SI21 a\

@ Authorised by: ...... 0. i5 .-Wt4.n...... Date: ..!.?\?.\.%.\ ...... (Study Director)

Distribution: as protocol

Form No. 26-COM Version No.: 1 Page No.: 1 of 1 _-- DIMETHYL ADIPATE .. R4T MICRONUCLEUS TEST

Contain NO CBI

-. .. . .-... i 'I

CONFIDENTLAL SOA 001/004850

DIMETHYL ADIPATE

RAT MICRONUCLEUS TEST

Sponsor Research Laboratory

SOCMA Huntingdon Life Sciences Ltd.

1850 M Street NW ' Woolley Road Suite 700 Akonbury Washington Huntingdon DC 20036 Cambridgeshire USA PE28 4HS ENGLAND

Report issued 16 May 200 1 Page 1 of 49 SOA 00 1/004850

CONTENTS

Page

COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS...... 4

QUALITY ASSURANCE STATEMENT...... 5

RESPONSIBLE PERSONNEL...... 6

SUMMARY ...... 7

INTRODUCTION...... 8

TEST SUBSTANCE...... 10

EXPERIMENTAL PROCEDURE...... 11

ASSESSMENT OF RESULTS ...... 15

MAINTENANCE OF RECORDS ...... 16

RESULTS...... 17

CONCLUSION ...... 18

REFERENCES ...... 19

TABLES

1. Summary of results and statistical analysis ...... 20

2 . Results for individual animals ...... 21

3 . Animal bodyweights ...... 23

:2 SOA 001/004850

Page

APPENDICES

1. Micronucleustest - Clinical signs and mortalities...... 25

2. Historical vehicle control values...... 27

3. Historical positive control values ...... 28

4. Administration of Dimethyl Adipate by inhalation to rats ...... 29 SOA 001/004850

COMPLIANCE WIT" GOOD LABORATORY PRACTICE STANDARDS

The study described in this report was conducted in compliance with the following Good Laboratory Practice standards and I consider the data generated to be valid.

The United Kingdom Good Laboratory Practice Regulations I999 (Statutory Instrument No 3 106).

OECD Principles of Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM(98)17.

EC Commission Directive 199911IEC of 8 March 1999(Official Journal No L 77/8).

US EPA, (TSCA), Title 40 Code of Federal Regulations Part 792, 1989.

The expiry date of the test substance was the Sponsor's responsibility.

r.E."% \SULMtw a=\. Christine E. Mason, B.Sc. (Hons.), Date Study Director, Huntingdon Life Sciences Ltd.

:4: SOA 001/004850

The following inspectionsand audits have been carried out in relation to this study.

Study Phase Date of Inspection Date of Reporting

Protocol Audit I December 2000 1 December 2000

Study Based Inspections TerqinaI procedures 20 December 2000 21 December 2000 Preparation of bone marrow smears 20 December 2000 21 December 2000 Study documentation 2 I December 2000 21 December 2000

Process Based Inspections Housing and environment 2 October 2000 13 October 2000 Test substance control 2 October 2000 13 October 2000 Exposure 2 October 2000 13 October 2000 Sampling 2 October 2000 13 October 2000 Clinical signs 2 October 2000 13 October 2000 Bodyweights 6 October 2000 13 October 2000 Feeding 6 October 2000 13 October 2000 Records 6 October 2000 13 October 2000 Slide scoring 10 January 2001 10 January 200 1

Report Audit 14 May 2001 14 May 2001

Protocol Audit: An audit of the protocol for this study was conducted and reported to the Study Director and Company Management as indicated above.

Study Based Inspections: Inspections and audits of phases of this study were conducted and reported to the Study Director and Management as indicated above.

Report Audit: This report has been audited by the Quality Assurance Department. This audit was conducted and reported to the Study Director and Company Management as indicated above.

The methods, procedures and observations were found to be accurately described and the reported results of this study to reflect the raw data.

< P c ,B m Group Manawr, Department of Quality Assurance, Huntingdon Life Sciences Ltd.

:5 SOA 001/004850

Christine E. Mason, B.Sc.(Hons.), Study Director. Department of Genetic Toxicology.

Lincoln Pritchard, B.Sc.(Hons.), Study Supervisor, Department of Genetic Toxicology.

Terry Kenny, B.Sc.(Hons.), Senior Study Manager, Department of Toxicological Sciences.

Stuart Cracknell, B.Sc. (Hons.), Aerosol Technologist, Department of ToxicologicaI Sciences.

Graham F. Healey, B.Sc., MSc., A.R.C.S., Head of Department, Department of Statistics.

:6 SOA 001/004850

SUMMARY

This study was designed to assess the potential induction of micronuclei by Dimethyl adipate in bone marrow cells of the Fischer 344 rat. Animals were treated for two six hour periods of whole body inhalation exposure, to the test substance at dose levels of 0.5, 1.O and 2.0 mg/l expressed in terms of the weight of test substance per unit volume of chamber air.

Incidences of weight loss were recorded throughout the test but these were small and not considered to be significant. Clinical signs observed for animals dosed with Dimethyl adipate included partially closed eyes, smacking mouth, lethargy, piloerection, walking on toes, oily fur, yellow staining in urinogenital region, brown staining around snout and wet fur in urinogenital region. No adverse clinical signs were obtained for the negative control treated animals over the duration of the test.

No statistically significant increases in the frequency of micronucleated immature erythrocytes and no substantial decrease in the proportion of immature erythrocyteswere observed in rats treated with Dimethyl adipate and killed 24 hours after two six hour periods of whole body inhalation exposure, compared to vehicle control values (P>O.O1 in each case).

The positive control compound, cyclophosphamide, produced large, highly significant increases in the frequency of micronucleated immature erythrocytes(P

It is concluded that Dimethyl adipate did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered by whole body inhalation exposure in this in vivo test procedure.

:7 SOA 001/004850

INTRODUCTION

The purpose of this study was to assess the potential of Dimethyl adipate to induce mutagenic effects in rats following whole body exposure using an in vivo cytogenetic system (Boller and Schmid 1970, MacGregor el a2 1987, Mavoumin et al 1990). The inhalation route was selected for use in this test as the most likely route of human exposure.

The procedures used were based on the recommendations of the following guidelines:

. OECD Guideline for the Testing of Chemicals. (1 997) Genetic Toxicology: Mammalian Erythrocyte Micronucleus Test, Guideline 474. EC Commission Directive 2000/32/EC Annex 4C - B.12. Mutagenicity - In vivo mammalian erythrocyte micronucleus test. No. L 13650. US EPA (1 998) Health Effects Test Guidelines. OPPTS 870.5395 Mammalian erythrocyte micronucleus test. EPA 712-C-98-226.

The bone marrow micronucleus test, originally developed by Matter and Schmid (1971), is a widely employed and internationaIly accepted short-term assay for identification of genotoxic effects (chromosome damage and aneuploidy) associated with mutagens and carcinogens (Mavournin et al 1990). This in vivo system allows consideration of various factors including pharmacokinetics, metabolism and DNA repair which cannot be accurately modelled in an in vitro system. Young adult rats are chosen for use because of the high rate of cell division in the bone marrow, because of the wealth of background data on this species, and because of 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 (Howell-Jolly bodies) in the cytoplasm of these cells. Micronuclei are seen in a wide variety of cells, but erythrocytes are chosen for examination since micronuclei are not obscured by the main nucleus and are therefore easily detected in this cell type (Boller and Schmid 1970).

Micronucleated immature erythrocytes appear in the bone marrow approximately 24 hours after induction of chromosome damage. These immature erythrocytes can be differentiated by a variety of staining techniques which rely on their relatively high content of residual RNA. Using the Feulgen/H&E method, they stain slate-blue while mature erythrocytes (which contain little RNA) are counterstained orange. An increased incidence of micronucleated immature erythrocytes is indicative of recent exposure to a chromosome-damagingagent. A simultaneousmarked increase in the incidence of micronucleated mature erythrocytes is not expected and may be indicative of micronucleus-like artifacts (Schmid 1976).

:8: SOA 001/004850

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 immature erythrocytes; to compensate for this, peripheral blood is shunted into the bone marrow (von Ledebur and Schmid 1973). If the proportion of immature erythrocytes is found to be significantly less than the control value, this is taken as being indicative of toxicity.

The protocol was approved by Huntingdon Life Sciences Management on 25 February 2000, by the Sponsor on 18 May 2000 and by the Study Director on 22 November 2000.

The in-life phase of the study and statistical analysis were performed at the Department of Inhalation Toxicology and the Department of Statistics, Huntingdon Life Sciences Ltd., Huntingdon, Cambridgeshire, PE28 4HS,England. Slide scoring and reporting were performed at the Department of Genetic Toxicology, Huntingdon Life Sciences Ltd., Eye, Suffolk, IP23 7PX, England.

Experimental start date (analytical procedure): 1 December 2000

Experimental completion date (slides decoded): 25 January 200 1

9: SOA 001/004850

TEST SUBSTANCE

Identity: Dimethyi adipate

Appearance: Clear liquid

Storage conditions: Room temperature, in the dark

Batch number: 5806339-A

Expiry date: Sponsor’s responsibility; sufficiently stable for the duration of the study

Purity: 98.824%

Date received: 16 October 2000

Supplier: DuPont

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

: 10: SOA 00 1/004850

EXPERIMENTAL PROCEDURE

ANIMALS

All animals in this study were Fischer 344 outbred albino rats between six and eight weeks old, males weighed between 151.0 and 172.4 grams and females between 126.1 and 144.1 grams on despatch from Charles River UK Ltd, Margate, Kent, England.

On arrival the weight of the animals was checked and found to be acceptable. The animals were assigned to groups by random selection from the container and uniquely tail marked. Each group was kept, with the sexes separated, in cages and maintained in a controlled environment, with the temperature set at 21i2"C and relative humidity set at 50*10%. The room was illuminated by artificial light for 12 hours per day. Animals were provided with pelleted expanded rat and mouse No. 1 maintenance diet (SQC grade obtained from Special Diets Services Ltd., Witham, Essex, UK) and tap water ad Zibitum except diet and water were withheld during the period of inhaIation exposure. Food and tap water are routinely analysed for quality at source. Dietary contaminants are not suspected of having any significant effect on parameters measured in this test in this laboratory at any time over the last ten years. All animals were acclimatised for a minimum of 12 days and examined daily.

POSITIVE CONTROL COMPOUND

Cyclophosphamide, obtained from Sigma Chemical Co Ltd, batch number 87H0207; was used as the positive control compound. It was prepared as a solution in purified water, batch number OOIOlB26, at a concentration of 2 mg/ml just prior to administration.

TREATMENT PROCEDURE AND ATMOSPHERE ANALYSIS

Full details of the treatment procedure and atmosphere analysis are presented in Appendix 4 (Aministration of Dimethyl adipate by inhalation to rats).

DATES OF DOSING

Micronucleus test: 1 st exposure: 18 December 2000 2nd exposure: 19 December 2000

MICRONUCLEUS TEST

: 11 : SOA 001/004850

The experimental design is shown below:

Group Treatment Exposure Number of rats (mg/l) Male Female I Air 5 5

2 Dimethyl adipate 0.5 5 5 3 Dimethyl adipate 1.o 5 5 4 Dimethyl adipate 2.0 5 5

5 Cyclophosphamide 20 (mgkg) 5 5

ROUTE OF ADMINISTRATION The inhalation route was selected as the possible route of accidental exposure in man.

Exposure levels: The exposure levels were selected in consultation with the Sponsor taking account of all available data, including the results' of preliminary studies, any acute inhalation and repeat dose inhalation toxicity study in rats.

Groups and exposure levels: The allocation of animals to the experimental groups, group numbers and exposure levels are shown above.

Exposure: The test atmospheres for Groups 2-4 were administered by inhalation as described below.

Exposure duration: Two six-hour exposures on consecutive days were used for all animals in the negative control and treatment groups.

Exposure chambers: Animals were exposed in whole-body exposure chambers constructed from stainless steel and glass. The internal volume of each chamber was approximately 750 litres.

Air was introduced into each exposure chamber at a total rate of 150 litres'per minute.

The flow through each chamber was approximately 12 air changes an hour; normally sufficient to maintain oxygen concentration above 19% v/v, temperature approximately 22 (i 1) "C and relative humidity between 40-60%.

The exposure chamber was maintained 1-1 0 mm H,O below ambient pressure.

Animals were housed singly in stainless steel mesh compartments during exposure.

Generation of test atmosphere: The liquid test material was delivered to a concentric jet atomiser and generated as droplets into a stream of dry air for administration to the rats by inhalation while held in whole body exposure chambers. The target chamber concentrations were achieved by metering the test substance from polypropylene syringes mounted on syringe drivers. The atmospheres produced by the atomisers were further diluted with air to give the final chamber concentrations.

: 12: SOA 001/004850

PROCEDURE The animals were removed from their cages and placed into the chamber appropriate to the treatment group. A separate chamber was used for each group. The air supplies to the generators and chambers were turned on.

With the animals in position in the chamber, exposure commenced from the moment when generation commenced. Operation of the exposure system was monitored frequently during the period of exposure.

During exposure the chamber atmosphere was sampled to determine the concentration of test material on at.least 3 occasions during each exposure.

The nominal rate of aerosol production was calculated by recording the amount of test substance delivered to the generation system during the exposure. The usage over the six hours exposure was divided by the total aidow through the chamber. Any losses during the generation process were quantified and included in the calculation of the nominal concentration.

The control animals were exposed to clean air only in an identical exposure chamber to that used for the test groups. MONITORING OF CHAMBER CONDITIONS Method of analysis of the test substance in air: By chemical analysis of samples of chamber air.

Airflow: The total airflow through the chamber was 150 litredminute and all airflows were monitored continuously using rotameters. The airflow was recorded at 30-minute intervals.

Chamber temperature and relative humidity: Chamber temperature and calculated .relative humidity were monitored continuously using a weddry bulb hygrometer and recorded at 30-minute intervals.

PREPARATION OF BOME MARROW SMEARS Following dosing, the animals were examined regularly by visual assessment and any mortalities or clinical signs of reaction were recorded. Five males and five females were sacrificed from the negative control and each of the test substance groups 24 hours after completion of the second exposure period. The positive control group was sacrificed 24 hours after dosing.

The animals were killed by cervical dislocation following carbon dioxide inhalation and both femurs dissected out from each animal. The femurs were cleared of tissue 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 10 mi Hanks’ balanced salts solution by aspiration through a 21 g needle fitted to a syringe. The resulting cell suspensions were centrifuged at 1000 rpm (I50 x g) for 5 minutes and the supernatant discarded. Each resulting cell pellet was resuspended in 2 rnl of filtered foetal calf serum before being sedimented by centrifugation. The supernatant was discarded and 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). Several smears were prepared from each femur. 1

SOA 00 1/004850

The slides were fixed and stained as described in the following schedule:

1. Fixed for 10 minutes in SLR grade methanol.

. 2. Hydrolysed in Bouin's fluid at room temperature for 30 hours. 3. Washed three times in purified water (5 minutes per wash). 4. Stained in SchifFs reagent for one hour at room temperature. 5. Washed three times in purified water (5 minutes per wash). 6. Counter-stained for ten minutes in very dilute (approximately 0.06 g/l) aqueous Eosin yellowish. 7. Washed for five minutes in purified water. 8. Stained for 30 minutes in Mayer's Haemalum diluted 9 volumes: 1 volume with aqueous acridine orange solution in purified water (1mg/ml). 9. Rinsed in purified water. 10. Rinsed in running tap water. 1 I. Washed for 5 minutes in purified water. 12. Air-dried. 13. Slides were mounted with coverslips using DPX mountant. 14. The mountant was allowed to air dry until hardened.

NB All stains and Bouin's fluid were filtered immediately prior to use to remove particulate material.

Micronuclei are identified by the following criteria:

Large enough to discern morphological characteristics Should possess a generally rounded shape with a clearly defined outline Should be deeply stained and similar in colour to the nuclei of other cells - not black Should lie in the same focal plane as the cell Lack internal structure, ie they are pyknotic There should be no micronucleus-Iike debris in the area surrounding the cell The proportion of immature erythrocytes for each animal was assessed by examination of at least 1000 erythrocytes. A record of the number of micronucleated mature erythrocytes observed during assessment of this proportion was also kept as recommended by Schmid (1976).

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ASSESSMENT OF RESULTS

The results for each treatment group were compared with the results for the concurrent control group using non-parametric statistics. Non-parametric statistical methods were chosen for analysis of results because: 'They are suited to analysis of data consisting of discrete/integervalues with ties such . as the incidence of micronucleated immature erythrocytes. The methods make few assumptions about the underlying distribution of data and therefore the values do not require transformation to fit a theoretical distribution (where data can be approximately fitted to a normal distribution, the results of non parametric analysis and classical analysis of variance are very similar). 'Outliers' are frequently found in the proportion of immature erythrocytes for both control and treated animals; non-parametric analysis based on rank does not give these values an undue weighting. Unless there is a substantial difference in response between sexes (which occurs only rarely) results for the two sexes are combined to facilitate interpretation and maximise the power of statistical analysis.

For incidences of micronucleated immature erythrocytes, exact one-sided p-values are calculated by permutation (StatXact, CYTEL Software Corporation, Cambridge, Massachussetts). Comparison of several dose levels is made with the concurrent control using the Linear by Linear Association test for trend, in a step-down fashion if significance is detected (Agresti et al. 1990); for individual inter group comparisons (ie the positive control group) this procedure simplifies to a straightforward permutation test (Gibbons 1985). For assessment of effects on the proportion of immature erythrocytes, equivalent permutation tests based on rank scores are used, ie exact versions of Wilcoxon's sum of ranks test and Jonckheere's test for trend.

A positive response is normally indicated by a statistically significant dose-related increase in the incidence of micronucleated immature erythrocytes for the treatment group compared with the concurrent control group (PO.O 1) 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 dose-related decrease in the proportion of immature erythrocytes (PxO.0 1).

: 15: SOA 00 I /OO4SjO

MAINTENANCE OF RECORDS

All raw data, samples and specimens arising from the performance of this study will remain the property of 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. SOA 001/004850

RESULTS

CHAMBER ATMOSPHERE GENERATION

Analysed concentrations were in general agreement with target concentrations although slightly greater variation was seen between individual sample values than is normally found. The exposure mean concentrations of Dimethyl adipate were 0.577, 0.921 and 1.721 mg/l compared with target levels-of0.5, 1.O and 2.0 mg/l respectively expressed in terms of the weight of test substance per unit volume of atmosphere. Full details of the results of analysis are presented in Appendix 4.

MICRONUCLEUSTEST The highest dose level selected for the test by the Sponsor was 2.0 mg/l and although the maximum tolerated dose as evidenced by clinical signs was not achieved, a higher concentration would have produced higher concentrations of droplets to vapour. In addition the respirability of.the droplet aerosol, i.e. particles less than 7 p.m, would decrease and the mass median aerodynamic diameter of the aerosol would decrease, resulting in the dosage administered to the animal being reduced.

CLINICAL SIGNS AND MORTALITIES No mortalities were obtained in the micronucleus test.

Clinical signs for animals treated with the test substance are detailed in Appendix 1; Signs included partially closed eyes, smacking mouth, lethargy, piloerection, walking on toes, oily fur, yellow staining in urinogenital region, brown staining around snout and wet fur in urinogenital region. No adverse clinical signs were obtained for the negative control treated animals over the duration of the test. Incidences of weight loss were recorded throughout the test but these were small and not considered to be significant. MICRONUCLEATED IMMATURE ERYTHROCYTE COUNTS (MIE)

The test substance did not cause any statistically significant increases in the number of micronucleated immature erythrocytes [P>O.O I].

Cyclophosphamide caused large, highly significant increases in the frequency of micronucleated immature erythrocytes [P

The test substance did not cause any substantial increases in the incidence of micronucleated mature erythrocytes.

: 17:

. .. , I^ , _1 " _" SOA 001/004850

PROPORTION OF IMMATURE ERYTHROCYTES (Yo IEmE + ME)

The test substance failed to cause any significant decreases in the proportion of immature erythrocytes [P>O.OI].

Cyclophosphamide caused statistically significantdecreases in the proportion [P

CONCLUSION

No statistically significant increases in the frequency of micronucleated immature erythrocytes and no substantial decrease in the proportion of immature erythrocytes were observed in animals treated with Dimethyl adipate for two six hour periods of whole body inhalation exposure, compared to negative control values (P>O.Ol in each case).

: 18: SOA 001/004850

AGRESTI, A., MEHTA, C.R. and PATEL, N.R. (1 990) Exact inference for contingency tables with ordered categories. Journal of the American Statistical Association, 85,453. BOLLER, K. and SCHMID, W. (I 970) Chemical mutagenesis in mammals. The bone marrow of the Chinese hamster as an in vivo test system. HaematologicaI findings after treatment with Trenimon (translation). Humangenetik, 11,34.

CYTEL (1995) Stafiact 3 for Windows: Statistical Sofiare for Exact Nonparametric Inference. Cytel-SofbvareCorporation, NC, USA.

GIBBONS, J.D. (1 985) Nonparametric Statistical Injerence, 2nd edition, Marcel Dekker, New York.

JONCKHEERE, A.R (1 954) A distribution-free k-sample test against ordered alternatives. Biometrics, 41, 133-145.

KRUSKAL, W.H. and WALLIS, W.A (1952) Use of Ranks in One-Criterion Variance Analysis. Journal of the American Statistical Association, 47,583-621.

KRUSKAL, W.H. and WALLIS, W.A (1953) Errata for Kruskal-Wallis (1952). Journal of the American Statistical Association, 47,583-62 1.

MacCREGOR, J.T., HEDDLE, J.A., HIE, M., MARGOLIN, B.H., RAMEL, C., SALAMONE, M.F., TICE, R.R. and WILD, D. (1987) Guidelines for the conduct of micronucleus assays in mammalian bone marrow erythrocytes. Mutation Research, 189, 103.

MATTER, B. and SCHMID, W. (197 1) Trenimon-induced chromosomal damage in bone marrow cells of six mammalian species, evaluated by the micronucleus test. Mutation Research, 12,417. MAVOURNM. K.H., BLAKEY, D.H.,CIMMO, M.C.,SALAMONE, M.F. and HEDDLE, J.A. (1 990) 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.

MOWSON, V. and ASHBY, J. (1995) High resolution rodent bone marrow micronucleus assays of 1.kdimethylhydrazine :implication of systemic toxicity and individual responders. Mutagenesis, 10, 129.

SAS INSTITUTE (1989) SASSTAT User’s Guide, Version 6, Fourth Edition, Vol.2. SAS Institute Inc., Cap, NC, USA.

SCHMID, W. (1976) The micronucleus test for cytogenetic analysis. In: HOLLANDER, A. (ed.) Chemical Mutagens. Principles and Methods for their Detection, Vol. 4, p.3 1. Published by Plenum Press. New York. von LEDEBUR, M. and SCHMID. W. (1973) The micronucleus test. Methodological aspects. Mutation Research, 19, 109.

WILCOXON, F. (1945). Individual comparisons by ranking methods. Biometrics Bulletin, 1, 80 83.

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TABLE 1

Summary of results and statistical analysis

Sampling Treatment Exposure level % ie/(ie+me) .f Incidence mie Incidence mme time (rngll) (mean) (group mean)

54 Hours a Negative control - 34 I .4 0.3 Dimethyl adipate 0.5 34 0.4 1.2 1.o 38 1.2 0-6 2.0 37 1.3 0.9 24 Hours Cyclophosphamide 20 mg/kg 25** 1;.o*** 1.5

% ie/(ie+me) Proportion of immature erythrocytes mie Number of micronucleated cells observed per 2000 immature erythrocytesexamined mme Number of micronucleated cells calculated per 2000 mature erythrocytes a 24 hours after completion of the second exposure (48 hours from completion of first exposure)

Results of statistical analysis using the appropriate nonparametric method of analysis based on permutation (one-sided probabilities): *** P < 0.001 (highly significant) ** P co.01 (significant) otherwise P =- 0.01 (not significant)

Occasional apparent errors off 1% may occur due to rounding of values for presentation in the table 1

SOA 001/004850

TABLE 2 ResuIts for individual animals Treatment Exposure level Animal ie % ie/(ie+me) Incidence me Incidence (mg/l) number mie mme Negative - M 201 479 48 3 525 0 control M 202 401 39 2 638 1 M 203 436 43 3 5 89 0 M 204 394 38 2 632 0 M 205 319 31 1 708 0 F 206 23 1 22 0 800 0 F 207 232 23 1 785 0 F 208 408 38 0 672 0 F 209 371 36 I 655 0 F 210 260 25 1 787 0 Dimethyl 0.5 M211 375 37 0 647 0 adipate M 212 437 39 0 689 0 M 213 378 36 0 660 0 M 214 39 1 36 0 685 0 M 215 367 35 I 682 0 F 216 279 27 0 750 0 F 217 338 33 0 686 0 F 218 306 30 1 730 1 F 219 418 41 2 594 2 F 220 264 26 0 748 1 Dimethyl 1.o M 221 362 35 2 685 0 adipate M 222 395 39 2 627 1 M 223 438 43 0 587 0 M 224 419 40 1 626 0 M 225 408 41 1 592 0 F 226 352 35 2 . 660 0 F 227 402 40 I 603 1 F 228 364 35 1 674 0 F 229 378 36 1 671 0 F 230 370 35 ' 1 674 0 Dimethyl 2.0 M 231 518 49 0 546 0 adi pate M 232 465 42 1 647 0 M 233 405 36 1 717 0 M 234 324 31 2 707 0 M 235 377 37 2 636 1 F 236 489 42 2 670 1 F 237 333 32 1 696 0 F 238 362 36 0 645 1 F 239 307 27 1 826 0 F240. 394 39 ' 3 623 0 ie Number of immature erythrocytes observed per I000 cells scored YO;e/(ie+me) Proportion of immature erythrocytes mie Number of micronucleated cells observed per 2000 immature erythrocytes me Total number of mature erythrocytes recorded per 1000 cells scored mme Number of micronucleated mature erythrocytes observed

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TABLE 2 - (continued) Results for individual animals 1 Treatment Dosage Animal ie YOie/(iei-me) Incidence me Incidence 1 mp/kg number mie mme CP 20 M 241 361 35 27 665 1 M 242 263 25 20 776 0 M 243 360 29 15 880 0 M 244 240 22 17 83 8 1 M 245 320 27 21 859 1 F 246 223 21 8 860 0 F 247 232 21 11 889 I F 248 280 24 8 863 0 F 249 229 21 10 844 2 F 250 246 24 13 764 0 CP Cyclophosphamide ie Number of immature erythrocytes observed per 1000 cells scored YOie/(ie+me) Proportion of immature erythrocytes mie Number of micronucleated cells observed per 2000 immature erythrocytes me Total number of mature erythrocytes per 1000 cells scored mme Number of micronucleated mature erythrocytes observed I

SOA OOI/O04850

TABLE 3 Animal bodyweights Animal Treatment Exposure Bodyweighr (9) number (mg/l) Allocation to study At start of first At termination exposure period Individual Mean Individual Mean Individual Mean 2 sd 2 sd sd 201 M Negativecontrol- - 202 1972 11 221 213f9 219* 210 2 9 202 M 209 22 I 216* 203 M 198 213 212* 204 M 180 198 195* .205 M 196 214 210* 206 F 157 147 2 6 156* 149k 6 154* 146 2 6 207 F 149 149 146* 208 F 141 148 146* 209 F 142 140' 136* 210 F 147 150 146* 21 1 M Dimethyl adipate 0.5 189 1982 14 205 214 k 13 205 212 f 13 2 12 M 202 222 221* 213 M 198 216 214* 214 M 182 19s 193* 215 M 218 230 227* 2 16 F 157 14827 163 155 6 159' 151 +.6 217 F 149 152 148* 218 F 150 160 156* 219 F I45 151 147* 220 F 139 148 144* 22 I M Dimethyl adipate 1.o 215 206f 13 233 228 ,+ 14 227, 223 2 12 222 M 204 224 218* 223 M 221 242 235* 224 M 203 234 230* 225 M 188 206 205* 226 F 148 148 ,S 2 155 155 52 149* 150 i- 3 227 F 152 155 154' 228 F 148 158 I54* 229 F 146 151 147* 230 F 148 155 I48* 231 M Dimethyl adipate 2.0 189 192 k 16 210 212 k 18 202* 204 i 19 232 M I88 208 199* 233 M 176 190 183* 234 M 219 240 234* 235 M 186 213 204* 236 F 140 I46 2 5 146 150 3,: 139* 144 2 3 237 F 151 153 147* 238 F 147 152 I46* 239 F 148 151 147* 240 F 142 I50 143* id Standard deviation * Denotes weight loss from previous weighing

:23 : SOA 001/004850

TABLE 3 - continued Animal bodyweights

Animal Treatment Exposure Bodyweight (9) number (m&g) Allocation to study At treatment At termination

Individual Mean Individual Mean Individual Mean fsd 2 sd k sd 241 M CP 20 206 1962 17 222 217-C 18 219* 214 i 18 242 M 181 203 201* 243 M 190 212 209* 244 M 220 246 243* 245 M 182 20 I 200*

246 F 152 14856 152 150 & 3 150* 148 2 3 247 F 156 153* 152* 248 F 148 152 150* 249 F 143 147 144* 250 F 143 147 145*

CP Cyclophosphamide sd Standard deviation * Denotes weight loss from previous weighing

: 24 : SOA 001/004850

APPENDIX 1

Micronucleus test - Clinical signs and mortalities (1st exposure period)

r 1 Treatment I Dimethyl adipate 1.o 2.0

Approx. M F M F M F . time (hr :min)

~~ 0 :30 0 PC PC PC, SM PC, SM 1 :30 0 PC PC PC, SM PC, SM 3 :oo 0 PC PC PC, L PC, L 3 :30 0 PC, L PC, L PC, L PC, L 4 :00 0 PC, L PC, L PC, L, P PC, L, P 5 :30 L PC, L PC, L PC, L, P PC, L, P 6 :00 L PC, L PC, L PC, L, P PC, L,P 6:38 0 6 :39

6 :42 ys, BS,OF YS,BS,OF 7 :05 0 7 :06

7 : 07 YS,BS, OF YS,BS, OF 23 :31”* 23 :34* 2(W) I Mortalities I 015 015 015 015 015 015 Approx. time: Calculated from start of commencement of generation for firs :xposure period.

NB No adverse clinical signs were noted for the vehicle control group throughout the experiment. 0 No reaction detected. Type of reaction: BS Brown staining around snout, L Lethargic, OF Oily fur, P Piloerection, PC Partially closed eyes. SM Smacking mouth, WF Wet fur in urinogenital region, WT Walking on toes. YS Yellow staining in urinogeniral region . Clinical signs shown refer to all animals within that dose group and sex, except where x(...), x denoting the number of animals displaying the clinical sign(s) within the brackets

: 25 : SOA 001/003850

APPENDIX 1 (continued)

Micronucleus test - Clinical signs and mortalities (2nd exposure period)

Treatment Dimethyl adipate Exposure 0.5 1.o 3.0 (mgfl) Approx. M F M F M F time (hr-: min) . 0:o 0 0 0 0 PC PC 1 :30 0 0 PC PC PC PC 3 :O 0 0 PC PC PC,P,L PC,P,L 4 :30 0 0 PC. L PC.L PC,P, L PC,P, L 5 :30 L L PC,L PC,L PC,P. L PC,P,L 6:O L L PC,L PC,L PC,P, L PC,P,L 6 :34 0 0 6 :37 OF OF 6 :40 WT,BS,OF WT, BS,OF, 3(YS,WF) 7 :34 0 0 7 :35 OF OF 7 :36 WT, BS. OF WT, BS,OF, 3(YS,WF) Mortalities I 015 Of5 0:5 Q!j Of5 015

Approx. time: Calculated from start of commencement of generation for second exposure period. NB No adverse clinical signs were noted for the vehicle control sroup throughout the experiment. 0 No reaction detected. Type of reaction: BS Brown staining around snout, L Lethargic?OF Oily fur, P Piloerection, PC Partially closed eyes, WF Wet fur in urinogenital region, WT Walking on toes. Clinical signs shown refer to all animals within that dose group and sex, except where x( ...), x denoting the number of animais displaying the clinical sign(s) within the brackets

:26 : SOA 00 1IO04850

APPENDIX 2

Historical negative controlvalues (1 February 1999 - 31 January 2001) Frequency of micronucleated immature erythrocytes (individual animals)

0 1 2 3 4 5 6 7 8 Number of micronucleated immature erythrocytes (MIEper 2000) Individual mean 0.94

Historical negative controlvalues (1 February 1999 - 31 January 2001) Frequency of micronucleatedimmature erythrocytes (Group mean values)

,,130. I

0.0-0.2 0.30.5 0.Ei-0.8 0.9-1.1 1.2-1.4 1.51.7 1.8-2.0 2.1-2.3 2.42.6 Number of micronucleated immature erythrocytes (MIEper 2000) Group man 0.71

: 27 : SOA 00 1/004850

APPENDIX 3

Historical positive control values (1 February 1999 - 31 January 2001) Frequency of micronucleated immature erythrocytes (Individual animals) Cyclophosphanddeat 20 rykg

0-20 21-40 41-60 61-80 81-100 101-120 121-140 Number of micronucleated immature erythrocytes (MIEper 2000 cells) Indkidual man 48.03

Historical positive control values (1 February 1999 - 31 January 2001) Frequency of micronucleated immature erythrocytes (Group mean values) Cyclophosphamdeat 20 mglkg

0-10 11-20 21-30' 31-40 41-50 51-60 61-70 71-80 81-90 91-100 Number of rn icronucleated immature erythrocytes (MIEper 2000) Group mean 49.2

:28 : SOA 001/004850

APPENDIX 4

ADMINISTRATION OF DI[METHYL ADIPATE

BY INNALATION TO RATS

AUTHOR S. Cracknell

: 29 : c

Rmb ,,. SOA 00 1 /004850

APPENDM 4 (continued)

TEST SUBSTANCE AND ADMINISTRATION

TEST SUBSTANCE

The test substance, which was also identified as Dimethyl adipate (batch number J806339-A), was supplied as a clear liquid in a glass container (net contents 3986 g) and was received at these laboratories on 16 October 2000. Information from the Sponsor indicated that the test substance was of 98.824% purity and was sufficiently stable for use in this study. The Dimethyl adipate was administered to the rats as a liquid droplet aerosol.

ADMINISTRATION

The liquid test material was delivered to a concentric jet atomiser and generated as droplets into a stream of dry air for administration to the rats by inhalation while held in whole body exposure chambers. The target chamber concentrations were achieved by metering the test substance fiom polypropylene syringes mounted on syringe drivers. The atmospheres produced by the atomisers were further diluted with air to give the final chamber concentrations.

The flow of air to the atmosphere generation systems, and the diluent air supply was calibrated using a precision-made tapered glass tube flowmeter (generation air) or a dry type gas meter (diluent air) during the preliminary phase of the study. Airflow through the generation systems was monitored throughout each of the exposures using in-line tapered tube gas flowmeters.

The usage of Dimethyl adipate was determined, for each day of treatment, for each of the three test groups.

EXPOSURE SYSTEM

Each exposure system comprised a 0.75 m3 whole body inhalation exposure chamber, a stainless steel concentric jet atomiser, a syringe driver, a glass elutriator column, diluent air control valves, a compressed air supply and control valves to each generator and in-line airflow monitoring flowmeters. During periods of atmosphere generation, the test material was dispensed from single- use disposable syringes. c : 30 : SOA 00 1/004850

APPENDIX 4 (continued)

Schematic diagrams of the atmosphere generation system and an exposure chamber system are presented in Figures A and B. The component parts of the systems are described in further detail below:

Atmosphere generation

Each test group inhalation chamber used on the study was connected to an atmosphere . generation system supplying a flow of the appropriate dilution of Dimethyl adipate droplet aerosol. Each exposure system comprised a whole body inhalation exposure chamber, a concentric jet atomiser, a glass elutriator and a diluent air control system.

The test material delivery system for each group comprised a polypropylene syringe located on a syringe driver (Precidor, Model 5003). The liquid test material was delivered to the inlet of the concentric jet atomiser via a PolyTetraFluoroEthylene (PTFE) needle. The syringe driver settings required to achieve the target concentrations were established during the preliminary phase of the study. The atmosphere exiting each atomiser was delivered into a glass column with a volume of approximately I1 Iitres (elutriator) and further diluted with air that entered via a pair of inlet ports adjacent to the atomiser in the base of the unit. The test atmospheres exited the elutriator through a flexible duct located at the centre top of the column and were delivered directly to the top of the whole body exposure chambers.

A compressor supplied air to the atomisers and diluent lines. The air was filtered to remove any residual particulate and was dried (dew point -2OC).

The syringes were refilled with Dimethyl adipate as required during the exposure periods. On each day of exposure, the volume of test material dispensed to each exposure system was determined.

Inhalation chamber

The exposure chambers were of stainless steel and glass construction and consisted of a cuboidal body fitted with a pyramidal base and top. The internal volume of each chamber was approximately 0,75m3.At the apex of the upper pyramidal figure was the tangentially mounted air duct. Immediately below this was a perforated canister, which ensured equal distribution of the test atmosphere within the chamber.

Access to the chamber was through the front of the box section via a hinged door with a glass panel and stainless steel frame. The door was sealed using moulded rubber sealing strip.

Exposure cages constructed of stainless steel mesh were suspended on a fiamework arranged on four levels. Each level held four cages, with each cage capable of housing 4 rats individually. This gave a total animal exposure capacity of 64 rats. In this investigation 10 animal compartments were used. SOA 001/004850

APPENDIX 4 (continued)

A wet and dry alcohol bulb thermohygrometer, used to monitor chamber temperature and relative humidity, was suspended in the chamber, visible through the glass panelled door.

The pyramidal base of each chamber was fitted with a 2-inch drain. The drain connected with a common drainage system via a ball valve.

A square tubular exhaust plenum, 3 inches in diameter and perforated along the ventral surface, was situated in the pyramidal base. This connected to the main extract system.

A Magnehelic pressure gauge (0-100mm water gauge) was connected with each chamber by a nylon tube. This was mounted on the eiutriator trolley and was used to monitor the atmosphere pressure inside the chamber, relative to the exposure room.

Extract flow was adjusted using gate valves mounted in the extract ducting berween the chamber and filters. The internal pressure within each chamber was maintained at approximately-2 to -4 mm H,O below ambient when operational.

The negative control animals were exposed using a similar system to that used for the test groups, but received compressed air only.

PROCEDURE

The rats were removed from their cages and placed in ascending cage sequence from the front left hand side to back right hand side in the individual compartments of the exposure cages. All of the animals were located on level 2 of the chamber with the males on the left side of the chamber.

The syringe pumps were switched on and the exposure start time recorded.

c : 32 : SOA 00 1/004850

APPENDIX 4 (continued)

During the exposure (6 hours), samples were taken after approximately 1,3 and 5 hours to determine the chamber concentration of Dimethyl adipate. Each sample was removed through a modified blanking plug in a port on the level used to expose the animals.

At the end of 6 hours generation, the syringe drivers supplying test substance were switched off. The generator and diluent airflows were turned off and the chambers allowed to clear. Clearance air was allowed to enter each chamber through an open sampling port in the chamber wall. At the end of the clearance period, the rats were unloaded fiom the chambers into their respective holding cages. The chambers were then washed to remove animal waste.

TARGET CONCENTRATIONS The target concentrations of Dimethyl adipate were as follows:

Group Designation Concentration (mg4 2 (Low dose) 0.5 3 (Inter dose) I .O 4 (Hish dose) 2.0

The target concentrations were selected in consultation with the Sponsor, following the review of available data.

EXPOSURE CHAMBER CONDITIONS Analysis of chamber concentrationsof Dimetbyl adipate

The samples of chamber air were collected by drawing a previously selected volume of chamber atmosphere through a sintered glass bubbler containing I ,4-dioxane as a trapping agent. Sample collection was performed by inserting the inlet of the bubbler through a port located at the animal exposure level on the side wall of the chamber. The atmosphere samples were taken at a calibrated flow of 2 litredminute using a laboratory pump. The air volume of each sample collected was measured using and in-line wet type gas meter.

Investigations performed during the preliminary phase of the study confirmed satisfactory trapping of the test atmospheres with the equipment employed. Breakthrough to a second bubbler trap in series was found to be 0% (not detected) of the total collected and consequently the use of a single trap for the main study was considered satisfactory.

Air samples were collected in sequence from each of the three exposure chambers containing the test atmospheres (chambers 2 to 4 sampled sequentially). The Aerosol Technology and Analysis Section of the inhalation Studies Group developed the method of analysis and details of the analytical procedures used are given in Appendix A.

Analysis of droplet size distribution of Dimetbyl adipate

A single sample for the determination of the droplet size of Dimethyl adipate in the chamber atmospheres was taken from each test chamber over the two days of treatment. The atmosphere samples were collected at a calibrated flow of 2 litreslminute using a laboratory pump. The air volume of each sample collected was measured using an in-line wet type gas meter. - -

SOA 001/004850

APPENDIX 4 (continued)

Air sample collection

The air samples were collected in sequence from chambers 2 to 4.

Airflow

The air flow into each chamber was monitored continuously using tapered tube rotameters and recorded at approximately 30-minute intervals throughout each exposure. A documented check on the chamber negative pressure was also made at 30-minute intervals.

Temperature

The wet and dry chamber temperatures in each exposure chamber were recorded at approximately 30 minute intervals during each exposure. The relative humidity (RH%)in each chamber was derived from these data.

CALCULATIONS

Chamber concentration

The mass of test material collection in each sample was determined by Gas Chromatography (GC) and the aerosol concentration derived from the mass of Dimethyl adipate found and the air volume sampled.

Droplet size distribution

The collection characteristics of the Marple sampler under the conditions of use in this study are as follows:

Impactor stage 3 4 5 6 7 8 Filter Cut-point* (pm) 9.8 6.0 3.5 1.55 0.93 0.52 0 * Aerodynamic equivalent particle diameter for spherical particles of unit mass density in air at 25°C and 760 mm Hg.

The material collected on each substrate was extracted and subjected to GC analysis.

: 34 : I

SOA 001/004850

APPENDIX 4 (continued)

RESULTS

CHAMBER CONCENTRATION

Analysed concentration of Dimethyl adipate

The data are presented as follows:

Table A Individual and daily mean concentrations

The target and study mean concentrationsof Dimethyl adipate are presented below:

Group Concentration (mgfl) Target Analysed 2 (Low dose) 0.5 0.577 3 (Inter dose) 1.o 0.921 4 (High dose) 2.0 1.721

Analysed concentrations were in general agreement with target concentrations although sIightly greater variation was seen between individual sample values than is normally found and somewhat greater than the variation seen during preliminary generation trials without animals. The coefficients of variation of the individual samples over the two days of treatment were 32.4, 13.9 and 1 1.4 YOfor Groups 2, 3 and 4 respectively. It is possible that the presence of animals in the chamber through a combination of the additional surface area provided by the animal's bodies and the water vapour contributed to the chamber air by the breathing action of the animals increased the degree of vapour condensation and therefore increased the relative proportion of aerosol in the test atmosphere. Impaction of the aerosol on exposure system surfaces together with aggregation of droplets to a size which was no longer aerostable would reduce the generation efficiency as measured by the nominal to analysed concentration ratio. In a dynamic system the degree of condensation, aerosol impaction and droplet aggregation will vary with time and this probably accounts for the variation in chamber concentration over the 6 hour exposure period. The reduction of generation efficiency resulted in achieved concentrations at the Intermediate and High level being slightly lower than target levels. However, the differences between analysed and target levels were small and are considered not to have affected the outcome of the study. . .. , ' ,I '......

SOA 001/004850

Nominal concentration of Dimethyl adipate

The data are presented in Table B and are summarised below:

Group Study mean nominal Am concentration (mg/I) ("/o) 2 (Low dose) 1.345 41.9 3 (Inter. dose) 2.04 1 45.2 4 (High dose) 4.329 40.4

*IN=( Analysed concentration Nominal concentration

The nominal concentrations were calculated from the mass of DimethyI adipate used during the exposures together with the chamber calibrated airflow (150 l/min) and the period of atmosphere generation (360 min).

Individual exposure measurements of the nominal concentration showed consistent relationship with the analysed concentration for each occasion of atmosphere generation. The efficiency of generation was similar for the three target concentrationsemployed.

PARTICLE SIZE DISTRIBUTION Results of the particle size'determinations are presented in Table C.

Droplet size determination samples collected on one occasion from each test chamber during the study confirmed that there was a high proportion of inhalable aerosol in the exposure atmospheres. The expected trend of increasing particle size with ascending concentration was apparent in the results, however, the observed variation between the three test groups was small and is not considered to have influenced the outcome of the study.

CHAMBER TEMPERATUREAND RELATIVE HUMIDITY The daily mean chamber temperatures and relative humidities are presented in Table D.

: 36 : SOA 00 I /004850

APPENDIX 4 (continued)

FIGURE A

Schematic diagram of the aerosol generation system

Dispensing syringe Syringe driver Test material delivery pipe Aerosol generation air supply Aerosol outlet to elutriator Elutriator vessel Diluent air supply Diluent air inlet Aerosol outlet to inhalation exposure chamber

: 37 : SOA 00 1/004850

APPENDIX 4 (continued)

FIGURE B

Schematic of the exposure system used to expose rats

Key A Glass elutriator G Exhaust plenum B Air flow control and chamber monitoring H Drain C Dispersion device I Gate valve D Exposure chamber (0.75 m’) J Pre-filter E Animal exposure cages K Powered extract filter F Sampling port L Main exhaust

: 38 : SOA 001/004850

APPENDIX 4 (continued)

TABLE A

Chamber concentrations of Dimethyl adipate - individual and mean values

Group Exposure Sample Chamber concenuation Daily mean sd number number identity (mgm (mgm -7 1 SI 0.423 52 0.584 0.629 0.2323 53 0.881

2 s1 [2.795]" S2 0.423 0.498 S3 0.572 Mean 0.577 Sd 0.1 87 cv (Yo) 32.4 3 SI 0.72 I S2 1.ooo 0.916 0.1691 S3 1.026

s1 [ 1.2971a s2 0.994 0.929 S3 0.864 Mean 0.92I sd 0.1283 cv (Oh) 13.9 4 1 SI 1.700 S2 I .407 1.613 0.1795 53 I .733

2 s1 16.0401 S2 1.918 1.882 - S3 1.845 Mean 1.721 sd 0.1958 cv (Yo) 11.4 d Standardd liation CV Coefficient of variation (sd x 100fmean) a Samples excluded from mean as considered to have been contaminated during processing

:39 :

- - SOA 001/004850

APPENDIX 4 (continued)

TABLE B

Exposure nominal concentrations of Dimethyl adipate

Exposure Vol used Wt used Concentration (mg/l) AN ratio (ml) (8) Nominal Analysed (%I 1 76 80.56 1.492 0.629 42.2 2 61 64.66 1.197 0.498 41.6 Mean 1.345 0.564 41.9

Exposure Vol used Wt used Concentration (mg/l) AM ratio (mu (8) Nominal Analysed ("/) 1 104 I 110.24 2.04 1 0.9 16 44.9 2 104 I 110.24 1 2.041 0.929 1 45.5

Exposure Vol used Wt used Concentration (mg/1) AN ratio (mu (9) Nominal I Analysed ("/.I I 222 235.32 4.358 1 1.613 37.0 2 219 1 232.14 I 4.299 I 1.882 I 43,s

Wt used = Volume used (ml)x density (1.06. gml) AM Analysed to nominal ratio calculated as Analysed concentration /.Nominal concentration x 100

:40 : SOA 001/004850

APPENDIX 4 (continued)

TABLE C

Droplet size distrihution of Dimethyl adipate - individual sample and stage values

~ Group % of total collected on each stage Particle size number 3 4 5 6 7 8 FiIter MMAD og YO (pm> < 7pm 2 (Lowdose) 1.7 10.4 50.2 30.2 1.7 0.0 5.8 2.8 2.14 88 3 (Interdose) 4.5 12.3 38.4 38.3 3.7 0.8 2.0 3.3 2.08 84 4 (High dose) 5.5 14.6 31.1 X.5 8.6 0.5 1.2 3.4 2.05 84

41 : , ,I

SOA 001/004850

APPENDIX 4 (continued)

TABLE D

(Air control) I (LOW dose) I (Inter. Dose) 1 (High dose) TemD ,I RH I TemD I RH i TemP 1 RH I TemD 1 RH~ _. 1 20.5 52 20.5 33 19.7 29 20.8 33 2 20.4 27 20.5 32 19.6 29 20.8 33 Mean 20.5 30 20.5 33 19.7 29 20.8 33

:42 :

% I

SOA 001/004850

APPENDIX 4 (continued)

APPENDLX A

COMPOUND SPECIFIC INHALATION ANALYTICAL PROCEDURE FOR DIMETHYL ADIPATE AND DIMETHYL GLUTARATE

The analysis of Dimethyl adipate and Dimethyl giutarate in air sample substrate

The method outlined in this document has been validated and is considered fit for the purpose of monitoring test atmospheres in an Inhalation Toxicology study.

This document details the basic procedures for the analysis of Dimethyl adipate (DMA) and Dimethyl glutarate (DMG) collected using solvent traps (bubblers) and Marple cascade impactors. 1,4-Dioxane is used as the trapping solvent for the analyte. The resulting solutions of approximate concentration 50 to 1000 pg/ml are quantified by GC. Study specific amendments and additions will be detailed within a supplementarydocument.

NOTE Throughout this document, the symbol 9 indicates that the relevant information is included in a Study specific supplement.

I EFFECTIVE DATE: 1 8 December 2000 Test substance

Dimethyl adipate, dimethyl hexanedioate, CsH]404, molecular weight 174.2, has the following structure: 0

Appearance Colourless liquid

Storage Room temperature, dark

Dimethyl glutatrate, dimethyl pentanedioate, C7H 1204, molecular weight 160.2, has the following structure:

Appearance Colourless liquid c Storage Room temperature, dark : 43 : SOA 001/004850

APPENDIX 4 (continued)

APPENDIX A - continued

Equipment

Balance and data printer Sartorius R200D with YDP-02

Dispenser Bibby 2- 1Om1

Diluter Hamilton Microlab 500 Series

Ultrasonic bath Decon FS200b

General laboratory glassware

Consumables

Scintillation vials Packard ca 20 ml capacity Instruments BV

Autosampler vials, septa and Fisher Scientific 2 ml glass vials caps Ltd

Reagents

1,4-Dioxane Sigma Aldrich HPLC grade

Preparation of samples for analysis

Transfer the solution from a bubbler sample to a volumetric flask (Q ml). Rinse the bubbler with further dioxane and add this to the volumetric flask.

Transfer the sample substrate from the cascade impactor to an appropriately labelled container. A volume of extraction solvent (dioxane) is added immediately using the dispenser, ensuring the substrate is submerged. The container is then sealed. The stainless steel substrates are gently swirled and left to stand for a minimum of 10 mins. The filters are placed into an ultrasonic bath on full power for 10 mins. The volumes of extraction solvent added to each sample are as detailed in the study specific supplement.

AI! samples will be transferred to appropriately labelled scintillation vials prior to storage.

: 44 : SOA 001/004850

. APPENDIX 4 (continued)

APPENDIX A - continued

Preparation of standard solutions

Weigh approximately 100 mg of Dimethyl adipate and 1 OOmg of Dimethyl glutarate (to 0.1 mg) into a volumetric flask (100 ml), dissolve in dioxane (10 ml), mix thoroughly and make up to volume with dioxane to provide standard SI. Using the diluter, prepare standards at the required concentrations, as detailed in the study specific supplement.

Storage of standards and samples

The maximum storage periods for the various sample types are detailed below:

Type Storage conditions Maximum storage period Samplessolutions ca 4OC 7 Days Standard solutions ca 4°C 7 Days

Calibration and quantification

For each injection of the sample measure the peak area response and determine the amount present in the sample using the equation below: -.

Amount(pg) = (A-Uxv S

Where A = Peak area response of Dimethyl adipate or Dimethyl glutarate in the sample chromatogram S = Slope of calibration line derived from calibration data I = Intercept of calibration line derived from calibration data V = Dilution volume of sample (ml); = extraction volume x dilution factor

: 45 : SOA 001/004850

APPENDIX 4 (continued)

APPENDIX A - continued

Chromatographic conditions

Analytical column PE-5 dimethyl silicone FSWCOT 0.25 dF, 5m x 0.25 mm . Carrier gas He, 1 ml/min Septum purge 3 rnl/min Air 450 ml/min Hydrogen 45 ml/min Injection volume 2 4 Split vent 20 Split ratio 20: 1 Injector temperature 150DC Column temperature 12O0C Detector FID 25OoC,range 1, attenuation I Retention time Dimethyl glutarate and Dimethyl adipate approximately 0.6 and 0.9 minutes respectively. Quality assurance measures

When the method is established on a chromatographic system six injections of a standard will be used to verify performance of the system. The parameters and acceptance criteria are set out below; Parameter Typical value Acceptance criteria DMA DMG , Plate count (USP) 12510 8086 >8O% Tailing factor (USP) 1.20 1.25 i20 vo Measurement repeatability (n=6) 2.2% 2.5% <5 yo expressed as coefficient of variation

: 46 :

I , , SOA 001/004850

APPENDIX 4 (continued)

APPENDIX A - continued

A quality check standard must follow every 6 concentration samples (8 particle size sample stages) for the analysis to be regarded as valid. The results of the quality check standards must lie within the QC tolerance limits.

A quality check standard of low concentrationwill be run to verify the LOQ for the run. The LOQ for the run will be regarded as the concentration of the lowest acceptable quality check standard.

Summary of method validation

The raw data for the method validation is located in study SON00 1.

Comparison of test blanks, standards and test samples showed that the analyte was well resolved from any potential interfering peak.

Precision data showed coefficients of variation for Dimethyl adipate of less than 2.5% with solutions in the range of 1000 to 50 pg/ml increasing to 6% in the range 20 to 10 &mI. Precision data showed coefficients of variation for dimethyl glutarate of less than 3% with solutions in the range of 1000 to 100 pg/ml increasingto 6% in the range 50 to 10 pg/ml.

Least squares regression analysis with a l/concentration2weighting of the peak area response against concentration of Dimethyl adipate (50 to 1000 pg/ml) produced a correlation coefficient of 0.999924 and relative errors less than 4% in the range 1000 to 50 pg/ml.

Least squares regression analysis with a 1/concentration2weighting of the peak area response against concentration of Dimethyl adipate (1 0 to 50 pg/ml) produced a correlation coefficient of I .OOOOOO and relative errors less than 1% in the range 10 to 50 &ml. The Limit of Quantification (LOQ)for Dimethyl adipate will be set by the lowest acceptable check standard, however, the LOQ and Limit of Detection (LOD) are potentially as low as 6.01 and 1.98 pg/ml respectively (calculated statisticallyusing the standard deviation obtained for a solution of concentration 10 pg/ml).

Least squares regression analysis with a I /concentrationzweighting of the peak area response against concentration of Dimethyl glutarate (10 to 50 pg/ml) produced a correlation coefficient of 0.999999 and relative errors less than 1% in the range 10 to 50 pg/ml. The Limit of Quantification (LOQ)for Dimethyl adipate will be set by the lowest acceptable check standard, however, the LOQ and Limit of Detection (LOD) are potentially as low as 5.96 and 1.9.7 &mi respectively (calculated statisticallyusing the standard deviation obtained for a solution of concentration 10 pg/ml).

Standards of Dimethyl adipate and Dimethyl glutarate in dioxane in the range 50 to 1000 pg/ml stored at approximately 4°C for 7 days and subsequently anaiysed against fresh standards showed concentrations within 5% of their nominal concentrations except at concentrations approaching 50pig/ml, the Limit of Quantification, where concentrations within 10% of their nominal were observed.

: 47 : SOA 001/004850

APPENDIX 4 (continued)

APPENDIX A -continued GAS CHROMATOGRAPHSIN INHALATION STUDIES GROUP AT 4 OCTOBER 2000

System Components of gas chromatography system No. Manufacturer Model No. Description 1 Hewlett Packard 589014 Chromatograph with capillary inlets, heated manual gas sampling valve, ECD and FID. Hewlett Packard G1513A Autoinjector) Hewlett Packard 18596CX Controller}6890 Series Autosampler Hewlett Packard G 15 12AX Turntable) Thermoquest1 SP4500 AID interface Thermoquest PC1000 Integration software 2 Pye Unicam PU4550 Chromatograph with gas valve and FID. Pye Unicam PU4700 Autosampler Thermoquest SP4500 A/D interface Thennoquest PC1000 Integration software 3 Shimadzu GC-14A Chromatograph with FID. Shimadzu AOC- i 400 Autosampler Shimadzu AOC- 14 Autoinjector Shimadzu Split injection system Thermoquest SP4500 A/D interface Thennoquest PC1000 Integration software 6 Shimadzu GC-14A Chromatograph with FID. Shimadzu MGS-4 Automated gas valve Shimadzu SPL-14A Split injection system Shimadzu CR4-A Inte,orator 7 Shimadzu GC- 14A Chromatograph with FID. Shimadzu MGS-4 Automated gas valve Shimadzu SPL-I 4A Split injection system Shimadzu CR4-A Inteeorator 8 Hewlett Packard 5890A Chromatograph with capillary inlets, heated automatic gas sampling valve and FID. Hewlen Packard 18593B Autoinjector) Hewlett Packard 18596CX Controller) 7673 Autosampler Hewlen Packard G 15 12AX Turntable} Thermoquest SP4500 A/D interface Thennoquest PC 1000 Integration software 9 Perkin Elmer Autosystem Automatic Chromatograph with programmable XL splitlless capillary injector, heated automatic gas sampling valve and FID. ECD Electron capture detector FID Flame ionisation detector

Thermoquest, formerly Thermo Separation Products and Spectra Physics

: 48 : SOA 001/004850

APPENDIX 4 (continued)

SOA/OOl - STUDY SPECIFIC SUPPLEMENT TO THE INHALATIbN ANALYTICAL PROCEDURE FOR DIMETHYL ADIPATE I)IBiETM’GCVT~TE 1 ,

This supplement details additions and amendments to the procedure to be used for the GC assay of Dimethyl adipate and Dimethyl glutarate obtained from air samples coIlected on the above study.

The assay, incorporating the additions and amendments, is suitable for the analysis of Dimethyl adipate and Dimethyl glutarate, in solution, at concentrationswithin the range of 50 to 1000 pg/ml.

Details given in this supplement supersede those in the compound specific IAP.

I EFFECTIVE DATE : I 8 December 2000 Analytical standard

Name Dimethyl adipate Batch number 5806339-A Purity 98.824% Expiry date Not known Supplier Sponsor

Name Dimethyl giutarate Batch number H93 1363-A Purity 99.61% Expiry date Not known Supplier Sponsor

Method of sample extraction

Volume (ml) of solvent required in sample extraction Sample Group 2 Group 3 Group 4 Bubbler 25 . 25 25 Cascade impactor stage 3 - 8 5 5 5 Cascade impactor stage F 5 5 5

Preparation of standard solutions

Prepare standard solutions in the nominal range 10 to 1000 &ml.

Calibration and Quantification

Calibration of the instrument is performed using standard solutions in the range 50 to 1000 pg/ml or in the range 10 to 50 pg/ml.

Chromatographs

The analysis is performed using chromatograph 9.

49 : /

J -. . - . ._

...... lL. _