RMLAtk



Audited Draft Report

|Study Title |Mutation at the thymidine kinase (tk) locus of mouse lymphoma L5178Y cells (MLA) |

| |using the MicrotitreR fluctuation technique |

|Test Articles |Aluminium hydroxide |

| |Aluminium chloride |

|Author |M Ballantyne BSc |

|Sponsor |European Aluminium Association AISBL – REACH Consortium |

| |12, Avenue de Broqueville |

| |B - Brussels |

|Study Monitor |Eirik Nordheim |

|Test Facility |Covance Laboratories Ltd |

| |Otley Road, Harrogate |

| |North Yorkshire HG3 1PY |

| |ENGLAND |

|Covance Client Identifier |1006855 |

|Covance Study Number |8221367 |

|Report Issued |June 2010 |

|Page Number |1 of 83 |

Study Director Authentication

and GLP Compliance Statement

Aluminium hydroxide and Aluminium chloride: Mutation at the thymidine kinase (tk) locus of mouse lymphoma L5178Y cells (MLA) using the MicrotitreR fluctuation technique

I, the undersigned, hereby declare that the work was performed under my supervision and that the findings provide a true and accurate record of the results obtained.

The study was performed in accordance with the agreed protocol and with Covance Laboratories Limited Standard Operating Procedures, unless otherwise stated, and the study objectives were achieved.

The study was conducted in compliance with the United Kingdom Good Laboratory Practice Regulations 1999, Statutory Instrument No. 3106 as amended by the Good Laboratory Practice (Codification Amendments Etc.) Regulations 2004 and the OECD Principles on Good Laboratory Practice (revised 1997, issued January 1998) ENV/MC/CHEM (98) 17.

| | | |

|M Ballantyne BSc | |Date |

|Study Director |

Quality Assurance Statement

Aluminium hydroxide and Aluminium chloride: Mutation at the thymidine kinase (tk) locus of mouse lymphoma L5178Y cells (MLA) using the MicrotitreR fluctuation technique

This study has been reviewed by the Quality Assurance Unit of Covance Laboratories Ltd. and the report accurately reflects the raw data. The following inspections were conducted and findings reported to the Study Director (SD) and associated management.

Critical procedures, which are performed routinely in an operational area, may be audited as part of a "process" inspection programme. This can be in addition to phases scheduled on an individual study basis. Selected process inspections conducted and considered applicable to this study are included below.

In addition to the inspection programmes detailed below, a facility inspection programme is also operated. Details of this programme, which covers all areas of the facility annually (at a minimum), are set out in standard operating procedures.

Audit dates to be included in the final report.

| | | |

|Representative | |Date |

|Quality Assurance Unit |

reviewing Scientist's Statement

Aluminium hydroxide and Aluminium chloride: Mutation at the thymidine kinase (tk) locus of mouse lymphoma L5178Y cells (MLA) using the MicrotitreR fluctuation technique

I, the undersigned, hereby declare that I have reviewed this report in conjunction with the Study Director and that the interpretation and presentation of the data in the report are consistent with the results obtained.

| | | |

|M Lloyd BSc | |Date |

|Scientist |

Responsible Personnel

Aluminium hydroxide and Aluminium chloride: Mutation at the thymidine kinase (tk) locus of mouse lymphoma L5178Y cells (MLA) using the MicrotitreR fluctuation technique

The following personnel were responsible for key elements of the study:

|Study Director |M Ballantyne |

|Laboratory Supervisor |A-M Massip |

|Study Monitor 1 |E Nordheim |

1 Located at European Aluminium Association AISBL – REACH Consortium, Brussels.

Archive Statement

Aluminium hydroxide and Aluminium chloride: Mutation at the thymidine kinase (tk) locus of mouse lymphoma L5178Y cells (MLA) using the MicrotitreR fluctuation technique

All primary data, or authenticated copies thereof, specimens and the final report will be retained in the Covance Laboratories Limited archives for one year after issue of the final report. At the end of the specified archive period the Sponsor will be contacted to determine whether the data should be returned, retained or destroyed on their behalf. Sponsors will be notified of the financial implications of each of these options at that time.

Contents

Study Director Authentication and GLP Compliance Statement 2

QUALITY ASSURANCE STATEMENT 3

REVIEWING SCIENTIST'S STATEMENT 4

RESPONSIBLE PERSONNEL 5

ARCHIVE STATEMENT 6

CONTENTS 7

SUMMARY 9

INTRODUCTION 14

MATERIALS 16

TEST ARTICLE - ALUMINIUM HYDROXIDE 16

Test article - Aluminium chloride 17

Controls 19

Metabolic activation system 20

Growth media 21

Cell cultures 21

METHODS 22

CYTOTOXICITY RANGE-FINDER EXPERIMENT 22

Investigative Experiment – 24 hour treatments with aluminium hydroxide suspensions 23

Mutation assays 24

Analysis of results 27

RESULTS 31

TOXICITY; ALUMINIUM HYDROXIDE 31

Mutation; aluminium hydroxide 36

Toxicity; aluminium chloride 37

Mutation; aluminium chloride 40

CONCLUSION 42

REFERENCES 43

APPENDICES 45

APPENDIX 1 ALUMINIUM HYDROXIDE: RAW PLATE COUNTS AND DATA ANALYSIS FOR EXPERIMENT 1 IN THE ABSENCE OF S-9 (3 HOUR TREATMENT) 46

Appendix 2 Aluminium hydroxide: Raw plate counts and data analysis for Experiment 1 in the presence of S-9 (3 hour treatment) 50

Appendix 3 Aluminium hydroxide: Raw plate counts and data analysis for Experiment 2 in the absence of S-9 (3 hour treatment) 54

Appendix 4 Aluminium hydroxide: Raw plate counts and data analysis for Experiment 2 in the presence of S-9 (3 hour treatment) 58

Appendix 5 Aluminium chloride: Raw plate counts and data analysis for Experiment 1 in the absence of S-9 (3 hour treatment) 62

Appendix 6 Aluminium chloride: Raw plate counts and data analysis for Experiment 1 in the presence of S-9 (3 hour treatment) 66

Appendix 7 Aluminium chloride: Raw plate counts and data analysis for Experiment 2 in the absence of S-9 (24 hour treatment) 70

Appendix 8 Aluminium chloride: Raw plate counts and data analysis for Experiment 2 in the presence of S-9 (3 hour treatment) 74

Appendix 9 Quality control statements for S-9 78

Appendix 10 Certificates of analysis 82

Appendix 11 Minor deviations from protocol 83

Summary

Aluminium hydroxide and aluminium chloride were assayed for their ability to induce mutation at the tk locus (5-trifluorothymidine [TFT] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9).

All aluminium chloride treatments in this study were performed using formulations in water.

Solubility trials and investigations failed to identify any vehicle in which aluminium hydroxide could be dissolved that was compatible with the assay system. All treatments with this test article were therefore performed in this study using aluminium hydroxide formulations as a suspension in 0.5% methylcellulose (0.5% MC). Because treatment was via a suspension and not a solution, test cells and suspension particles were therefore differentially separated using a Percoll density gradient centrifugation step followed by cell recovery from the Percoll/treatment suspension interphase. This permitted removal of most if not all the test article particles from the cell suspension, and therefore from the resulting cell cultures and platings, however the Aluminium hydroxide treatment concentrations stated may be considered as nominal as the actual exposure of the cells (in terms of dissolved test article) is unknown. Whilst this method was considered acceptable and compatible with 3 hour treatments in the absence and presence of S-9, this methodology did not prove compatible with 24 hour treatments.

A 3 hour treatment incubation period was used for all experiments performed in the presence of S-9. In the absence of S-9, the Range-Finder Experiments with both test articles were performed using 3 and 24 hour treatment incubation periods. For Aluminium chloride treatments in the absence of S-9, Experiment 1 was performed using a 3 hour treatment incubation and Experiment 2 was performed using a 24 hour treatment incubation. For Aluminium hydroxide treatments in the absence of S-9, Experiments 1 and 2 were both performed using a 3 hour treatment incubation.

Aluminium hydroxide

Due to the inclusion of modified methodology (Percoll density gradient centrifugation step) employed for the Aluminium hydroxide treatments in order to permit testing of test article suspensions in this assay system, all Cytotoxicity Range-Finder Experiment treatments with Aluminium hydroxide in this study were also plated and scored for mutation and positive control cultures also included.

In the cytotoxicity Range-Finder Experiment, 3 hour treatment, concentrations (as suspensions in 0.5% MC) ranging from 24.38 to 780 (g/mL were tested, in the absence and presence of S-9. The top concentration, 780 (g/mL, was the maximum concentration selected to be within the range where particulate test article was observed, and corresponds to 10 mM had the test article been tested as a solution. At the highest concentration tested (780 (g/mL), relative total growth (RTG) values were 92% and 66% in the absence and presence of S-9 respectively, i.e. only slight or moderate toxicity was induced.

A cytotoxicity Range-Finder Experiment was attempted with a 24 hour treatment in the absence of S-9, in which concentrations (as suspensions in 0.5% MC) ranging from 3.047 to 780 (g/mL were tested. At the highest concentration tested (780 (g/mL), the RTG value was 61%. However, notable reductions in suspension growth (SG) were observed with these 24 hour treatments across the concentration range, as were sporadic (not treatment concentration-related) increases in mutant frequency (MF). These results were interpreted as being due to particles of the test article interfering with normal cell growth at all concentrations tested. Accordingly, it was considered that despite the Percoll density gradient centrifugation step, included post treatment to remove the test material from the assay cultures, particles of the test material had inhibited cell growth during the prolonged 24 hour treatment period. These effects sufficiently confounded the ability for these prolonged treatments to provide a reliable assessment of the cytotoxicity and genotoxicity of the test material under such treatment conditions that a 24 hour treatment period using a test article suspension was not considered an appropriate or valid treatment methodology. Both Mutation Experiments with Aluminium hydroxide were therefore conducted using 3 hour treatments in the absence and presence of S-9.

Accordingly, for Experiment 1, concentrations ranging from 6.094 to 780 (g/mL, were tested in the absence and presence of S-9. All concentrations were considered acceptable to determine viability and TFT resistance, and the highest treatment concentration (780 (g/mL) gave 101% and 93% RTG in the absence and presence of S-9 respectively, i.e. there was no detectable cytotoxic effect.

In Experiment 2, concentrations ranging from 6.094 to 780 (g/mL in the absence and presence of S-9 were tested. All concentrations were considered acceptable to determine viability and TFT resistance, and the highest treatment concentration (780 (g/mL) gave 92% and 62% RTG in the absence and presence of S-9 respectively, i.e. slight to moderate cytotoxic effects were induced.

Negative (vehicle and untreated) and positive control treatments were included in each Mutation Experiment in the absence and presence of S-9. Mutant frequencies in negative control cultures were all considered acceptable, and clear increases in mutation were induced by the positive control chemicals methyl methane sulphonate (without S-9) and benzo[a]pyrene (with S-9). Therefore the study was accepted as valid.

The mutant frequencies of the concentrations of aluminium hydroxide plated were all less than the sum of the mean control mutant frequency plus the global evaluation factor (GEF). A significant linear trend was observed following Experiment 2 treatments in the presence of S-9 only, but this observation alone (without any corresponding increases in mutant frequencies approaching the GEF) is not considered a biologically relevant observation, and this study was therefore considered to have provided a negative result for Aluminium hydroxide treatments in this assay system.

Aluminium chloride

In the cytotoxicity Range-Finder Experiment, 3 hour treatment, concentrations ranging from 41.69 to 1334 (g/mL (equivalent to 10 mM at the highest concentration tested) were tested, in the absence and presence of S-9. Precipitation of test article at the end of the treatment period was observed in all cultures treated at 83.38 (g/mL and above, and this was therefore the highest concentration analysed for cytotoxicity, which gave 134% and 76% RTG in the absence and presence of S-9 respectively, i.e. negligible to slight induction of cytotoxicity.

In the cytotoxicity Range-Finder Experiment, 24 hour treatment, concentrations ranging from 5.211 to 1334 (g/mL were tested in the absence of S-9. Precipitation of test article at the end of the treatment period was observed in all cultures treated at 83.38 (g/mL and above, and this was therefore the highest concentration analysed for cytotoxicity, which gave 64% RTG, i.e. moderate induction of cytotoxicity.

Accordingly, for Experiment 1, concentrations ranging from 3.125 to 100 (g/mL were tested in the absence and presence of S-9. Precipitation of test article persisting to the end of the treatment period was observed at 50 (g/mL and above in the absence and presence of S-9. This concentration was therefore the highest concentration selected to determine viability and TFT resistance, which gave 163% and 120% RTG in the absence and presence of S-9 respectively, i.e. no induction of cytotoxicity.

In Experiment 2, concentrations ranging from 5 to 120 (g/mL in the absence of S-9 (24 hour treatment), and from 5 to 80 (g/mL in the presence of S-9 (3 hour treatment), were tested. Precipitation of test article persisting to the end of the treatment period was observed at 120 (g/mL in the absence of S-9 and at 50 (g/mL and above in the presence of S-9. These were therefore the highest concentrations selected for analysis, which gave 97% and 94% RTG in the absence and presence of S-9 respectively, i.e. negligible induction of cytotoxicity.

Negative (vehicle) and positive control treatments were included in each Mutation Experiment in the absence and presence of S-9. Mutant frequencies in negative control cultures fell within acceptable ranges, and clear increases in mutation were induced by the positive control chemicals methyl methane sulphonate (without S-9) and benzo[a]pyrene (with S-9). Therefore the study was accepted as valid.

The mutant frequencies of the concentrations plated were all less than the sum of the mean control mutant frequency plus the global evaluation factor (GEF) with a negative linear trend, indicating a negative result for Aluminium chloride in this assay system.

Conclusion

It is concluded that Aluminium chloride did not induce mutation at the tk locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to precipitating concentrations in two independent experiments, in the absence and presence of a rat liver metabolic activation system (S-9). A 3 hour treatment incubation period was used for all treatments in the presence of S-9 and Experiment 1 treatments in the absence of S-9, and a 24 hour treatment incubation period used for Experiment 2 treatments in the absence of S-9.

When tested in the same test system, Aluminium hydroxide did not induce mutation at the tk locus of L5178Y mouse lymphoma cells when tested under the modified treatment conditions employed in this study. These modified conditions included treatments using test article suspensions (in 0.5% MC) at concentrations up to 780 (g/mL in two independent experiments, using a 3 hour treatment incubation period in the absence and presence of a rat liver metabolic activation system (S-9) throughout. This top concentration was the maximum selected to be within the range where particulate test article was observed, and corresponded to 10 mM had the test article been tested as a solution. The assay methodology was modified by the inclusion of a Percoll differential centrifugation step and cell recovery after the treatment incubation period, in order to remove as much of the particulate test material as possible.

INTRODUCTION

The tk (thymidine kinase) locus in mouse lymphoma L5178Y cells is capable of detecting both gene mutations and chromosome aberrations. The mutation system works by placing treated cells under selective pressure so that only mutant cells are able to survive. The tk locus is autosomal and the L5178Y cell line is heterozygous (tk+/-), producing the enzyme thymidine kinase. This enzyme is a salvage enzyme for nucleic acid breakdown products but if a toxic base analogue (5-trifluorothymidine, TFT) is present in the medium, the enzyme will incorporate the analogue into the cells. Thus the cells die unless the enzyme is rendered inactive, by mutation for example. Resistance to TFT results from a lack of thymidine kinase (TK) activity. Thus, the mutants (tk-/-) are able to survive, divide and form colonies in the presence of TFT.

Two types of TFT-resistant mutant colonies are selected and these are designated large colonies and small (slow-growing) colonies. Molecular analysis has indicated that the large colonies tend to represent events within the gene (base-pair substitutions and deletions) whereas small colony mutants often involve large genetic changes frequently visible as chromosome aberrations ([[i]], [[ii]]). Thus, in this system, gene mutations within the tk gene (11 to 13 kilobases) and chromosomal events involving the gene may be detected. The tk system has a high spontaneous mutant frequency and because of the high numbers of cells that can be treated and sampled it is the most satisfactory mammalian cell mutation assay from the statistical point of view. Furthermore, a fluctuation protocol has been developed for use with the assay ([[iii]], [[iv]]). The protocol described here is consistent with the consensus agreements regarding protocol issues discussed during successive Mouse Lymphoma Workshops, ([[v]], [[vi]], [[vii]], [[viii]], [[ix]]).

The objective of this study was to evaluate the potential of Aluminium hydroxide and Aluminium chloride to induce forward mutations in the absence and presence of a rat liver metabolising system (S-9). The methodology in this study complies with OECD Test Guideline 476 (1997) [[x]], the UKEMS Guidelines (1990), the ICH Tripartite Harmonised Guideline on Genotoxicity S2A: Specific Aspects of Regulatory Tests (1995) [[xi]] and ICH Requirements for Registration of Pharmaceuticals for Human Use S2B, Genotoxicity: a Standard Battery for Genotoxicity Testing of Pharmaceuticals (Step 4, recommended for adoption 16 July 1997).

Aluminium hydroxide treatments in this study were performed using a modified assay methodology as treatments were performed using suspensions of test article rather than treatment solutions. A differential centrifugation step (followed by cell recovery) was included at the end of the treatment period in order that valid mutation data could be obtained with this assay system whilst employing treatments with a test article suspension rather than a solution.

This study was performed according to the protocol and two amendments, with the exception of the minor deviations detailed in Appendix 11, none of which in any way prejudiced the validity of the study.

The study was initiated on 7 December 2009. Experimental work started on 15 December 2009 and was completed on 11 May 2010. The study completion date is considered to be the date the Study Director signs the final report.

MATERIALS

Test article - Aluminium hydroxide

Aluminium hydroxide, batch number 40/2009, was a white powder with a molecular weight of 78. It was received on 2 December 2009 and stored at 15 to 25(C in the dark. Purity was stated as 99%, and the Sponsor indicated that this test article is inherently stable and has provided a nominal expiry date of 31 December 2010. The certificates of analysis, provided by the Sponsor, are presented in Appendix 10. The test article information and certificates of analysis provided by the Sponsor are considered an adequate description of the characterisation, stability and purity of the test article. Determinations of the stability and characteristics of the test article were the responsibility of the Sponsor.

Preliminary solubility data indicated that aluminium hydroxide was not soluble in any commonly used or assay-compatible vehicle, but formed a homogeneous suspension in 0.5% methylcellulose (0.5% MC). In order that as much as possible of the test article particles were removed from treatment culture (to avoid continued exposure of the test cells throughout the expression period and possibly even after cell plating) a modified methodology was employed. At the end of the treatment period test cells and suspension particles (treated cultures) were differentially separated using a Percoll density gradient centrifugation step followed by cell recovery from the Percoll/treatment suspension interphase. By using this modified methodology it was considered that valid mutation data could be obtained with this assay system whilst employing treatments with a test article suspension rather than a solution.

A maximum suspension concentration of 780 (g/mL was selected and employed for the cytotoxicity Range-Finder Experiment. This maximum concentration was selected as it corresponds to 10 mM had the test article been tested as a solution (the maximum recommended concentration according to current regulatory guidelines [11]) and to test into the range where particulate test article was observed. Concentrations for the mutation experiments were selected based on the results of this cytotoxicity Range-Finder Experiment.

Test article stock suspensions were prepared under subdued lighting by formulating Aluminium hydroxide in 0.5% MC with the aid of stirring and Silverson homogeniser, to give the maximum required treatment suspension concentration, although all Aluminium hydroxide concentrations may be considered as nominal as the actual exposure of the cells (in terms of dissolved test article) is unknown. Subsequent dilutions were made using 0.5% MC. The test article formulations were protected from light and used within approximately 2 hours of initial formulation as shown in Table 1.

Test article - Aluminium chloride

Aluminium chloride, batch number MKBB8304, was a white powder with a molecular weight of 133.34 and obtained directly from Sigma-Aldrich Chemical Co. It was received on 4 December 2009 and stored at 15 to 25(C in the dark. Purity was stated as 99.999% and the expiry date was given as 4 December 2010. The certificate of analysis provided by the supplier, is presented in Appendix 10. The test article information and certificate of analysis provided are considered an adequate description of the characterisation, purity and stability of the test article. Determinations of the stability and characteristics of the test article were the responsibility of the Sponsor.

Preliminary solubility data indicated that aluminium chloride was soluble in water for irrigation (purified water) at concentrations up to at least 30.50 mg/mL. The solubility limit in culture medium was in excess of 3050 (g/mL, as indicated by a lack of precipitation at this concentration. A maximum concentration of 1334 (g/mL was selected for the cytotoxicity Range-Finder Experiment, in order that treatments were performed up to 10 mM, the maximum recommended concentration according to current regulatory guidelines [11]. Concentrations for Experiment 1 were selected based on the results of this cytotoxicity Range-Finder Experiment.

Test article stock solutions were prepared under subdued lighting by formulating Aluminium chloride in purified water with the aid of vortex mixing, as required to give the maximum required treatment solution concentration. The stock solutions were membrane filter-sterilised (Pall Acrodisc 32 filter, 0.2 (m pore size) and subsequent dilutions made using purified water. The test article solutions were protected from light and used within approximately 2 hours of initial formulation as shown in Table 1:

Table 1: Aluminium hydroxide Concentrations Tested

|Experiment | S-9 |Treatment period |Concentration of treatment |Final concentration ((g/mL) |

| | |(hours) |suspension(mg/mL) | |

| | | | | |

|Range-Finder |( and + |3 |0.2438 |24.38 |

| | | |0.4875 |48.75 |

| | | |0.9750 |97.5 |

| | | |1.950 |195 |

| | | |3.900 |390 |

| | | |7.800 |780 |

| | | | | |

| |( |24 |0.03047 |3.047 |

| | | |0.06094 |6.094 |

| | | |0.1219 |12.19 |

| | | |0.2438 |24.38 |

| | | |0.4875 |48.75 |

| | | |0.9750 |97.5 |

| | | |1.950 |195 |

| | | |3.900 |390 |

| | | |7.800 |780 |

| | | | | |

|1 and 2 |( and + |3 |0.06094 |6.094 |

| | | |0.1219 |12.19 |

| | | |0.2438 |24.38 |

| | | |0.4875 |48.75 |

| | | |0.975 |97.5 |

| | | |1.95 |195 |

| | | |3.90 |390 |

| | | |7.80 |780 |

| | | | | |

|Investigative experiment |( |24 |0.03 |3 |

| | | |0.24 |24 |

| | | |0.96 |96 |

| | | |7.80 |780 |

| | | | | |

Table 2: Aluminium chloride Concentrations Tested

|Experiment | S-9 |Treatment period|Concentration of treatment |Final concentration ((g/mL) |

| | |(hrs) |solution (mg/mL) | |

| | | | | |

|Range-Finder |( and + |3 |0.4169 |41.69 |

| | | |0.8338 |83.38 |

| | | |1.668 |166.8 |

| | | |3.335 |333.5 |

| | | |6.670 |667 |

| | | |13.34 |1334 |

| | | | | |

| |

|Table continued overleaf |

|Range-Finder |( |24 |0.05211 |5.211 |

| | | |0.1042 |10.42 |

| | | |0.2085 |20.85 |

| | | |0.4169 |41.69 |

| | | |0.8338 |83.38 |

| | | |1.668 |166.8 |

| | | |3.335 |333.5 |

| | | |6.67 |667 |

| | | |13.34 |1334 |

| | | | | |

|1 |( and + |3 |0.03125 |3.125 |

| | | |0.0625 |6.25 |

| | | |0.125 |12.5 |

| | | |0.25 |25 |

| | | |0.50 |50 |

| | | |1.00 |100 |

| | | | | |

|2 |( |24 |0.05 |5 |

| | | |0.10 |10 |

| | | |0.20 |20 |

| | | |0.40 |40 |

| | | |0.60 |60 |

| | | |0.80 |80 |

| | | |1.00 |100 |

| | | |1.20 |120 |

| | | | | |

| |+ |3 |0.05 |5 |

| | | |0.10 |10 |

| | | |0.15 |15 |

| | | |0.20 |20 |

| | | |0.25 |25 |

| | | |0.30 |30 |

| | | |0.40 |40 |

| | | |0.50 |50 |

| | | |0.60 |60 |

| | | |0.70 |70 |

| | | |0.80 |80 |

| | | | | |

It may be noted that the 24 hour treatments with Aluminium chloride in the absence of S-9 in Experiment 2 were repeated due to failure to achieve an acceptable concentration level in the initial experiment. All data presented in this report are from this repeat assay.

Controls

Negative controls comprised treatments with the vehicle 0.5% MC or purified water for Aluminium hydroxide and Aluminium chloride respectively, diluted 10-fold in the treatment medium. The positive control chemicals were supplied and used as shown in the table below (100-fold dilution).

For Aluminium hydroxide, further ‘untreated’ negative controls were included (media only added), and additional positive controls were included which were not subjected to the additional Percoll density gradient differential centrifugation and cell recovery steps. Untreated negative controls (media only added) were also performed and subjected to the Percoll density gradient differential centrifugation and cell recovery steps. All these additional controls were designed to provide information on the compatibility of the Percoll differential centrifugation and cell recovery steps, by allowing any effects on cell numbers, toxicity and mutation frequency to be assessed.

Table 3: Positive Controls

|Chemical*** |Stock* concentration (mg/mL) |Final concentration ((g/mL) |S-9 |

| | | | |

|Methyl methane sulphonate |1.50 |15.0** |- |

|(MMS) |2.00 |20.0** |- |

| | | | |

|Benzo[a]pyrene |0.050 |0.50 |+ |

|(B[a]P)a |0.100 |1.00 |+ |

| |0.200 |2.00 |+ |

| |0.300 |3.00 |+ |

| | | | |

|* All solutions were prepared in anhydrous analytical grade dimethyl sulphoxide (DMSO). B[a]P solutions, if not used |

|immediately, were stored as frozen aliquots at –80ºC nominal in the dark. |

|** For Experiment 2 in the absence of S-9 (24 hour treatment) for Aluminium chloride, final MMS concentrations of 5 and |

|7.5 (g/mL were tested. |

|*** Obtained from Sigma-Aldrich Chemical Co, Poole, UK. |

|a Final concentrations of 0.5 and 1 (g/mL of B[a]P were tested in treatments of Aluminium hydroxide in the presence of S-9 |

|only. |

Metabolic activation system

The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA, where it was prepared from male Sprague Dawley rats induced with Aroclor 1254. The batches of MolToxTM S-9 were stored frozen in aliquots at -80°C nominal prior to use. Each batch was checked by the manufacturer for sterility, protein content, ability to convert known promutagens to bacterial mutagens and cytochrome P-450-catalyzed enzyme activities (alkoxyresorufin-O-dealkylase activities). The quality control statements, relating to the batches of S-9 preparation used, are included in Appendix 9 of this report.

Treatment was carried out both in the absence and presence of S-9, prepared in the following way:

Glucose-6-phosphate (180 mg/mL), (-nicotinamide adenine dinucleotide phosphate (NADP) (25 mg/mL), potassium chloride (KCl) (150 mM) and rat liver S-9 were mixed in the ratio 1:1:1:2. For all cultures treated in the presence of S-9, a 1 mL aliquot of the mix was added to each cell culture (19 mL) to give a total of 20 mL. The final concentration of the liver homogenate in the test system was 2%. Cultures treated for 3 hours in the absence of S-9 received 1 mL KCl (150 mM).

Growth media

RPMI 1640 media were prepared as follows:

Table 4: Growth Media

| |Final concentration in: |

| |RPMI A |RPMI 10 |RPMI 20 |

| | | | |

|Horse serum |0% v/v |10% v/v |20% v/v |

|(heat inactivated) | | | |

| | | | |

|Penicillin / |100 units/mL / |100 units/mL / |100 units/mL / |

|streptomycin |100 (g/mL |100 (g/mL |100 (g/mL |

| | | | |

|Amphotericin B |2.5 (g/mL |2.5 (g/mL |2.5 (g/mL |

| | | | |

|Pyruvic acid |0.2 mg/mL |0.2 mg/mL |0.2 mg/mL |

| | | | |

|Pluronic |0.5 mg/mL |0.5 mg/mL |- |

| | | | |

Heat inactivated horse-serum was used in order to eliminate a factor which degrades TFT [[xii]].

Cell cultures

The master stock of L5178Y tk+/- (3.7.2C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Covance Laboratories Ltd. were stored as frozen stocks in liquid nitrogen. Full details of the supplier are documented in central records. Each batch of frozen cells was purged of tk- mutants, checked for spontaneous mutant frequency and that it was mycoplasma free. For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5% (v/v) CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of flasks.

METHODS

Cytotoxicity Range-Finder Experiment

Treatment and post-treatment dilution of cell cultures for the cytotoxicity Range-Finder Experiments with each test article were as described below for the Mutation Experiments. However, single cultures only were used and positive controls were not included for the Aluminium chloride assay. The final treatment culture volume was 20 mL. In the absence of S-9, 3 and 24 hour treatment incubation periods were used, in the presence of S-9 a 3 hour treatment incubation was used.

Following 3 hour treatment, cells were centrifuged (200 g) for 5 minutes, washed with tissue culture medium and then resuspended in 50 mL RPMI 10.

Following 24 hour treatment, cultures were centrifuged (200 g) for 5 minutes, washed and resuspended in 20 mL RPMI 10. Cell densities were determined using a Coulter counter and adjusted to 2 x 105 cells/mL.

For the aluminium hydroxide treatments and associated controls only, cultures were also subject to plating for TFT resistance and scoring for mutation, as detailed below for the mutation assays.

The aluminium hydroxide treatments and associated controls (except those specified in the control section) were also subject to the following differential centrifugation and cell recovery steps at the end of the treatment phase:

Following the appropriate treatment period, cultures were centrifuged (200 g) for 5 minutes). The resulting cell pellets (with or without test article particles) were resuspended in 0.5 mL RPMI 10 and gently overlaid onto 10 mL 65% Percoll (6.5 mL 100% stock Percoll:3.5 mL sterile water) in a 15 mL tube to create distinct Percoll and cell suspension layers. The culture tube was then washed with a further 0.5 mL RPMI 10, to ensure maximal cell recovery, and this was carefully added to the culture overlay maintaining the layer integrity. Cultures were centrifuged (400 g) for 20 minutes (the centrifuge brake was disabled to minimise any cell layer disruption). L5178Y cells were harvested from the media:Percoll interface by removal of the top 2-3 mL of contents from each tube (see Minor deviations from protocol, Appendix 11). The removed 2-3 mL aliquot from each tube was transferred to a fresh 50 mL centrifugation tube. 40 mL RPMI 10 was added to each tube and centrifuged (200 g) for 7 minutes, and the supernatant (containing the Percoll) discarded. The resulting cell pellets were then treated in the same way as the post treatment wash off.

All cultures were incubated at 37±1°C for 1 day, recounted and diluted to 2 x 105 cells/mL. Cultures were incubated for a further day, counted and adjusted to 8 cells/mL and, for each concentration, 0.2 mL was plated into each well of a 96-well microtitre plate for determination of viability (plating efficiency). All 96-well plates were incubated at 37±1°C in a humidified incubator gassed with 5% v/v CO2 in air for 7 to 11 days. Wells containing viable clones were identified by eye using background illumination and counted.

There was an apparent dilution error with the viability plating for the Aluminium chloride vehicle control culture of the 3 hour treatments in the absence of S-9. This resulted in a zero viability plate count for this control treatment, and therefore no RTG value could be calculated. In order to provide a baseline toxicity measure (100% RTG value) against which other treatment concentrations could be compared, the cell count and viability data from the lowest treatment concentration (41.69 µg/mL) were considered a relevant substitute for the vehicle control data (ie to provide a 100% RTG baseline), see Minor deviations from protocol, Appendix 11.

Changes in osmolality of more than 50 mOsm/kg and fluctuations in pH of more than one unit may be responsible for an increase in mutant frequencies ([[xiii]], [[xiv]]). Osmolality and pH measurements on post-treatment incubation medium were taken in the cytotoxicity Range-Finder Experiment for both Aluminium hydroxide and Aluminium chloride.

Investigative Experiment – 24 hour treatments with Aluminium hydroxide suspensions

Following sporadic and non-concentration related increases in mutation frequency values for 24 hour treatments in the absence of S-9 in the range-finder experiment with aluminium hydroxide, a further investigative experiment was conducted to better assess the physical effect 24 hour treatments with suspensions of aluminium hydroxide may have had, with a view to establishing whether these physical effects were sufficiently confounding to prevent reliable data being obtained when conducting 24 hour treatments with particulate test material.

Treatments were performed as previously described for the range-finder experiment (24 hour treatments in the absence of S-9 only), using duplicate cultures and with only negative vehicle controls and no positive controls. At various times during the 24 hour treatment period (at approximately 3, 6 and 24 hours after the start of the treatment period) the treatment cultures were sampled and examined microscopically for cell morphology, the presence/adherence of any particles to the test cells, and any other noteworthy observations.

Similar sampling and observations were also made following the Percoll differential centrifugation step and on Day 1 and Day 2 of the expression period. Cells were subjected to cell counts during the expression period, but were not plated for either toxicity or mutation assessment. No cell count, toxicity or end-point data are reported for these treatments.

Mutation assays

Treatment of cell cultures

For 3 hour treatments in Experiment 1 in the absence and presence of S-9, and in Experiment 2 in the presence of S-9 (and also in the absence of S-9 for Aluminium hydroxide), at least 107 cells in a volume of 17 mL tissue culture medium (cells in RPMI 10 diluted with RPMI A [no serum] to give a final concentration of 5% serum) were used. For Experiment 2 in the absence of S-9 (24 hour treatment - Aluminium chloride only) at least 4 x 106 cells in a volume of 18 mL RPMI 10 were used. The cell suspensions were placed in a series of appropriate sterile disposable containers which were gassed with 5% v/v/ CO2 in air. For all treatments 2 mL vehicle, untreated control, test article or 0.2 mL positive control solution plus 1.8 mL of water were added. For 3 hour treatments, S-9 mix or 150 mM KCl was added, as described. Each treatment, in the absence or presence of S-9, was in duplicate (single cultures only used for positive control treatments) and the final treatment culture volume was 20 mL.

3 hour treatment: After 3 hours incubation at 37±1°C with gentle agitation, cultures were centrifuged (200 g) for 5 minutes, washed and resuspended in 50 mL fresh RPMI 10 medium. For the aluminium hydroxide treatments (and associated controls), the additional steps for differential centrifugation using Percoll described previously were included.

24 hour treatment: After static incubation at 37±1°C for 24 hours, cultures were centrifuged (200 g) for 5 minutes, washed and resuspended in fresh RPMI 10 medium (20 mL). Cell densities were determined using a Coulter counter and, where sufficient cells survived, adjusted to 2 x 105 cells/mL.

Cells were transferred to tissue culture flasks for growth throughout the expression period. The solubility of the test article in culture was assessed, by eye, at the beginning and end of treatment.

It should be noted that initial 24 hour treatments in the absence of S-9 with Aluminium chloride in Experiment 2 were performed up to a maximum treatment concentration of 80 (g/mL. As no precipitation was observed following the treatment period at any of the concentrations tested, these treatments were considered invalid as they failed to test up to an acceptable limit, and the experiment was terminated at the end of the treatment period (with no toxicity or mutation data generated). These treatments are therefore not reported further. Repeat 24 hour treatments in the absence of S-9 with Aluminium chloride were performed, up to a higher maximum treatment concentration of 120 (g/mL, and it is the data from these repeat treatments that are reported as the Aluminium chloride 24 hour data in the absence of S-9 for Experiment 2.

Expression period

Cultures were maintained in flasks for a period of 2 days during which the tk-/- mutation would be expressed. During the expression period, subculturing was performed as required with the aim of not exceeding 1 x 106 cells per mL and, where possible, retaining at least 1 x 107 cells/flask. From observations on recovery and growth of the cultures during the expression period, the following cultures were selected to be plated for viability and TFT resistance:

Table 5: Cultures selected for mutation assessment - aluminium hydroxide

|Experiment 1 ((g/mL) |Experiment 2 ((g/mL) |

|- S-9 |+ S-9 |- S-9 |+ S-9 |

|0 |0 |0 |0 |

|UTC |UTC |UTC |UTC |

|6.094 |6.094 |6.094 |6.094 |

|12.19 |12.19 |12.19 |12.19 |

|24.38 |24.38 |24.38 |24.38 |

|48.75 |48.75 |48.75 |48.75 |

|97.5 |97.5 |97.5 |97.5 |

|195 |195 |195 |195 |

|390 |390 |390 |390 |

|780 |780 |780 |780 |

| | | | |

|MMS 15 |B[a]P 0.5 |MMS 15 |B[a]P 0.5 |

|MMS 20 |B[a]P 1 |MMS 20 |B[a]P 1 |

| |B[a]P 2 | |B[a]P 2 |

| |B[a]P 3 | |B[a]P 3 |

| | | | |

UTC Untreated controls

Table 6: Cultures selected for mutation assessment - aluminium chloride

|Experiment 1 ((g/mL) |Experiment 2 ((g/mL) |

| | | | |

|- S-9 3 hour |+ S-9 |- S-9 24 hour |+ S-9 |

| | | | |

|0 |0 |0 |0 |

|3.125 |3.125 |5 |5 |

|6.25 |6.25 |10 |10 |

|12.5 |12.5 |20 |15 |

|25 |25 |40 |20 |

|50 |50 |60 |25 |

| | |80 |30 |

| | |100 |40 |

| | |120 |50 |

| | | | |

|MMS 15 |B[a]P 2 |MMS 5 |B[a]P 2 |

|MMS 20 |B[a]P 3 |MMS 7.5 |B[a]P 3 |

| | | | |

Plating for viability

At the end of the expression period, cell concentrations in the selected cultures were determined using a Coulter counter and adjusted to give 1 x 104/mL in readiness for plating for TFT resistance. Samples from these were diluted to 8 cells/mL as follows (see also Minor deviations from protocol, Appendix 11):

Table 7: Plating for Viability

| |Initial cell |Dilution |Intermediate cell |Dilution |Final cell conc |

| |conc (A) | |conc (B) | |(C) |

| | |mL |mL Medium | |mL |mL RPMI 20 | |

| | |A | | |B | | |

| | | | | | | | |

|Viability |1 x 104/mL |0.5 |9.5 |5 x 102/mL |0.8 |50 |8/mL |

| | | | | | | | |

Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells averaging 1.6 cells per well). The plates were incubated at 37±1(C in a humidified incubator gassed with 5% v/v CO2 in air until scoreable (8 to 9 days). Wells containing viable clones were identified by eye using background illumination and counted.

Plating for TFT resistance

At the end of the expression period, the cell densities in the selected cultures were adjusted to 1 x 104/mL. TFT (300 (g/mL) was diluted 100-fold into these suspensions to give a final concentration of 3 (g/mL. Using an eight-channel pipette, 0.2 mL of each suspension was placed into each well of four 96-well microtitre plates (384 wells at 2 x 103 cells per well). Plates were incubated at 37(1(C in a humidified incubator gassed with 5% v/v CO2 in air until scoreable (12 to 14 days) and wells containing clones were identified as above and counted. In addition, the number of wells containing large colonies and the number containing small colonies were scored for the negative and positive controls.

Analysis of results

All calculations were performed either manually or by computer using validated software.

Suspension Growth (SG) is a measure of the growth in suspension during treatment and the expression period.

SG was calculated as follows:

[pic][pic]

NB for three hour treatments "a" is assumed to equal 1.

Usually the denominators for "b" and "c" are 2 x 105 cells/mL. However, if cytotoxicity causes the cell count to be lower than 2 x 105 cells/mL following treatment and/or if the cells do not grow during part of the expression period, it can be lower. In these cases, the respective cell count values were entered into the calculation above.

Relative suspension growth (RSG) is a measure of the growth in suspension during treatment and the expression period relative to the mean control.

RSG was calculated as follows overleaf:

[pic]

Viability is the measure of the cells' ability to clone i.e. Cloning efficiency (CE).

CE was calculated as follows:

For microtitre plate tests, calculations are based on P(0), the proportion of wells in which a colony has not grown:

[pic]

The CE for each culture was calculated according to the following calculation:

[pic]

* Number of cells per well is 1.6 cells per well on average on all viability plates.

Relative Total Growth (RTG) is the measure of cytotoxicity relative to the control, that takes into account all cell growth, and cell loss during the treatment period and the 2 day expression period (RSG), and the cells' ability to clone 2 days after treatment (viability).

RTG was calculated as follows:

[pic]

Mutant frequency (MF) was calculated as follows:

MF = [pic]

* Number of cells per well is 2000 cells per well on average on all mutant plates.

Small and large colony mutant frequencies were calculated in an identical manner, using the relevant number of empty wells for small and large colonies, as appropriate.

The significance of increases in mutant frequencies (total wells with clones), by comparison with concurrent controls and the global evaluation factor (GEF), was assessed according to the recommendations of the Mouse Lymphoma Workgroup, Aberdeen, 2003 [8]. The control mutant frequency was compared with each test article treatment and the data were checked for a linear trend in mutant frequency with treatment concentration using weighted regression. The test for linear trend is one-tailed, therefore negative trend was not considered significant.

For microwell assays, the GEF is defined as 126 mutants per 106 viable cells.

Acceptance criteria

The assay was considered valid if all the following criteria were met:

1. The mean mutant frequencies in the negative (vehicle) control cultures fell within the normal range (50 to 170 mutants per 106 viable cells)

2. At least one positive control should show either an absolute increase in mean total MF of at least 300 x 10-6 (at least 40% of this should be in the small colony MF), or an increase in small colony mutant frequency of at least 150 x 10-6 above the concurrent vehicle control

3. The RTG for the positive controls should be greater than 10%

4. The mean cloning efficiencies of the negative controls from the Mutation Experiments were between the range 65% to 120% on Day 2

5. The mean suspension growth of the negative controls from the Mutation Experiments was between the range 8 to 32 following 3 hour treatments or between 32 and 180 following 24 hour treatments

6. There should be no excessive heterogeneity between replicate cultures.

Evaluation criteria

For valid data, a test article was considered to be mutagenic in this assay if:

1. The MF of any test concentration exceeded the sum of the mean control mutant frequency plus GEF.

2. The linear trend test was positive.

A test article was considered as positive in this assay if both of the above criteria were met.

A test article was considered as negative in this assay if neither of the above criteria were met.

Results which only partially satisfied the assessment criteria described above were considered on a case-by-case basis.

RESULTS

Toxicity; aluminium hydroxide

In the cytotoxicity Range-Finder Experiment, 3 hour treatment, six concentrations were tested in the absence and presence of S-9 ranging from 24.38 to 780 (g/mL. The latter was the maximum concentration selected to be within the range where particulate test article was observed and corresponding to 10 mM had the test article been tested as a solution. Upon addition of the test article to the cultures, particulate test article was observed (with the naked eye) at the highest concentration tested in the absence and presence of S-9 (780 (g/mL). Following the 3 hour treatment incubation period, particulate test article was observed by eye at the highest five concentrations tested in the absence of S-9 (48.75 to 780 (g/mL), and at the highest four concentrations test in the presence of S-9 (97.5 to 780 (g/mL). All cultures in the absence and presence of S-9 were retained as the test article was treated as a suspension, and observations relating to the presence of particulate test article were therefore not considered as dose-limiting effects. The highest concentration tested was 780 (g/mL in the absence and presence of S-9, which gave 92% and 66% RTG respectively. The RTG and MF values are shown in Table 8. There were no clear differences between the RTG and MF values of the vehicle and untreated controls, indicating that the vehicle was compatible with the assay system (0.5% MC). There was generally slightly greater toxicity (as indicated by a lower RTG value) for the controls subject to the Percoll differential centrifugation and cell recovery steps compared to the corresponding controls not subject to these steps, indicating that these steps may cause some slight toxicity, but these effects were considered minimal and acceptable and there was no appreciable effect on MF values.

Table 8: RTG Values - 3 hour Range-Finder Experiment, aluminium hydroxide

|Treatment |-S-9 |Treatment |+S-9 |

|(µg/mL) | |(µg/mL) | |

| |%RTG |MF§ | |%RTG |MF§ |

|0 | |100 |79.30 | |0 | |100 |105.42 | |

|UTC | |89 |77.63 | |UTC | |87 |97.36 | |

|UTC (Std) | |158 |47.28 | |UTC (Std) | |128 |89.60 | |

|24.38 | |94 |101.77 | |24.38 | |82 |75.63 | |

|48.75 |PP |109 |71.44 | |48.75 | |64 |107.87 | |

|97.5 |PP |89 |78.78 | |97.5 |PP |69 |122.10 | |

|195 |PP |77 |84.21 | |195 | |X |X | |

|390 |PP |97 |57.90 | |390 |PP |68 |91.09 | |

|780 |P, PP |92 |89.21 | |780 |P, PP |66 |78.61 | |

|Linear trend |NS |Linear trend |NS |

|MMS | | | | |B[a]P | | | | |

|15 | |45 |624.04 | |2 | |2 |1878.36 | |

|20 | |27 |1153.67 | |3 | |1 |1800.46 | |

|15 (Std) | |62 |865.01 | |2 (Std) | |12 |1411.83 | |

|20 (Std) | |50 |1175.25 | |3 (Std) | |2 |1202.02 | |

§ 5-TFT resistant mutants/106 viable cells 2 days after treatment

Std Standard (Non-Percoll modified) treatments not subject to the density gradient separation steps

%RTG Percentage Relative Total Growth

NS Not significant

P Particulate test article observed at time of treatment

PP Particulate test article observed following treatment incubation period

X No cells present. Assumed technical error during treatment - no data for this concentration

In the cytotoxicity Range-Finder Experiment, 24 hour treatment, nine concentrations were tested in the absence of S-9 ranging from 3.047 to 780 (g/mL. Upon addition of the test article to the cultures, particulate test article was observed (with the naked eye) at the highest concentration tested (780 (g/mL). Following the 24 hour treatment incubation period, particulate test article was observed at the highest five concentrations tested (48.75 to 780 (g/mL). All cultures in the absence and presence of S-9 were retained as the test article was treated as a suspension, and observations relating to the presence of particulate test article were therefore not considered as dose-limiting effects. The highest concentration tested was 780 (g/mL, which gave 61% RTG. The RTG values are shown in Table 9. Here the slight increase in toxicity with the controls subject to the Percoll differential centrifugation and cell recovery steps compared to the corresponding controls not subject to these steps described above for the 3 hour treatments was again evident, but again these effects were sufficiently minimal as to be considered as acceptable, and there was no appreciable effect on MF values. There were however clear effects on the MF values for the Aluminium hydroxide treatments, with elevated (but not concentration-related) increases occurring, and there were also notable reductions in suspension growth (compared to the negative controls) across most Aluminium hydroxide treatment concentrations.

Table 9: RTG Values - 24 hour Range-Finder Experiment, aluminium hydroxide

|Treatment |-S-9 |

|(µg/mL) | |

| |%RTG SG |MF§ |

|0 | | 100 96.10 |108.57 | |

|UTC (Std) | | 123 102.92 |67.21 | |

|UTC | | 108 87.37 |64.88 | |

|3.047 | | 63 71.46 |169.63 | |

|6.094 | | 71 61.66 |267.52 |# |

|12.19 | | 67 65.74 |189.22 | |

|24.38 | | 58 51.62 |231.61 | |

|48.75 |PP | 63 57.72 |174.85 | |

|97.5 |PP | 53 51.97 |263.25 |# |

|195 |PP | 63 55.94 |199.36 | |

|390 |PP | 89 85.15 |108.83 | |

|780 |P, PP | 61 47.60 |99.10 | |

|Linear trend |NS |

|MMS | | | | |

|5 | | 26 37.95 |1311.94 | |

|7.5 | | 15 29.81 |2176.75 | |

|5 (Std) | | 32 50.11 |1761.31 | |

|7.5 (Std) | | 18 36.34 |2133.50 | |

§ 5-TFT resistant mutants/106 viable cells 2 days after treatment

%RTG Percentage Relative Total Growth (adjusted by Day 0 factor for 24 hour treatment)

SG Suspension growth

NS Not significant

# The MF of the test concentration exceeds the sum of the mean control MF plus GEF

Std Standard (Non-Percoll modified) treatments not subject to the density gradient separation steps

P Particulate test article observed at time of treatment

PP Particulate test article observed following treatment incubation period

Due to the sporadic and non-dose-related increases in mutation frequency (combined with reductions in SG and RTG values across the treatment concentration range) for 24 hour treatments in the absence of S-9 in the Range-Finder experiment with aluminium hydroxide, a further investigative experiment was conducted to better assess the physical effect 24 hour treatments with suspensions of aluminium hydroxide may have had, with a view to establish whether these physical effects were sufficiently confounding to prevent reliable data being obtained when conducting 24 hour treatments with particulate test material. When cell cultures were treated with Aluminium hydroxide at concentrations up to 780 µg/mL, and examined microscopically approximately 3, 6 and 24 hours after the start of treatment, no effect on the cell morphology could be discerned with these treatments, nor was any adherence of the particulate test article to the cells noted. Similar observations were made following the Percoll differential centrifugation step and after 1 and 2 days of the expression period, with no effect on cell morphology or adherence of particulate test material to the test cells noted.

Although the investigative experiment failed to demonstrate any effects on the cell morphology with 24 hour treatments using aluminium hydroxide suspensions in 0.5% MC, corresponding treatments in the Cytoxicity Range-Finder Experiment provided results (including notable reductions in SG) consistent with the particulate nature of the test article interfering with normal cell growth at all concentrations tested, despite the differential centrifugation steps employed. Accordingly, it was considered that despite the modifications made to the post treatment methodology to remove the particulate test material from the assay cultures, the particulate nature of the test material interfered with the growth of the cells during the 24 hour treatment period. These effects confound the ability for these prolonged treatments to provide a reliable assessment of the cytotoxicity and genotoxicity of the test material under such treatment conditions, and a 24 hour treatment period using a test article suspension is not considered an appropriate or valid treatment methodology. Experiment 2 treatments with aluminium hydroxide were therefore conducted using 3 hour treatments in the absence and presence of S-9.

No marked changes in osmolality or pH were observed in the 3 and 24 hour Range-Finder Experiment at the highest concentration tested (780 (g/mL) as compared to the concurrent vehicle controls (individual data not reported).

Accordingly, in Experiment 1, eight concentrations ranging from 6.094 to 780 (g/mL were tested in the absence and presence of S-9. Upon addition of the test article to the cultures, particulate test article was observed (with the naked eye) at the highest concentration tested (780 (g/mL). Following the 3 hour treatment incubation period, particulate test article was observed at the highest seven concentrations tested in the absence and presence of S-9 (12.19 to 780 (g/mL). All cultures in the absence and presence of S-9 were retained and subject to the Percoll differential centrifugation and cell recovery steps. The highest concentration tested was 780 (g/mL in both the absence and presence of S-9, which gave 101% and 93% RTG, respectively (see Table 10).

In Experiment 2, eight concentrations ranging from 6.094 to 780 (g/mL in the absence and presence of S-9 were again tested. Upon addition of the test article to the cultures, particulate test article was observed at the highest concentration tested in the absence and presence of S-9 (780 (g/mL). Following the 3 hour treatment incubation period, particulate test article was observed at the highest seven concentrations tested in the absence of S-9 (12.19 to 780 (g/mL), and at the highest six concentrations tested in the presence of S-9 (24.38 to 780 (g/mL). All cultures in the absence and presence of S-9 were retained and subject to the Percoll differential centrifugation and cell recovery steps. The highest concentration tested was 780 (g/mL in both the absence and presence of S-9, which gave 92% and 62% RTG, respectively (see Table 10).

Table 10: Summary of mutation data; aluminium hydroxide

Experiment 1 (3 hour treatment in the absence and presence of S-9)

|Treatment |-S-9 |Treatment |+S-9 |

|(µg/mL) | |(µg/mL) | |

| |%RTG |MF§ | |%RTG |MF§ |

|0 | |100 |64.78 | |0 | |100 |59.07 | |

|UTC | |118 |64.83 | |UTC | |117 |59.47 | |

|6.094 | |111 |56.02 | |6.094 | |100 |45.44 | |

|12.19 |PP |133 |61.34 | |12.19 |PP |102 |48.94 | |

|24.38 |PP |120 |59.34 | |24.38 |PP |98 |58.03 | |

|48.75 |PP |140 |54.81 | |48.75 |PP |105 |52.67 | |

|97.5 |PP |106 |61.54 | |97.5 |PP |103 |47.28 | |

|195 |PP |119 |54.94 | |195 |PP |95 |56.35 | |

|390 |PP |120 |49.17 | |390 |PP |100 |50.76 | |

|780 |P, PP |101 |58.15 | |780 |P, PP |93 |53.51 | |

|Linear trend |NS |Linear trend |NS |

|MMS | | | | |B[a]P | | | | |

| | | | | |0.5 | |87 |131.34 | |

| | | | | |1 | |82 |317.47 | |

|15 | |51 |659.14 | |2 | |8 |1047.69 | |

|20 | |38 |755.10 | |3 | |29 |878.38 | |

| | | | |

| |%RTG |MF§ | |%RTG |MF§ |

|0 | |100 |53.44 | |0 | |100 |43.62 | |

|UTC | |85 |61.05 | |UTC | |102 |56.05 | |

|6.094 | |94 |59.14 | |6.094 | |94 |57.14 | |

|12.19 |PP |101 |63.23 | |12.19 | |106 |42.23 | |

|24.38 |PP |96 |55.94 | |24.38 |PP |105 |45.21 | |

|48.75 |PP |104 |61.32 | |48.75 |PP |91 |53.57 | |

|97.5 |PP |91 |57.20 | |97.5 |PP |76 |42.24 | |

|195 |PP |89 |54.48 | |195 |PP |88 |77.32 | |

|390 |PP |91 |55.21 | |390 |PP |86 |57.90 | |

|780 |P, PP |92 |70.52 | |780 |P, PP |62 |65.18 | |

|Linear trend |NS |Linear trend |** |

|MMS | | | | |B[a]P | | | | |

|15 | |48 |441.10 | |0.5 | |87 |126.19 | |

|20 | |32 |593.79 | |1 | |73 |228.41 | |

| | | | | |2 | |51 |525.12 | |

| | | | | |3 | |27 |883.47 | |

| | | |

|0 |NE |100 |

|41.69 |100* |76 |

|83.38 PP |134 |76 |

%RTG Percentage Relative Total Growth

PP Precipitate observed following treatment incubation period

ND Not evaluated – a zero viability plate count meant that no RTG value could be calculated

* Data calculated and used as though it were vehicle control data, in order to provide a baseline to compare other treatment concentration data with, as no vehicle control RTG data was available.

In the cytotoxicity Range-Finder Experiment, 24 hour treatment, nine concentrations were tested in the absence of S-9 ranging from 5.211 to 1334 (g/mL. Upon addition of the test article to the cultures, precipitate was observed at the highest two concentrations tested (667 and 1334 (g/mL). Following the 24 hour treatment incubation period, precipitate was observed at the highest five concentrations tested (83.38 to 1334 (g/mL). The lowest concentration at which precipitate was observed at the end of the treatment incubation period was retained and higher concentrations were discarded. The highest concentration tested was 83.38 (g/mL, which gave 64% RTG. The RTG values are shown in Table 12.

No marked changes in osmolality or pH were observed in the 3 and 24 hour Range-Finder Experiments at the highest concentration tested (83.38 (g/mL) as compared to the concurrent vehicle controls (individual data not reported).

Table 12: RTG Values - 24 hour Range-Finder Experiment, aluminium chloride

|Treatment |-S-9 |

|(µg/mL) |% RTG |

|0 |100 |

|5.211 |67 |

|10.42 |63 |

|20.85 |70 |

|41.69 |64 |

|83.38 PP |64 |

§ 5-TFT resistant mutants/106 viable cells 2 days after treatment

%RTG Percentage Relative Total Growth (adjusted by Day 0 factor for 24 hour treatment)

NS Not significant

PP Precipitate observed following treatment incubation period

Accordingly, in Experiment 1, six concentrations ranging from 3.125 to 100 (g/mL were tested in the absence and presence of S-9. Following the 3 hour treatment incubation period, precipitate was observed at the highest two concentrations tested in the absence and presence of S-9 (50 and 100 (g/mL). The lowest concentration at which precipitate was observed at the end of the treatment incubation period in the absence and presence of S-9 was retained and higher concentrations were discarded. The highest concentration selected was 50 (g/mL in the absence and presence of S-9, which gave 163% and 120% RTG, respectively (see Table 13).

In Experiment 2, eight concentrations ranging from 5 to 120 (g/mL in the absence of S-9 (24 hour treatment) and eleven concentrations ranging from 5 to 80 (g/mL in the presence of S-9 (3 hour treatment) were tested. Following the 3 and 24 hour treatment incubation periods, precipitate was observed at the highest concentration tested in the absence of S-9 (120 (g/mL), and at the highest four concentrations tested in the presence of S-9 (50 to 80 (g/mL). All cultures were retained in the absence of S-9, and the lowest concentration at which precipitate was observed at the end of the treatment incubation period in the presence of S-9 was retained and higher concentrations were discarded. The highest concentrations selected were 120 (g/mL in the absence of S-9 and 50 (g/mL in the presence of S-9, which gave 97% and 94% RTG respectively (see Table 13).

Table 13: Summary of mutation data; aluminium chloride

Experiment 1 (3 hour treatment in the absence and presence of S-9)

|Treatment |-S-9 |Treatment |+S-9 |

|(µg/mL) | |(µg/mL) | |

| |%RTG |MF§ | |%RTG |MF§ |

|0 | |100 |91.54 | |0 | |100 |70.46 | |

|3.125 | |116 |70.68 | |3.125 | |133 |64.03 | |

|6.25 | |181 |56.47 | |6.25 | |161 |59.90 | |

|12.5 | |183 |68.44 | |12.5 | |130 |58.97 | |

|25 | |165 |74.50 | |25 | |134 |56.35 | |

|50 |PP |163 |82.67 | |50 |PP |120 |69.97 | |

|Linear trend |NS |Linear trend |NS |

|MMS | | | | |B[a]P | | | | |

|15 | |74 |699.96 | |2 | |50 |934.97 | |

|20 | |43 |1095.71 | |3 | |13 |1650.41 | |

| | | | |

| |%RTG |MF§ | |%RTG |MF§ |

|0 | |100! |52.63! | |0 | |100 |99.69 | |

|5 | |85 |48.60 | |5 | |113 |78.60 | |

|10 | |78 |41.47 | |10 | |95 |94.77 | |

|20 | |101 |42.54 | |15 | |94 |91.54 | |

|40 | |94 |48.29 | |20 | |99 |110.31 | |

|60 | |110 |38.88 | |25 | |105 |75.33 | |

|80 | |118 |33.66 | |30 | |83 |113.59 | |

|100 | |110 |36.18 | |40 | |83 |81.95 | |

|120 |PP |97 |52.27 | |50 |PP |94 |97.03 | |

|Linear trend |NS |Linear trend |NS |

|MMS | | | | |B[a]P | | | | |

|5 | |31 |962.16 | |2 | |32 |1077.54 | |

|7.5 | |22 |1246.55 | |3 | |27 |887.11 | |

Footnotes overleaf:

§ 5-TFT resistant mutants/106 viable cells 2 days after treatment

%RTG Percentage Relative Total Growth (adjusted by Day 0 factor for 24 hour treatment)

NS Not significant

PP Particulate test article observed following treatment incubation period

! Based on one replicate only

Mutation; aluminium chloride

A summary of the results for Experiments 1 and 2 is shown in Table 13. The individual plate counts observed and the data analysis in each experiment are shown in Appendix 5 and Appendix 6 for Experiment 1 in the absence and presence of S-9 respectively, and in Appendix 7 and Appendix 8 for Experiment 2 in the absence and presence of S-9 respectively. The acceptance criteria were met and the study was therefore accepted as valid.

In Experiments 1 and 2, the mutation frequency of the concentrations plated were all less than the sum of the mean control mutation frequency plus the global evaluation factor (GEF) with a no significant linear trends, indicating a negative result.

In addition, for the negative and positive controls, the number of wells containing small colonies and the number containing large colonies were scored. Thus the small and large colony MF could be estimated and the proportion of small mutant colonies could be calculated (Appendix 5 to Appendix 8). For the negative controls, the proportion of small colony mutants in the absence and presence of S-9 ranged from 44% to 60% in Experiment 1 and from 51 % to 58% in Experiment 2. Marked increases in the number of both small and large colony mutants were observed following treatment with the positive control chemicals MMS and B[a]P.

CONCLUSION

It is concluded that Aluminium chloride did not induce mutation at the tk locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to precipitating concentrations in two independent experiments, in the absence and presence of a rat liver metabolic activation system (S-9). A 3 hour treatment incubation period was used for all treatments in the presence of S-9 and Experiment 1 treatments in the absence of S-9, and a 24 hour treatment incubation period used for Experiment 2 treatments in the absence of S-9.

When tested in the same test system, Aluminium hydroxide did not induce mutation at the tk locus of L5178Y mouse lymphoma cells when tested under the modified treatment conditions employed in this study. These modified conditions included treatments using test article suspensions (in 0.5% MC) at concentrations up to 780 (g/mL in two independent experiments, using a 3 hour treatment incubation period in the absence and presence of a rat liver metabolic activation system (S-9) throughout. This top concentration was the maximum selected to be within the range where particulate test article was observed, and corresponded to 10 mM had the test article been tested as a solution. The assay methodology was modified by the inclusion of a Percoll differential centrifugation step and cell recovery after the treatment incubation period, in order to remove as much of the particulate test material as possible.

REFERENCES

Appendices

Appendix 1

Aluminium hydroxide: Raw plate counts and data analysis for Experiment 1 in the absence of S-9 (3 hour treatment)

Table 14: Aluminium hydroxide: Experiment 1 in the absence of S-9 (3 hour treatment) - Cell counts and wells with clones/plate

|Treatment |Cell Counts |Viability ( |Resistant mutants ( |

|((g/mL) |24h $ 48h $ |(Day 2) |(Day 2) |

|0 |A |5.53 |9.38 |72 |70 |9 |7 |13 |11 |

| |B |5.46 |8.45 |73 |82 |9 |15 |10 |13 |

|UTC |A |6.25 |9.08 |80 |72 |11 |15 |10 |14 |

| |B |6.01 |8.90 |80 |71 |12 |6 |10 |13 |

|6.094 |A |5.30 |9.56 |82 |77 |10 |8 |11 |9 |

| |B |5.28 |9.18 |71 |79 |9 |12 |13 |11 |

|12.19 PP |A |5.90 |9.04 |80 |83 |12 |12 |13 |12 |

| |B |5.77 |9.78 |71 |84 |10 |15 |14 |9 |

|24.38 PP |A |5.62 |9.54 |74 |77 |10 |10 |15 |7 |

| |B |5.83 |9.69 |76 |78 |16 |7 |9 |11 |

|48.75 PP |A |5.97 |9.59 |81 |78 |9 |9 |6 |12 |

| |B |5.99 |9.62 |80 |80 |10 |11 |16 |15 |

|97.5 PP |A |5.26 |9.10 |79 |77 |8 |6 |10 |9 |

| |B |5.47 |9.59 |73 |73 |9 |16 |15 |13 |

|195 PP |A |5.78 |9.66 |72 |71 |7 |9 |9 |8 |

| |B |6.06 |9.86 |79 |76 |12 |12 |9 |9 |

|390 PP |A |5.90 |9.22 |74 |73 |13 |12 |4 |8 |

| |B |6.18 |9.57 |77 |77 |6 |7 |9 |10 |

|780 P, PP |A |5.51 |8.93 |81 |74 |8 |14 |8 |11 |

| |B |5.15 |9.89 |68 |73 |10 |6 |10 |11 |

|Positive control MMS ((g/mL) |

|15 |A |4.33 |9.04 |66 |51 |51 |54 |57 |45 |

|20 |A |4.81 |6.91 |58 |51 |59 |50 |51 |50 |

| | | | | |

|0 | |A |12.97 |84.09 |0.96 |65.41 |

| | |B |11.53 |102.91 |1.04 |63.43 |

|UTC | |A |14.19 |98.04 |1.22 |71.15 |

| | |B |13.37 |96.50 |1.14 |58.51 |

|6.094 | |A |12.67 |110.06 |1.23 |47.34 |

| | |B |12.12 |94.99 |1.01 |65.61 |

|12.19 PP | |A |13.33 |118.14 |1.39 |57.78 |

| | |B |14.11 |102.91 |1.28 |64.88 |

|24.38 PP | |A |13.40 |96.50 |1.14 |60.02 |

| | |B |14.12 |101.24 |1.26 |58.65 |

|48.75 PP | |A |14.31 |110.06 |1.39 |44.72 |

| | |B |14.41 |111.98 |1.42 |64.97 |

|97.5 PP | |A |11.97 |104.62 |1.10 |42.94 |

| | |B |13.11 |89.30 |1.03 |83.16 |

|195 PP | |A |13.96 |85.35 |1.05 |52.64 |

| | |B |14.94 |102.91 |1.35 |56.28 |

|390 PP | |A |13.60 |90.68 |1.08 |55.87 |

| | |B |14.79 |101.24 |1.32 |42.97 |

|780 P, PP | |A |12.30 |102.91 |1.11 |54.86 |

| | |B |12.73 |82.85 |0.93 |61.14 |

|15 MMS | |A |9.79 |58.75 |0.51 |659.14 |

|20 MMS | |A |8.31 |52.42 |0.38 |755.10 |

| | | | | | | |

A,B Replicate cultures

§ TFT resistant mutants/106 viable cells 2 days after treatment

SG Suspension growth; calculated as increase in cell numbers from end of treatment period to end of expression period (i.e. over Day 1 and Day 2)

%V % Day 2 viability

RTG Relative Total Growth = (Individual SG / Mean control SG) x (Individual %V / Mean control %V)

MF Mutant frequency

UTC Untreated controls

P Particulate test article observed at time of treatment

PP Particulate test article observed following treatment incubation period

Table 16: Aluminium hydroxide: Experiment 1 in the absence of S-9 (3 hour treatment) - Summary of results

|Treatment | |%V |%RTG |MF§ |

|((g/mL) | |(Day 2) | | |

|0 | |92.80 |100 |64.78 |

|UTC | |97.26 |118 |64.83 |

|6.094 PP | |102.07 |111 |56.02 |

|12.19 PP | |110.06 |133 |61.34 |

|24.38 PP | |98.82 |120 |59.34 |

|48.75 PP | |111.02 |140 |54.81 |

|97.5 PP | |96.50 |106 |61.54 |

|195 PP | |93.52 |119 |54.94 |

|390 PP | |95.74 |120 |49.17 |

|780 P, PP | |92.08 |101 |58.15 |

|Positive control (MMS) |

|Treatment |%V |%RTG |MF§ |

|((g/mL) |(Day 2) | | |

|15 | |58.75 |51 |659.14 |

|20 | |52.42 |38 |755.10 |

| | | | | |

%V % Day 2 viability

%RTG Mean relative total growth

§ TFT resistant mutants/106 viable cells 2 days after treatment

MF Mutant frequency

UTC Untreated controls

P Particulate test article observed at time of treatment

PP Particulate test article observed following treatment incubation period

Table 17: Aluminium hydroxide: Experiment 1 in the absence of S-9 (3 hour treatment) - Linear Trend

|Slope |-6.43E-09 |

|Variance |1.11E-16 |

|b2/Sb |0.372 |

b²/Sb Test for linear trend, slope b and its variance Sb

Table 18: Aluminium hydroxide: Experiment 1 in the absence of S-9 (3 hour treatment) - Large and small colony plate counts

|Treatment |Small colonies # |Large colonies # |

|((g/mL) |(Day 2) |(Day 2) |

|0 |A |5 |3 |7 |7 |4 |4 |6 |4 |

| |B |5 |10 |5 |7 |4 |5 |5 |6 |

|UTC |A |5 |7 |3 |9 |6 |9 |7 |5 |

| |B |6 |3 |6 |5 |6 |3 |4 |8 |

|15 MMS |A |38 |41 |39 |28 |13 |13 |19 |17 |

|20 MMS |A |48 |40 |32 |38 |11 |10 |19 |12 |

| | | | | | | | | | |

# Positive wells per plate. Total number of wells scored is 96

A,B Replicate cultures

UTC Untreated controls

Table 19: Aluminium hydroxide: Experiment 1 in the absence of S-9 (3 hour treatment) - Mutant frequencies

|Treatment |Small colonies |Large Colonies |Proportion |

|((g/mL) | | |small |

| | | |colony |

| | | |mutants |

| |Mutants | |Mutants | | |

| |Ym |Nm |MF§ |Ym |Nm |MF§ | |

|0 | |719 |768 |35.5 |730 |768 |27.3 |0.56 |

|UTC | |724 |768 |30.3 |720 |768 |33.2 |0.48 |

|15 MMS | |238 |384 |407.1 |322 |384 |149.9 |0.70 |

|20 MMS | |226 |384 |505.7 |332 |384 |138.8 |0.75 |

| | | | | | | | | |

§ TFT resistant mutants/106 viable cells 2 days after treatment

MF Mutant frequency

Ym Wells without colonies (mutant)

Nm Total wells (mutant)

UTC Untreated controls

Appendix 2

Aluminium hydroxide: Raw plate counts and data analysis for Experiment 1 in the presence of S-9 (3 hour treatment)

Table 20: Aluminium hydroxide: Experiment 1 in the presence of S-9 (3 hour treatment) - Cell counts and wells with clones/plate

|Treatment |Cell Counts |Viability ( |Resistant mutants ( |

|((g/mL) |24h $ 48h $ |(Day 2) |(Day 2) |

|0 |A |4.67 |9.79 |72 |80 |11 |7 |17 |9 |

| |B |4.84 |9.85 |81 |80 |13 |11 |13 |9 |

|UTC |A |5.20 |10.02 |78 |87 |8 |11 |13 |13 |

| |B |5.77 |9.57 |75 |75 |10 |8 |7 |22 |

|6.094 |A |5.17 |9.80 |76 |82 |11 |6 |10 |8 |

| |B |4.95 |10.56 |69 |74 |8 |7 |5 |9 |

|12.19 PP |A |5.45 |10.34 |70 |79 |5 |9 |10 |8 |

| |B |5.94 |10.18 |73 |65 |9 |8 |7 |6 |

|24.38 PP |A |5.59 |10.17 |68 |69 |9 |13 |9 |14 |

| |B |5.39 |9.93 |78 |74 |5 |9 |9 |6 |

|48.75 PP |A |5.61 |10.15 |77 |73 |10 |10 |11 |13 |

| |B |5.35 |10.28 |73 |73 |7 |8 |6 |6 |

|97.5 PP |A |5.07 |10.17 |80 |75 |8 |10 |6 |8 |

| |B |5.17 |10.10 |74 |75 |8 |9 |7 |12 |

|195 PP |A |5.64 |10.37 |77 |76 |5 |10 |6 |7 |

| |B |4.73 |9.91 |68 |70 |12 |7 |16 |10 |

|390 PP |A |4.24 |9.84 |75 |78 |4 |14 |4 |9 |

| |B |5.09 |10.08 |79 |81 |14 |7 |12 |14 |

|780 P, PP |A |4.66 |10.11 |73 |73 |12 |11 |11 |7 |

| |B |5.18 |10.79 |68 |78 |8 |9 |4 |8 |

|Positive control B[a]P ((g/mL) |

|0.5 |A |4.10 |9.71 |79 |79 |21 |31 |24 |19 |

|1 |A |4.27 |10.33 |80 |68 |41 |40 |46 |43 |

|2 |A |1.52 |4.82 |55/95 |51 |59 |59 |65 |68 |

|3 |A |2.33 |8.79 |67 |63 |72 |71 |72 |58 |

| | | | | |

|0 | |A |11.43 |98.04 |0.91 |62.07 |

| | |B |11.92 |113.97 |1.10 |55.98 |

|UTC | |A |13.03 |122.60 |1.30 |50.83 |

| | |B |13.80 |94.99 |1.06 |68.72 |

|6.094 | |A |12.67 |108.20 |1.11 |44.17 |

| | |B |13.07 |85.35 |0.91 |46.00 |

|12.19 PP | |A |14.09 |93.52 |1.07 |46.52 |

| | |B |15.12 |79.28 |0.97 |51.30 |

|24.38 PP | |A |14.21 |78.14 |0.90 |79.76 |

| | |B |13.38 |98.04 |1.07 |40.05 |

|48.75 PP | |A |14.24 |94.99 |1.10 |64.06 |

| | |B |13.75 |89.30 |1.00 |40.82 |

|97.5 PP | |A |12.89 |102.91 |1.08 |42.28 |

| | |B |13.05 |93.52 |0.99 |52.63 |

|195 PP | |A |14.62 |99.62 |1.18 |38.00 |

| | |B |11.72 |79.28 |0.75 |78.61 |

|390 PP | |A |10.43 |99.62 |0.84 |42.25 |

| | |B |12.83 |111.98 |1.17 |58.29 |

|780 P, PP | |A |11.78 |89.30 |0.85 |63.22 |

| | |B |13.97 |89.30 |1.01 |43.97 |

|0.5 B[a]P | |A |9.95 |108.20 |0.87 |131.34 |

|1 B[a]P | |A |11.03 |92.08 |0.82 |317.47 |

|2 B[a]P | |A |1.83 |50.60 |0.08 |1047.69 |

|3 B[a]P | |A |5.12 |70.65 |0.29 |878.38 |

| | | | | | | |

A,B Replicate cultures

§ TFT resistant mutants/106 viable cells 2 days after treatment

SG Suspension growth; calculated as increase in cell numbers from end of treatment period to end of expression period (i.e. over Day 1 and Day 2)

%V % Day 2 viability

RTG Relative Total Growth = (Individual SG / Mean control SG) x (Individual %V / Mean control %V)

MF Mutant frequency

UTC Untreated controls

P Particulate test article observed at time of treatment

PP Particulate test article observed following treatment incubation period

Table 22: Aluminium hydroxide: Experiment 1 in the presence of S-9 (3 hour treatment) - Summary of results

|Treatment | |%V |%RTG |MF§ |

|((g/mL) | |(Day 2) | | |

|0 | |105.50 |100 |59.07 |

|UTC | |107.28 |117 |59.47 |

|6.094 | |95.74 |100 |45.44 |

|12.19 PP | |86.00 |102 |48.94 |

|24.38 PP | |87.30 |98 |58.03 |

|48.75 PP | |92.08 |105 |52.67 |

|97.5 PP | |98.04 |103 |47.28 |

|195 PP | |88.63 |95 |56.35 |

|390 PP | |105.50 |100 |50.76 |

|780 P, PP | |89.30 |93 |53.51 |

|Positive control (B[a]P) |

|Treatment |%V |%RTG |MF§ |

|((g/mL) |(Day 2) | | |

|0.5 | |108.20 |87 |131.34 |

|1 | |92.08 |82 |317.47 |

|2 | |50.60 |8 |1047.69 |

|3 | |70.65 |29 |878.38 |

| | | | | |

%V % Day 2 viability

%RTG Mean relative total growth

§ TFT resistant mutants/106 viable cells 2 days after treatment

MF Mutant frequency

UTC Untreated controls

P Particulate test article observed at time of treatment

PP Particulate test article observed following treatment incubation period

Table 23: Aluminium hydroxide: Experiment 1 in the presence of S-9 (3 hour treatment) - Linear Trend

|Slope |1.74E-09 |

|Variance |1.08E-16 |

|b2/Sb |0.028 |

b²/Sb Test for linear trend, slope b and its variance Sb

Table 24: Aluminium hydroxide: Experiment 1 in the presence of S-9 (3 hour treatment) - Large and small colony plate counts

|Treatment |Small colonies # |Large colonies # |

|((g/mL) |(Day 2) |(Day 2) |

|0 |A |9 |4 |7 |4 |2 |3 |10 |5 |

| |B |6 |6 |8 |6 |7 |5 |5 |3 |

|UTC |A |5 |7 |9 |10 |3 |4 |4 |3 |

| |B |3 |4 |3 |13 |7 |4 |4 |9 |

|0.5 B[a]P |A |12 |19 |15 |10 |9 |12 |9 |9 |

|1 B[a]P |A |22 |24 |30 |23 |19 |16 |16 |20 |

|2 B[a]P |A |40 |32 |33 |41 |19 |27 |32 |27 |

|3 B[a]P |A |39 |38 |45 |42 |33 |34 |27 |16 |

| | | | | | | | | | |

# Positive wells per plate. Total number of wells scored is 96

A,B Replicate cultures

UTC Untreated controls

Table 25: Aluminium hydroxide: Experiment 1 in the presence of S-9 (3 hour treatment) - Mutant frequencies

|Treatment |Small colonies |Large Colonies |Proportion |

|((g/mL) | | |small |

| | | |colony |

| | | |mutants |

| |Mutants | |Mutants | | |

| |Ym |Nm |MF§ |Ym |Nm |MF§ | |

|0 | |718 |768 |31.9 |728 |768 |25.4 |0.56 |

|UTC | |714 |768 |34.0 |730 |768 |23.7 |0.59 |

|0.5 B[a]P | |328 |384 |72.8 |345 |384 |49.5 |0.59 |

|1 B[a]P | |285 |384 |161.9 |313 |384 |111.0 |0.58 |

|2 B[a]P | |238 |384 |472.7 |279 |384 |315.6 |0.58 |

|3 B[a]P | |220 |384 |394.2 |274 |384 |238.9 |0.60 |

| | | | | | | | | |

§ TFT resistant mutants/106 viable cells 2 days after treatment

MF Mutant frequency

Ym Wells without colonies (mutant)

Nm Total wells (mutant)

UTC Untreated controls

Appendix 3

Aluminium hydroxide: Raw plate counts and data analysis for Experiment 2 in the absence of S-9 (3 hour treatment)

Table 26: Aluminium hydroxide: Experiment 2 in the absence of S-9 (3 hour treatment) - Cell counts and wells with clones/plate

|Treatment |Cell Counts |Viability ( |Resistant mutants ( |

|((g/mL) |24h $ 48h $ |(Day 2) |(Day 2) |

|0 |A |4.74 |9.94 |77 |75 |9 |7 |10 |10 |

| |B |5.41 |7.88 |80 |78 |15 |10 |9 |10 |

|UTC |A |4.59 |8.45 |78 |75 |9 |13 |12 |7 |

| |B |4.90 |8.62 |76 |74 |11 |8 |12 |14 |

|6.094 |A |4.26 |9.03 |79 |79 |9 |14 |14 |13 |

| |B |4.91 |8.37 |80 |79 |15 |8 |7 |13 |

|12.19 PP |A |5.04 |8.65 |78 |78 |11 |13 |14 |14 |

| |B |4.79 |8.76 |77 |84 |12 |11 |15 |9 |

|24.38 PP |A |4.76 |9.46 |77 |77 |10 |6 |13 |5 |

| |B |4.67 |9.41 |78 |74 |4 |13 |10 |20 |

|48.75 PP |A |5.37 |9.66 |78 |72 |8 |9 |14 |15 |

| |B |4.91 |9.31 |74 |80 |9 |16 |8 |8 |

|97.5 PP |A |4.46 |8.91 |73 |77 |11 |8 |9 |12 |

| |B |4.75 |9.05 |82 |76 |14 |9 |15 |6 |

|195 PP |A |4.57 |9.22 |81 |78 |15 |10 |10 |8 |

| |B |4.56 |8.84 |73 |74 |9 |9 |11 |7 |

|390 PP |A |4.76 |9.22 |78 |71 |12 |10 |10 |8 |

| |B |4.74 |8.59 |75 |82 |8 |9 |12 |11 |

|780 P, PP |A |4.51 |9.61 |76 |80 |11 |15 |9 |7 |

| |B |4.81 |9.50 |68 |77 |14 |12 |14 |15 |

|Positive control MMS ((g/mL) |

|15 |A |4.07 |9.17 |57 |61 |36 |37 |44 |40 |

|20 |A |3.96 |8.23 |50 |48 |37 |38 |43 |40 |

| | | | | |

|0 | |A |11.78 |98.04 |1.00 |50.20 |

| | |B |10.66 |108.20 |1.00 |56.24 |

|UTC | |A |9.70 |99.62 |0.84 |56.67 |

| | |B |10.56 |94.99 |0.87 |65.61 |

|6.094 | |A |9.62 |108.20 |0.90 |64.47 |

| | |B |10.27 |110.06 |0.98 |53.95 |

|12.19 PP | |A |10.90 |104.62 |0.99 |69.54 |

| | |B |10.49 |113.97 |1.04 |57.28 |

|24.38 PP | |A |11.26 |101.24 |0.99 |45.78 |

| | |B |10.99 |98.04 |0.93 |66.59 |

|48.75 PP | |A |12.97 |94.99 |1.07 |67.16 |

| | |B |11.43 |101.24 |1.00 |55.76 |

|97.5 PP | |A |9.93 |94.99 |0.82 |57.90 |

| | |B |10.75 |108.20 |1.01 |56.24 |

|195 PP | |A |10.53 |110.06 |1.00 |53.95 |

| | |B |10.08 |90.68 |0.79 |54.28 |

|390 PP | |A |10.97 |93.52 |0.89 |58.81 |

| | |B |10.18 |106.38 |0.94 |51.70 |

|780 P, PP | |A |10.84 |104.62 |0.98 |55.36 |

| | |B |11.42 |87.96 |0.87 |87.87 |

|15 MMS | |A |9.33 |59.59 |0.48 |441.10 |

|20 MMS | |A |8.15 |44.64 |0.32 |593.79 |

| | | | | | | |

A,B Replicate cultures

§ TFT resistant mutants/106 viable cells 2 days after treatment

SG Suspension growth; calculated as increase in cell numbers from end of treatment period to end of expression period (i.e. over Day 1 and Day 2)

%V % Day 2 viability

RTG Relative Total Growth = (Individual SG / Mean control SG) x (Individual %V / Mean control %V)

MF Mutant frequency

UTC Untreated controls

P Particulate test article observed at time of treatment

PP Particulate test article observed following treatment incubation period

Table 28: Aluminium hydroxide: Experiment 2 in the absence of S-9 (3 hour treatment) - Summary of results

|Treatment | |%V |%RTG |MF§ |

|((g/mL) | |(Day 2) | | |

|0 | |102.91 |100 |53.44 |

|UTC | |97.26 |85 |61.05 |

|6.094 | |109.12 |94 |59.14 |

|12.19 PP | |109.12 |101 |63.23 |

|24.38 PP | |99.62 |96 |55.94 |

|48.75 PP | |98.04 |104 |61.32 |

|97.5 PP | |101.24 |91 |57.20 |

|195 PP | |99.62 |89 |54.48 |

|390 PP | |99.62 |91 |55.21 |

|780 P, PP | |95.74 |92 |70.52 |

|Positive control (MMS) |

|Treatment |%V |%RTG |MF§ |

|((g/mL) |(Day 2) | | |

|15 | |59.59 |48 |441.10 |

|20 | |44.64 |32 |593.79 |

| | | | | |

%V % Day 2 viability

%RTG Mean relative total growth

§ TFT resistant mutants/106 viable cells 2 days after treatment

MF Mutant frequency

UTC Untreated controls

P Particulate test article observed at time of treatment

PP Particulate test article observed following treatment incubation period

Table 29: Aluminium hydroxide: Experiment 2 in the absence of S-9 (3 hour treatment) - Linear Trend

|Slope |1.08E-08 |

|Variance |1.32E-16 |

|b2/Sb |0.892 |

b²/Sb Test for linear trend, slope b and its variance Sb

Table 30: Aluminium hydroxide: Experiment 2 in the absence of S-9 (3 hour treatment) - Large and small colony plate counts

|Treatment |Small colonies # |Large colonies # |

|((g/mL) |(Day 2) |(Day 2) |

|0 |A |6 |4 |5 |6 |3 |3 |5 |4 |

| |B |7 |5 |5 |3 |8 |5 |4 |7 |

|UTC |A |4 |7 |6 |4 |5 |6 |6 |3 |

| |B |9 |7 |6 |6 |2 |1 |6 |8 |

|15 MMS |A |26 |22 |32 |25 |10 |15 |12 |15 |

|20 MMS |A |24 |23 |33 |23 |13 |15 |10 |17 |

| | | | | | | | | | |

# Positive wells per plate. Total number of wells scored is 96

A,B Replicate cultures

UTC Untreated controls

Table 31: Aluminium hydroxide: Experiment 2 in the absence of S-9 (3 hour treatment) - Mutant frequencies

|Treatment |Small colonies |Large Colonies |Proportion |

|((g/mL) | | |small |

| | | |colony |

| | | |mutants |

| |Mutants | |Mutants | | |

| |Ym |Nm |MF§ |Ym |Nm |MF§ | |

|0 | |727 |768 |26.7 |729 |768 |25.3 |0.51 |

|UTC | |719 |768 |33.9 |731 |768 |25.4 |0.57 |

|15 MMS | |279 |384 |268.0 |332 |384 |122.1 |0.67 |

|20 MMS | |281 |384 |349.8 |329 |384 |173.2 |0.65 |

| | | | | | | | | |

§ TFT resistant mutants/106 viable cells 2 days after treatment

MF Mutant frequency

Ym Wells without colonies (mutant)

Nm Total wells (mutant)

UTC Untreated controls

Appendix 4

Aluminium hydroxide: Raw plate counts and data analysis for Experiment 2 in the presence of S-9 (3 hour treatment)

Table 32: Aluminium hydroxide: Experiment 2 in the presence of S-9 (3 hour treatment) - Cell counts and wells with clones/plate

|Treatment |Cell Counts |Viability ( |Resistant mutants ( |

|((g/mL) |24h $ 48h $ |(Day 2) |(Day 2) |

|0 |A |4.87 |10.99 |68 |74 |4 |9 |7 |10 |

| |B |5.13 |10.29 |81 |79 |11 |4 |11 |6 |

|UTC |A |5.30 |11.05 |72 |72 |9 |8 |8 |10 |

| |B |4.98 |10.55 |79 |76 |10 |10 |8 |14 |

|6.094 |A |4.57 |10.32 |76 |67 |11 |13 |5 |9 |

| |B |5.50 |9.83 |79 |78 |9 |13 |8 |11 |

|12.19 |A |4.68 |10.18 |79 |79 |5 |6 |9 |7 |

| |B |5.33 |10.20 |78 |78 |6 |10 |13 |10 |

|24.38 PP |A |5.00 |10.38 |74 |77 |9 |7 |6 |12 |

| |B |5.02 |9.71 |82 |82 |7 |7 |15 |8 |

|48.75 PP |A |4.78 |10.13 |74 |79 |15 |8 |5 |9 |

| |B |4.89 |9.77 |80 |70 |11 |12 |10 |6 |

|97.5 PP |A |4.16 |10.24 |78 |71 |7 |5 |4 |9 |

| |B |4.57 |10.16 |73 |68 |7 |10 |5 |8 |

|195 PP |A |4.95 |10.62 |77 |67 |15 |13 |7 |15 |

| |B |5.10 |10.79 |71 |68 |12 |9 |11 |11 |

|390 PP |A |4.56 |10.77 |80 |67 |8 |7 |6 |10 |

| |B |3.92 |11.11 |77 |76 |16 |11 |10 |12 |

|780 P, PP |A |4.49 |9.75 (1.80)# |73 |77 |6 |11 |12 |10 |

| |B |4.49 |6.51 |68 |61 |6 |13 |8 |11 |

|Positive control B[a]P ((g/mL) |

|0.5 |A |4.18 |11.06 |80 |71 |17 |19 |29 |18 |

|1 |A |3.81 |11.94 |69 |72 |24 |31 |29 |37 |

|2 |A |3.21 |10.30 |70 |68 |62 |52 |46 |57 |

|3 |A |2.50 |8.84 |50 |71 |65 |67 |60 |64 |

| | | | | |

|0 | |A |13.38 |84.09 |0.88 |48.37 |

| | |B |13.20 |111.98 |1.15 |38.85 |

|UTC | |A |14.64 |86.64 |0.99 |55.15 |

| | |B |13.13 |102.91 |1.05 |56.28 |

|6.094 | |A |11.79 |85.35 |0.78 |61.04 |

| | |B |13.52 |106.38 |1.12 |53.07 |

|12.19 | |A |11.91 |108.20 |1.00 |33.69 |

| | |B |13.59 |104.62 |1.11 |51.18 |

|24.38 PP | |A |12.98 |96.50 |0.98 |48.04 |

| | |B |12.19 |120.33 |1.14 |42.10 |

|48.75 PP | |A |12.11 |99.62 |0.94 |50.85 |

| | |B |11.94 |94.99 |0.88 |56.37 |

|97.5 PP | |A |10.65 |93.52 |0.78 |35.99 |

| | |B |11.61 |82.85 |0.75 |49.09 |

|195 PP | |A |13.14 |86.64 |0.89 |80.50 |

| | |B |13.76 |80.45 |0.86 |73.81 |

|390 PP | |A |12.28 |90.68 |0.87 |46.41 |

| | |B |10.89 |99.62 |0.85 |68.52 |

|780 P, PP | |A |12.16 |94.99 |0.90 |56.37 |

| | |B |7.31 |69.65 |0.40 |74.81 |

|0.5 B[a]P | |A |11.56 |96.50 |0.87 |126.19 |

|1 B[a]P | |A |11.37 |82.85 |0.73 |228.41 |

|2 B[a]P | |A |8.27 |79.28 |0.51 |525.12 |

|3 B[a]P | |A |5.53 |62.18 |0.27 |883.47 |

| | | | | | | |

A,B Replicate cultures

§ TFT resistant mutants/106 viable cells 2 days after treatment

SG Suspension growth; calculated as increase in cell numbers from end of treatment period to end of expression period (i.e. over Day 1 and Day 2)

%V % Day 2 viability

RTG Relative Total Growth = (Individual SG / Mean control SG) x (Individual %V / Mean control %V)

MF Mutant frequency

UTC Untreated controls

P Particulate test article observed at time of treatment

PP Particulate test article observed following treatment incubation period

Table 34: Aluminium hydroxide: Experiment 2 in the presence of S-9 (3 hour treatment) - Summary of results

|Treatment | |%V |%RTG |MF§ |

|((g/mL) | |(Day 2) | | |

|0 | |96.50 |100 |43.62 |

|UTC | |94.25 |102 |56.05 |

|6.094 | |94.99 |94 |57.14 |

|12.19 | |106.38 |106 |42.23 |

|24.38 PP | |107.28 |105 |45.21 |

|48.75 PP | |97.26 |91 |53.57 |

|97.5 PP | |87.96 |76 |42.24 |

|195 PP | |83.47 |88 |77.32 |

|390 PP | |94.99 |86 |57.90 |

|780 P, PP | |81.04 |62 |65.18 |

|Positive control (B[a]P) |

|Treatment |%V |%RTG |MF§ |

|((g/mL) |(Day 2) | | |

|0.5 | |96.50 |87 |126.19 |

|1 | |82.85 |73 |228.41 |

|2 | |79.28 |51 |525.12 |

|3 | |62.18 |27 |883.47 |

| | | | | |

%V % Day 2 viability

%RTG Mean relative total growth

§ TFT resistant mutants/106 viable cells 2 days after treatment

MF Mutant frequency

UTC Untreated controls

P Particulate test article observed at time of treatment

PP Particulate test article observed following treatment incubation period

Table 35: Aluminium hydroxide: Experiment 2 in the presence of S-9 (3 hour treatment) - Linear Trend

|Slope |2.76E-08 |

|Variance |1.28E-16 |

|b2/Sb |5.929** |

b²/Sb Test for linear trend, slope b and its variance Sb

Significant: * P ................
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