An Investigation of The Effects of The Airwave TETRA Signal On Cellular Calcium and Brain Function

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An Investigation of the Effects of the Airwave TETRA

Signal on Cellular Calcium and Brain Function
R N Smith, J E H Tattersall, S C Bowditch, S J Holden, A C Green, Biomedical Sciences

I R Scott, P K Harrison, D A Low, S J R Smith, R I Grose & N C D Mifsud Dstl Porton Down
Salisbury
SP4 0JQ
Dstl/CR15728 Issue 1.0 © Crown copyright 2005 Dstl
27 September, 2005
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1
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This document was produced by Dstl for the Home Office under Order/ Contract reference CS652.
© Crown copyright
Defence Science and Technology Laboratory UK
Approval for wider use or release must be sought from:
Intellectual Property Department

Defence Science and Technology Laboratory
Porton Down, Salisbury, Wiltshire, SP4 0JQ.
Authorisation (Complete as applicable)

Name Signature Date
Project Manager R N Smith
Technical Reviewer G J Cooper
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Executive summary
Terrestrial Trunked Radio (TETRA) is the new telecommunications standard being rolled
out by Airwave 02 for use by the Police and Emergency Services in the UK. Airwave
operates on the Terrestrial Trunked Radio (TETRA) European Standard at 380 – 385 and
390 - 395 MHz; the system is digital with four time slots, and mobile radio terminals
transmit energy in pulses at a rate of 17.6 Hz.
The Defence Science and Technology Laboratory (Dstl) has undertaken a programme of
work on behalf of the Home Office entitled: “An Investigation of the Effects of the
Airwave TETRA Signal on Cellular Calcium and Brain Function”. The objectives of the
research are summarised as follows:
• To determine whether TETRA pulse modulated radiofrequency (RF) fields can affect
calcium levels and signalling in neurones and other cells.
• If effects on calcium physiology are found, to determine whether these differ from
effects produced by sinusoidally-modulated and non-modulated continuous wave
(CW) RF frequency exposure alone.
• To compare the effects of TETRA pulse modulated RF fields with those of

modulated and non-modulated CW RF exposure in an established in vitro model (rat
brain slice electrophysiology).
• To assess the potential for effects on human cognitive function from low level
exposure to TETRA fields.
From the conducted study the following conclusions have been drawn:
1. The results show no evidence of any consistent or biologically relevant effect of

TETRA fields on intracellular calcium in granule cells and cardiac myocytes at any
of the specific absorption rates (SAR) tested.
2. The results reported here do not provide support for the notion that TETRA-
modulated RF fields, up to the maximum SAR likely to be encountered by handset
users, affect intracellular calcium physiology in neurones or cardiac tissue.
3. From brain slice studies (specifically in the hippocampus), although changes in

population spike amplitude were observed at all SARs tested, TETRA fields
produced no greater effect than CW at equivalent SARs. Statistical comparisons
failed to detect significant differences between the effects of TETRA and those of
CW at any of the SARs tested.
4. From the human cognitive study; when compared to sham conditions, continuous
exposure of TETRA RF over a period of approximately one and a half hours does not
specifically result in a robust sustained effect on cognitive performance, self-reported
mood and anxiety state and perception of workload, in normal healthy males.
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Table of contents
Executive summary 3
1 Introduction 11
1.1 Background and Contractual 11
1.2 Previous Research 12
1.3 Objectives 13
1.4 Programme of Work 13
2 Equipment Procurement and Set-up 15
3 Modelling and Calculation of Specific Absorption Rate 18
4 Calcium Imaging Experiments 19
4.1 Introduction 19
4.2 Experimental Design 19
4.3 Results and Conclusions 19
4.4 Reporting 20
5 Studies on Hippocampal Slices 21
5.1 Introduction 21
5.2 Methodology 21
5.3 Results and Conclusions 22
5.3.1 Evoked Field Potentials 22
5.3.2 Effects on Epileptiform Activity 22
5.3.3 Thermal Imaging 22
5.3.4 RF induced heating in C.elegans 23
6 Cognitive Performance Studies 24
6.1 Introduction 24
6.2 Methodology 24

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6.3 Results 25
7 Conclusions 26
8 Acknowledgements 28
Appendix A NPL Calibration of Equipment 29
A-1 Calibration of Strip Line TEM cells 29
A-1.1 Equipment Description 29
A-1.2 Measurement Procedure 29
A-1.3 Uncertainties 30
A-1.4 Results 30
A-2 Dielectric Properties of Specimen Holder Materials 32
A-3 Measurements on Dstl Monopole Antenna 32
A-4 Discussion 33
Appendix B Experimental Assessment of Motorola Handsets 34
B-1 Determination of SAR Values 34
B-2 Computational Modelling of Exposure Systems 35
Appendix C Calcium Imaging Experiments 37
C-1 Introduction 37
C-2 Materials and Methods 37
C-2.1 Cerebellar Granule Cell Cultures 37
C-2.2 Rat Cardiac Myocyte Cultures 38
C-2.3 Calcium Imaging 39
C-2.4 Selection of Specific Absorption Rates (SARs) 40
C-2.5 Exposure to TETRA Fields 40
C-2.6 Data Analysis 40
C-2.7 Statistical Comparisons 41
C-2.8 Materials 41
C-3 Results 41
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C-3.1 Effects of TETRA RF on Resting Levels of [Ca2+]i in Granule Cells 41
C-3.2 Effects on [Ca2+]i Responses to Depolarising Stimuli in Granule Cells 42
C-3.3 Cardiac Myocytes 44
C-4 Discussion 45
C-5 References 48
Appendix D Studies on Hippocampal Slices 62
D-1 Introduction 62
D-2 Methods 62
D-2.1 Experimental Procedure 62
D-2.2 Selection of Specific Absorption Rates (SARs) 63
D-2.3 Exposure to TETRA Fields 64
D-2.4 Thermal Imaging 64
D-3 Results 65
D-3.1 Evoked Field Potentials 65
D-3.2 Effects of TETRA on Epileptiform Activity 66
D-3.3 RF-induced Heating in C.elegens 70
D-3.4 Future Work: Heating Effects 70
D-4 Discussion 71
D-5 Recommendations 72
Appendix E Human Cognitive Study 105
E-1 Introduction 105
E-2 Methodology 106
E-2.1 Participants 106
E-2.2 Design 107
E-2.3 Conditions 107
E-2.4 Test Day Procedures 108
E-2.5 Cognitive and Psychometric Performance Measures 110

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E-2.5.1 Simple and 2-Choice Reaction Time 110
E-2.5.2 Sustained Attention Response Task (SART) 110
E-2.5.3 Symbol Digit Modalities Test (SDMT) 110
E-2.5.4 Incidental Learning 111
E-2.5.5 Digit Span 111
E-2.5.6 Paced Visual Serial Addition Task (PVSAT) 111
E-2.5.7 Trails A and B 111
E-2.5.8 'n' Backs (Verbal and Spatial) 112
E-2.5.9 Visual Search 112
E-2.5.10 Visual Classification 112
E-2.5.11 Visual Recognition 113
E-2.5.12 Semantic Classification 113
E-2.5.13 Semantic Recognition 113
E-2.6 Subjective Measures 113
E-2.6.1 Positive and Negative Affect Schedule (PANAS) 113
E-2.6.2 State-Trait Anxiety Inventory (STAI) 113
E-2.6.3 NASA Task Load Index (NASA TLX) 114
E-2.6.4 Symptom Rating Scale 114
E-2.7 Psychometric Measures 114
E-2.7.1 Raven's Standard Progressive matrices (SPM) 114
E-2.7.2 National Adult Reading Test (NART) 115
E-2.7.3 Eysenck's Personality Questionnaire (EPQ-R) 115
E-2.8 Statistical Analysis 115
E-3 Results 115
E-3.1 Cognitive and Psychomotor Performance Measures 115
E-3.1.1 Simple Reaction Time 115
E-3.1.2 Choice Reaction Time (CRT) 116

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E-3.1.3 Sustained Attention Response Task (SART) 117
E-3.1.4 Symbol Digit Modalities Test 117
E-3.1.5 Incidental Learning 117
E-3.1.6 Digit Span 118
E-3.1.7 Paced Visual Serial Addition Task 118
E-3.1.8 Trails A and B 198
E-3.1.9 'n' Back Tests 120
E-3.1.10 Visual Search 123
E-3.1.11 Visual Classification 123
E-3.1.12 Visual Recognition 123
E-3.1.13 Semantic Classification 124
E-3.1.14 Semantic Recognition 124
E-3.2 Subjective Measures 124
E-3.2.1 Positive and Negative Affect Schedule (PANAS) 124
E-3.2.2 State-Trait Anxiety Inventory 125
E-3.2.3 NASA Task Load Index (NASA TLX) 125
E-3.2.4 Symptoms Rating Questionnaire 126
E-4 Discussion 126
E-5 Conclusions 129
E-6 References 129
Appendix F RFR dosimetry for cognitive study 132
F-1 Dosimetry Measurements on Exposure System Used 132
F-2 References 133
Appendix G Cognitive Study Test Battery Blocks 134
Appendix H Cognitive Study Keyboard Layout 135
Appendix I Positive and Negative Affect Schedules 136
Initial distribution 137

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1 Introduction
1.1 Background and Contractual
Terrestrial Trunked Radio (TETRA) is the new telecommunications standard being rolled
out by Airwave O2 for use by the Police and Emergency Services in the UK. Airwave
operates on the TETRA European Standard at 380 – 385 and 390 - 395 MHz; the system
is digital with four time slots, and radio terminals transmit energy in pulses at a rate of
17.6 Hz.
In the light of the above and with reference to the Report of the Independent Expert
Group on Mobile Phones1 (“The Stewart Report”) to systems operating at a frequency of
amplitude modulation of around 16 Hz, the Home Office requested that a scientific
review of the relevant issues be conducted.
In response to this concern, the Defence Evaluation and Research Agency (DERA) (now
Dstl – Defence Science and Technology Laboratory) was commissioned by the Home
Office to produce a short expert review of the reported effects on calcium outflow from
brain tissue (Tattersall, 2001)2. The review concluded that the reported effect on calcium
outflow, if it is real, is difficult to interpret in terms of its physiological impact and that
there was no evidence to suggest it would be harmful. In view of the precautionary
approach suggested by the Stewart Report, the Dstl review recommended that research be
undertaken to assess whether or not there are any particularly unusual characteristics of
17.6 Hz pulse modulated RF fields, with regard to biological effects.
Following a successful bid to the Home Office, Dstl initiated a programme of work in
May 2001 (under Contract Number CS652) to address a number of these concerns.
An amendment to Contract CS652 was issued January 2002, to cover the purchase of
additional equipment for further studies, defined by Amendment Number 2 (issued July
2002) covering the effects of TETRA on epileptiform activity and human cognitive
performance. Five further amendments have been negotiated, covering calibration of
equipment by the National Physical Laboratory (Amendment 3, March 2003), an
expansion of the scope of the human cognitive performance study (Amendment 4, June
2003), an extension of the contract duration (Amendment 5, January 2004), an
examination of potential electrode artefact issues (Amendment 6, February 2004;
currently in abeyance) and local (electrode) heating effects (Amendment 7, April 2004).
This report provides a summary of Dstl’s studies.
1
Independent Expert Group on Mobile Phones (2000). Mobile phones and health.
2
Tattersall, JEH, “The Biological Effects Of Modulated Radiofrequency Electromagnetic Fields Relevant To
TETRA: A Review of Published Studies on Calcium”, DERA/TR00186-1.1 (2001)

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1.2 Previous Research
Safety guidelines for human exposure to radiofrequency (RF) electromagnetic fields are
based on the well-understood heating effects produced by the fields (McKinlay et al.
1993, ICNIRP 1998). In contrast, the effects on biological systems of exposure to RF
fields at levels that do not produce thermal changes remain contentious. Although a
number of “non-thermal” effects have been reported, these have proved difficult to
replicate and have not been widely accepted by the scientific community. In particular,
such effects have not fulfilled the criteria required to be incorporated into exposure
standards (ICNIRP 1998, McKinlay et al. 2004).
A number of published studies have reported enhanced effects of RF fields subjected to

modulation in the ELF range on the efflux of radioactively labelled calcium ions from
brain tissue (for a review, see Advisory Group on Nonionising Radiation 2001). In
particular, a modulation frequency-dependent “window” (i.e. a narrow range of
modulation frequencies over which an effect is observed) has been suggested with a peak
around 16 Hz (Bawin and Adey 1976, Blackman et al. 1979). In addition, a number of
studies have suggested that there may also be “windows” of effective power density
which produce a greater effect than higher or lower power densities (Bawin et al. 1975,
Blackman et al. 1980a, Blackman et al. 1980b, Blackman et al. 1989). In contrast,
studies in other laboratories have failed to detect a significant change in calcium efflux
from brain explants in vitro (Shelton, Jr. and Merritt 1981, Merritt et al. 1982, Albert et
al. 1987).
The suggestion that RF exposure might perturb cellular Ca2+ handling raises concerns
because of the important role that Ca2+ plays in cellular regulation and signalling.
Intracellular calcium is a crucial and ubiquitous intracellular messenger regulating many
cellular proteins and enzymes involved in intracellular signalling cascades and cellular
homeostasis (Berridge et al. 2000). Moreover, Ca2+ is essential for nervous system
function for initiating neurotransmitter release and regulating excitability. Importantly,
uncontrolled elevations of intracellular Ca2+ concentrations can trigger cell death in many
cell types, with neurones being particularly susceptible. The UK Independent Expert
Group on Mobile Phones (IEGMP) concluded that: “If [effects on calcium efflux] occur
as a result of exposure to mobile phones, their implications for cell function are unclear
and no obvious health risk has been suggested. Nevertheless, as a precautionary
measure, amplitude modulation around 16 Hz should be avoided, if possible, in future
developments in signal coding” (Independent Expert Group on Mobile Phones, 2000).
The TETRA communications standard incorporates a pulsed modulation at 17.6 Hz, very
close to the 16Hz modulation which has been reported to enhance calcium efflux. In the
light of the IEGMP suggestion of a precautionary approach to such modulation, concern
has been expressed about the possible biological effect of this pulsed modulation.
A major drawback of previous calcium efflux studies is that it is difficult to determine

where the measured calcium comes from. Calcium ion efflux was never a standard,
widely-employed measure of nerve function (Advisory Group on Nonionising Radiation
2001). Calcium efflux from brain explants almost certainly involves a number of factors
other than release from neurones, including the release of calcium bound or adherent to
membranes and simply trapped in the interstices of the tissue. It is also likely to be highly
influenced by temperature (Blackman et al. 1991). Furthermore, the procedures used to

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load the brain explants with radioactively labelled calcium would have resulted in the
neurones becoming severely hypoxic, and probably non-viable; this is confirmed by the
observation that the RF effect was independent of metabolic processes (Bawin et al.
1975), which are essential for the control of intracellular calcium levels. All of this
makes the interpretation of such experiments very difficult, particularly with respect to
the possible physiological and health consequences of the RF-induced effect. A review
by the Nobel Laureate Paul Greengard concluded that “The effect of electromagnetic
radiation on calcium efflux is potentially interesting, but further progress should be
along more imaginative lines where the functional significance, if any, of this
observation can be studied” (Greengard et al. 1982).
1.3 Objectives
The objectives of the research are summarised as follows:
1. To determine whether TETRA pulse modulated RF fields can affect calcium levels
and signalling in neurones and other cells.
2. If effects on calcium physiology are found, to determine whether these differ from
effects produced by sinusoidally modulated and non-modulated continuous wave
RF frequency exposure alone.
3. To compare the effects of TETRA pulse modulated RF fields with those of

modulated and non-modulated CW RF frequency exposure in an established in
vitro model (rat brain slice electrophysiology).
4. To assess the potential for effects on human cognitive function from low level
exposure to TETRA fields.
1.4 Programme of Work
The technical programme for this study fell into seven areas, as follows:
Equipment Procurement and set-up.
Modelling and Calculation of Specific Absorption Rate (SAR).
Calcium Imaging Experiments.
Brain Slice Electrophysiology.
Effects on Epileptiform Activity.
Heating effects.
Human Cognitive Performance.
Details of the equipment set-up and calibration are presented in Section 2 of this report,
with information on the calculation of SAR reported in Section 3. Sections 4, 5 and 6
report the studies on calcium imaging, brain slice electrophysiology (including
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epileptiform activity and heating effects) and human cognitive performance, respectively.
Sections 7 and 8 present the conclusions and recommendations.

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2 Equipment Procurement and Set-up

Dstl has considerable experience, expertise and state-of-the-art experimental facilities
relating to radio-frequency exposure, electrophysiology and calcium imaging. All in vitro
exposures conducted within this programme have been performed in well-characterised
parallel plate waveguide systems (see Figure 1), used extensively by Dstl in radio
frequency studies.
Replication of the TETRA signal has necessitated the procurement and subsequent
installation and testing, of four new signal generators (type ESG-D E4432B, ex Agilent
Technologies Limited) and associated sensors, power meters, amplifiers and directional
couplers. A diagram of the equipment set-up is shown in Figure 2. The exact nature of
the output waveform from the signal generator (to match that of in-service hand-sets)
was agreed with the Home Office, as standard ETSI modulation at 380.8875 MHz.
The equipment used in these studies has been calibrated by the UK National Physical
Laboratory (NPL), to improve confidence in the results generated. NPL’s measurements
have included impedance, S-parameter and field calibration in (i) an empty transmission
line, with window blanked off (analytic case), (ii) a stripline with window and (iii)
stripline with window and tissue holder. In addition, a monopole field probe, supplied by
Dstl, has been calibrated and the complex permittivity of Perspex™ and PTFE samples
similar to those used to manufacture the tissue holders has been measured. Details of the
methodology and results obtained are presented in Appendix A.
Results confirmed the assumptions on the specific absorption rate (SAR) calibrations for
the experimental set-up (see Section 3).

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Figure 1: Dstl Stripline Assembly
A
20 mm 1 mm
7 mm
Sample chamber (see B and C)
Ground Plane
13 mm ∅ hole Copper conductor

In ground plane 50 mm
150 mm
75 mm
20 mm ∅ in 17 mm
Copper conductor
448 mm
638 mm
710 mm
B Y1 C
COPPER CONDUCTOR
COVERSLIP OF CELLS
PERFUSION MEDIUM
X X1
X CELL CHAMBER
(PTFE)
X1
GROUND PLANE
OBJECTIVE
IMMERSION OIL
Y
OUTFLOW
INFLOW
Y Y1
1 cm
Key
A: Stripline plan and dimensions
B: Plan view of cell chamber
C: Cross-sections of cell chamber

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Figure 2: TETRA Equipment schematic

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3 Modelling and calculation of Specific Absorption Rate (SAR)

The specific absorption rate (SAR) in Wkg-1 is the standard measure of how much RF
power is absorbed by a tissue. It is essential that this parameter is known for a typical
exposure to a TETRA Airwave handset, for incorporation of comparable exposures into
the biological experiments. For this work the SAR of a head model exposed to TETRA
has been determined by modelling and numerical calculation, at typical input powers;
this work was undertaken by Microwave Consultants Limited (MCL), by sub-contract
from Dstl.
Full details of the experimental assessment of an in-service Motorola TETRA handset

and computational modelling of Dstl’s brain slice exposure systems are presented in
Appendix B. Generated data have been employed in the experiments described in
subsequent sections.
The 1g and 10g average SARs have been measured across a head phantom, with the
handset in each of four standard test positions. Table 1 summarises the maximum
volume-averaged SARs from the Motorola MTP 300 handset supplied by the Home
Office.
Handset Position SAR (Wkg-1) SAR (Wkg-1)

1 g Average 10 g Average
“Cheek” Left 0.537 0.391
Maximum Right 0.396 0.281
(cortical) “Tilted” Left 0.570 0.396
SAR Right 0.465 0.327
“Cheek” Left 0.093 0.075
Hippocampus Right 0.048 0.037
SAR “Tilted” Left 0.022 0.040
Right 0.057 0.044
Table 1: Volume Averaged SARs at Position of Maximum SAR and Hippocampus Position
The measurements were made in the standard CENELEC assessment positions, namely
“cheek” and “tilted”, on the left and right-hand sides of the head. Within Table 1, the
“Maximum SAR” is the maximum 1g and 10g average SARs at any part of the head in
each of the stated positions. The maximum head SARs found using the CENELEC
procedures should correspond to the maximum cortical SAR, particularly for the 10g-
average data.

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4 Calcium Imaging Experiments
4.1 Introduction
This part of the study aimed to determine whether TETRA fields could affect
intracellular calcium signalling in excitable cells; two culture types were employed:
Rat Cerebellar Granule Cells: These neuronal cells were isolated from rat brains and
grown in cell culture. They are well characterised and representative of many types of
neurones; for example, they are electrically excitable and express a range of
neurotransmitter receptors and ion channels.
Cardiac Myocytes: These are electrically excitable, contractile heart muscle cells, which
show spontaneous and synchronous beating in culture. The contractions are associated
with oscillations in calcium concentrations in the cells.
4.2 Experimental Design
Intracellular calcium concentration ([Ca2+]i) was measured in cultured mouse cerebellar

granule cells and cardiac myocytes during exposure to TETRA fields (380.8875MHz
pulse modulated at 17.6Hz, 25% duty cycle, simulating a single slot voice mode). [Ca2+]i
was measured as fura-PE3, fluo-3 or fluo-4 fluorescence by digital image analysis. Flow
diagrams for the work plan are presented in Figure 3.
4.3 Results and Conclusions
Granule cells exposed at specific absorption rates (SARs) of 5, 10, 20, 50 or 400mWkg-1
showed no significant changes in resting [Ca2+]i. Increases in [Ca2+]i in response to
potassium-induced depolarisation were significantly different from sham controls in
TETRA-exposed cells, but the majority of the difference was attributable to initial
biological variation between cell cultures. No difference was found between fura-PE3
(UV excitation) and fluo-3 (visible light excitation) measurements in these cells; thus, the
lack of a RF effect could not be due to its abolition by UV light.
Exposure to TETRA (50 or 400 mWkg-1) had no significant effect on either the rate or
amplitude of spontaneous Ca2+ transients in cardiac myocytes. The cells showed normal
responses to salbutamol (50µM) and acetylcholine (10 µM).
Overall, these results showed no evidence of any consistent or biologically relevant

effect of TETRA fields on [Ca2+]i in granule cells and cardiac myocytes at any of the
SARs tested.
Further details are presented in Appendix C.

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4.4 Reporting
A paper on the work covered in this Section has been submitted to an international
journal.
Neurones (cerebellar granule cells)
High power TETRA, 20

min exposure
min exposure
K+ depolarisations -
Resting [Ca2+] - Fura-2
Fura-2
EFFECT NO NO
EFFECT
EFFECT EFFECT

Repeat with 4 levels of
min exposure
lower power TETRA,
10 min exposures* K+ depolarisations -
Fluo-3
Cardiac muscle cells

min exposure
[Ca2+] rhythm in beating
cells
EFFECT NO EFFECT
Repeat with low power

TETRA,
TETRA cf CW
20 mins
Figure 3: Flow diagrams of work plan

(*selected on the basis of reported “power windows”)

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5 Studies on Hippocampal Slices
5.1 Introduction
Previous work by Dstl (Tattersall et al, 2001) found that exposure to low intensity RF
fields (SAR less than 5mWkg-1) changed the amplitude of evoked extracellular field
potentials in slices of rat hippocampus maintained in vitro and could also affect
epileptiform activity induced by 4-aminopyridine (4-AP). The hippocampus is a part of
the brain associated with learning, memory, spatial awareness and epilepsy. The
experiments reported here were designed to use this model to compare the effects of the
TETRA signal and the unmodulated carrier wave, to determine whether the pulse
modulation had any additional effect on electrical activity in brain tissue.
The study was expanded in response to Recommendation 3 of the AGNIR Committee

(August 2001), which suggested the following: “The possibility that modulated RF fields
might somehow synchronise the activity of groups of coupled neurons, and hence
increase the likelihood of epileptic seizures could be investigated in isolated slices of
rodent hippocampus, and also in strains of animals that are especially prone to
epilepsy”.
To address this recommendation, three series of in vitro experiments on epileptiform

activity were proposed:
Population Spikes: The presence of additional population spikes in the evoked field
potential is indicative of repetitive firing of cells, a characteristic of epileptic behaviour.
4-aminopyridine: The AGNIR Committee report referred to the inhibition of 4-AP-

induced epileptiform activity by 700MHz unmodulated RF, described by Tattersall et al,
2001. Using this as a starting point, it was proposed to repeat these experiments using the
TETRA signal and compare the effects with the unmodulated carrier frequency and with
sham exposed control slices.
Extracellular Potassium: Seizures may also be triggered by raising the extracellular

concentration of potassium ions. To determine whether TETRA increases the likelihood
of seizures, experiments were designed to compare the development of seizure activity,
in different concentrations of potassium, for slices exposed to TETRA, unmodulated
carrier frequency and sham-exposure conditions.
5.2 Methodology
Further details of this work are presented in Appendix D.

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5.3 Results and Conclusions
5.3.1 Evoked Field Potentials
These experiments were performed on a total of 133 slices, with between nine and fifteen
slices in each experimental group. Slices were exposed to TETRA and CW fields at
SARs of 5, 10, 20 and 50 mWkg-1; corresponding sham exposures were also performed.
Since the aim was to compare the SAR-dependence of any effects between TETRA and
CW, the highest SAR of 400 mWkg-1 was not used, in order to reduce the problems of
interference of the fields with the electronics in the recording system.
In summary, although changes in Population Spike (PS) amplitude were observed at all
SARs tested, TETRA fields produced no greater effect than CW at equivalent SARs.
Statistical comparisons failed to detect significant differences between TETRA and CW
at any of the SARs tested (see Appendix D).
5.3.2 Effects on Epileptiform Activity
Initial experiments used 4-aminopyridine (4-AP), which blocks potassium channels, to

induce epileptiform activity in slices. This was used in a previous study on 700 MHz
fields (Tattersall et al, 2001); however, in this study it was found that the activity induced
by 4-AP was not regular enough to provide sufficient data for a statistically robust
comparison. Subsequent experiments used elevated concentrations of potassium in the
perfusion solution to induce epileptiform activity.
The initial experiments showed that exposure to CW or TETRA at this SAR (which is
nearly eight times higher than the maximum SAR of 50 mWkg-1 tested in the
experiments on evoked activity, and nearly 100 times higher than the 4.4 mWkg-1 used
by Tattersall et al, 2001) irreversibly inhibited the evoked field potential and spontaneous
activity in the slices. This was an unexpected result, which suggested that exposure to RF
fields at this SAR was dramatically affecting the electrophysiology of the brain slice
preparation. The possibility was suspected that the electrodes used to stimulate and
record from the slice were interacting with the RF field to produce artifactual effects.
This was explored by using a thermal imaging camera to identify potential changes in
temperature in the vicinity of the electrodes.
5.3.3 Thermal Imaging
With these experiments, it has been possible, for the first time, to carry out physical
dosimetry of the brain slice system during exposure to RF fields. Concurrently,
improvements in computational electromagnetics, such as Finite Difference Time
Domain (FDTD) techniques, have allowed the slice and stimulating electrodes to be
modelled in greater detail. Both approaches show that the metal stimulating electrode
produces a greater elevation of SAR in the tissue near its tip than previously expected.
This can result in heating near the tip of between 1° and 10°C at a nominal SAR of 400
mWkg-1. In addition, the thermal imaging data showed that heating can also occur at the
tip and in the shaft of the glass recording microelectrode, an unexpected result. The
thermal imaging measurements confirmed that there was no heating in the tissue at a
SAR of 400 mWkg-1, if the electrodes are not present.

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The heating produced in the electrodes by a field intensity used to produce a SAR of 400
mWkg-1 in the tissue was clearly sufficient to explain the abolition of epileptiform
activity in the hippocampal slice preparation. Experiments in which the electrodes were
removed during exposure and then replaced confirmed this. It is not yet clear what effect
this locally increased SAR and heating would have on physiological responses in the
tissue; however, there is a strong possibility that artefacts may be induced by both the
recording and stimulating electrodes, and this needs to be investigated further.
Previous experiments have shown that a bulk temperature rise of 1°C has no effect on
evoked responses in the brain slice (Tattersall et al, 2001), but these imposed rises were
much slower than those recorded in response to RF exposure and did not produce
temperature gradients within the tissue. The lower SARs (up to 50 mWkg-1) used in the
experiments on evoked responses in this programme would produce lower temperature
increases than those produced by the SAR of 400 mWkg-1, which abolished epileptiform
activity. Furthermore, there was no difference between the effects of TETRA and of the
unmodulated carrier wave, although the possibility that electrode-induced artefacts may
have masked any difference between the two cannot be excluded.
5.3.4 RF-induced heating in C. elegans
As a result of the thermal imaging study in brain slices, the Study Management Group
speculated whether it would be possible to detect differential heating induced by RF
fields in the nematode worm Caenorhabditis elegans, which might help to explain the
results reported by de Pomerai (Nottingham University). A limited study was undertaken
to investigate this.
Although the experiments appeared to show bulk heating of the agar block at 3.2W kg-1,
no differential heating of the worms themselves could be resolved.

UNCLASSIFIED
UNCLASSIFIED
6 Cognitive Performance Studies
6.1 Introduction
This part of the study incorporates human volunteer trials carried out to measure changes
in cognitive performance arising from exposure to TETRA handsets.
Current radio frequency radiation exposure guidelines are based upon well-established
thermal effects. However, recent research into digital (pulsed carrier wave) and analogue
(continuous wave) transmission fields at levels within the exposure guidelines have
indicated potential effects on human cognitive performance.
The study programme has been devised to build upon previous findings and
demonstrated effects on reaction time and some aspects of working memory. The human
volunteer trial was conducted using a counterbalanced, within subjects, repeated
measures experimental design. Volunteer performance on complex computer-based
cognitive and psychomotor tasks was monitored during exposure to TETRA and sham
control signals. The study utilised 40 volunteers.
6.2 Methodology
Four experimental TETRA handsets have been delivered for use within this programme.
These handsets, developed within the Mobile Telecommunications Health Research
(MTHR) programme3 specifically for studies of this nature, are custom built devices,
designed to produce exposures to humans which are representative of exposures from
genuine TETRA handsets (or other mobile communication devices). The handset
produces exposure characteristics (i.e. waveform and near field radiation) representative
of that generated by a genuine system. The devices are compatible with double-blind
type experimental investigations.
The aim of this study was to determine whether realistic exposure to TETRA RF affected
human cognitive performance. The study design incorporated a six-week period during
which the effects of TETRA (Left-sided) exposure and TETRA (Right-sided) exposure
on the performance of computer based cognitive tasks were assessed, and subsequently
compared to performance during Sham (Left-sided) and Sham (Right-sided) control
conditions.
Forty male volunteers were trained on a comprehensive battery of cognitive tasks, which
were performed on four test days (0930-1130hrs) during exposure to TETRA RF or sham
condition, each of which were separated by a period of one week. Subjective self-
assessments of mood-state, perceived workload and symptoms were also recorded.
3
Following the publication of the report by the Independent Expert Group on Mobile Phones a research programme, entitled the Link Mobile
Telecommunications and Health Research Programme (MTHR), was established to look into the possible health impact of Mobile
Telecommunications. Funds of around £7 million have been allocated to the programme by industry and Government.

UNCLASSIFIED
UNCLASSIFIED
6.3 Results
The results from this study indicate that acute exposure to TETRA RF (380-385 and 390-
395 MHz) does not significantly affect performance on a wide range of cognitive and
psychomotor tasks, as measured by accuracy, response latency and error type. However,
performance effects were noticed for three of the twenty-two tests employed: Semantic
Recognition; Verbal 0-back and Visual Recognition. Of these, post-hoc comparisons
identified a significant difference between TETRA (Right sided presentation) and the
corresponding Sham (Right sided presentation) comparator for the Semantic Recognition
task alone. The absence of any observed effect on the Digit Symbol Incidental Learning
and Digit Span tasks, which were incorporated to enable cross verification of memory
domain aspects of cognitive performance, highlight the requirement for this result to be
viewed with caution.
Further details are presented in Appendix E.

UNCLASSIFIED
UNCLASSIFIED
7 Conclusions
From the studies conducted the following conclusions have been drawn:
Calcium Imaging Experiments
• Overall, the results showed no evidence of any consistent or biologically relevant

effect of TETRA fields on [Ca2+]i in granule cells and cardiac myocytes at any of the
SARs tested.
• Granule cells exposed at specific absorption rates (SARs) of 5, 10, 20, 50 or

400mWkg-1 showed no significant changes in resting [Ca2+]i. Increases in [Ca2+]i in
response to potassium-induced depolarisation were significantly different from sham
controls in TETRA-exposed cells, but the majority of the difference was attributable
to initial biological variation between cell cultures. No difference was found between
fura-PE3 (UV excitation) and fluo-3 (visible light excitation) measurements in these
cells; thus, the lack of an RF effect could not be due to its abolition by UV light.
• Exposure to TETRA (50 or 400 mWkg-1) had no significant effect on either the rate
or amplitude of spontaneous Ca2+ transients in cardiac myocytes, but the cells showed
normal responses to salbutamol (50µM) and acetylcholine (10µM).
Studies on Hippocampal Slices
• Experiments were carried out to compare the effects of TETRA and unmodulated
(continuous wave, CW) fields on evoked field potential responses in slices of rat
hippocampus. The aim was to determine whether the pulse modulation of TETRA
caused enhanced effects on the excitability of the brain tissue. Changes in the
amplitude of the population spike response were observed at all SARs tested (5-
50mWkg-1), but TETRA fields produced no greater effect than CW at equivalent
SARs: statistical comparisons failed to detect significant differences between TETRA
and CW at any of the SARs tested. Furthermore, there was no TETRA-induced
increase in the occurrence of multiple population spikes in the evoked responses,
which would have been indicative of an epileptogenic effect.
• Further experiments were performed to evaluate the effect of 400mWkg-1 TETRA on

epileptiform activity induced in the slices by perfusion with elevated potassium
concentrations; however, these were confounded by RF-induced heating in the
stimulating and recording electrodes at the higher field intensity (measured using a
sensitive thermal imaging camera), which completely abolished the epileptiform
activity.

UNCLASSIFIED
UNCLASSIFIED
Human Cognitive Study
• From the cognitive study; when compared to sham conditions, continuous exposure
of TETRA RF over a period of approximately one and a half hours did not
specifically result in a robust sustained effect on cognitive performance, self-reported
mood and anxiety state and perception of workload, in normal healthy males.

UNCLASSIFIED
UNCLASSIFIED
8 Acknowledgements
The authors wish to acknowledge the support and advice provided by the Home Office
throughout the work programme. Additionally we are indebted to the advice supplied by
the TETRA Health and Safety Management Committee drawn from representatives from
the Medical Research Council, the University of Nottingham, the University of
California, the Health and Safety Executive, the Department of Health and Police
Information Technology Organisation (PITO).
We are grateful to the National Physical Laboratory for carrying out the measurements
on the exposure system and to Microwave Consultants Limited for the computational
assessments of SAR.

UNCLASSIFIED

An Investigation of The Effects of The Airwave TETRA Signal On Cellular Calcium and Brain Function

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iUNCLASSIFIED

An Investigation of the Effects of the Airwave TETRA

R N Smith, J E H Tattersall, S C Bowditch, S J Holden, A C Green, Biomedical Sciences

Approval for wider use or release must be sought from:

Intellectual Property Department

Authorisation (Complete as applicable)

Technical Reviewer G J Cooper

• To compare the effects of TETRA pulse modulated RF fields with those of

1. The results show no evidence of any consistent or biologically relevant effect of

3. From brain slice studies (specifically in the hippocampus), although changes in

Dstl/CR15728 Issue 1.0 Page 3 of 142

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Page 4 of 142 Dstl/CR15728 Issue 1.0

1.1 Background and Contractual 11

1.2 Previous Research 12

1.4 Programme of Work 13

2 Equipment Procurement and Set-up 15

3 Modelling and Calculation of Specific Absorption Rate 18

4 Calcium Imaging Experiments 19

4.2 Experimental Design 19

4.3 Results and Conclusions 19

5 Studies on Hippocampal Slices 21

5.3 Results and Conclusions 22

5.3.1 Evoked Field Potentials 22

5.3.2 Effects on Epileptiform Activity 22

5.3.3 Thermal Imaging 22

5.3.4 RF induced heating in C.elegans 23

6 Cognitive Performance Studies 24

Dstl/CR15728 Issue 1.0 Page 5 of 142

Appendix A NPL Calibration of Equipment 29

A-1 Calibration of Strip Line TEM cells 29

A-1.1 Equipment Description 29

A-1.2 Measurement Procedure 29

A-2 Dielectric Properties of Specimen Holder Materials 32

A-3 Measurements on Dstl Monopole Antenna 32

Appendix B Experimental Assessment of Motorola Handsets 34

B-1 Determination of SAR Values 34

B-2 Computational Modelling of Exposure Systems 35

Appendix C Calcium Imaging Experiments 37

C-2 Materials and Methods 37

C-2.1 Cerebellar Granule Cell Cultures 37

C-2.2 Rat Cardiac Myocyte Cultures 38

C-2.3 Calcium Imaging 39

C-2.4 Selection of Specific Absorption Rates (SARs) 40

C-2.5 Exposure to TETRA Fields 40

C-2.6 Data Analysis 40

C-2.7 Statistical Comparisons 41

C-3.1 Effects of TETRA RF on Resting Levels of [Ca2+]i in Granule Cells 41

C-3.2 Effects on [Ca2+]i Responses to Depolarising Stimuli in Granule Cells 42

C-3.3 Cardiac Myocytes 44

Appendix D Studies on Hippocampal Slices 62

D-2.1 Experimental Procedure 62

D-2.2 Selection of Specific Absorption Rates (SARs) 63

D-2.3 Exposure to TETRA Fields 64

D-2.4 Thermal Imaging 64

D-3.1 Evoked Field Potentials 65

D-3.2 Effects of TETRA on Epileptiform Activity 66

D-3.3 RF-induced Heating in C.elegens 70

D-3.4 Future Work: Heating Effects 70

Appendix E Human Cognitive Study 105

E-1 Introduction 105

E-2 Methodology 106

E-2.1 Participants 106