PRECLINICAL TEST - BACTERIAL REVERSE MUTATION TEST

2013 International Nuclear Atlantic Conference - INAC 2013 Recife, PE, Brazil, November 24-29, 2013 ASSOCIAÇÃO BRASILEIRA DE ENERGIA NUCLEAR - ABEN ISBN: 978-85-99141-05-2

PRECLINICAL TEST - BACTERIAL REVERSE MUTATION TEST

FOR 18

F-FLUOROCHOLINE PRODUCED IN CDTN

Bruno M. Mendes1, Ana Carolina A. Bispo

1, Danielle C. Campos

1 and Juliana B. Silva

1

1 Centro de Desenvolvimento da Tecnologia Nuclear (CDTN / CNEN – MG)

Av. Presidente Antônio Carlos, 6627.

31270-901 Belo Horizonte, MG

[email protected]

[email protected]

[email protected]

[email protected]

ABSTRACT

The choline labeled with fluorine-18 (18FCH) is being considered as a great importance radiopharmaceutical

due to its effective detection of many type of malignant neoplasm. The research related to 18F-fluorocholine

synthesis in CDTN was initiated in 2010. In order to obtain clinical research approval, as well as to register 18

FCH for marketing, safety and efficacy preclinical testing are required. The present work evaluated the

18FCH genotoxic potential through the bacterial reverse mutation test (Ames test) using Salmonella

typhimurium TA-98, TA-100, TA-1535 and TA-1537 strains and Escherichia coli WP2 uvrA strain. The reverse

mutation test in bacteria for fluorcolina was conducted in two stages. Initially the method was applied to “cold”

fluorocholine molecule (19FCH). Subsequently, the decayed product of 18

FCH synthesis was evaluated. The

first step was performed in order to examine the FCH molecule mutagenicity. The second was carried out to

determine the mutagenic potential of final product. All strains were tested in triplicate for each exposure

concentration, in the presence and absence of metabolic activation (S-9 mix - 10%). There were no statistically

significant increases in revertant colonies rate for any strains tested after their exposure to decayed 18

FCH or 19

FCH. The number of revertant colonies in positive controls was significantly higher than that observed in

negative controls. Based on results of this assay, 18

FCH and 19

FCH, at tested doses, were found to be non-

mutagenic in bacterial reverse mutation test.

1. INTRODUCTION

Fluorine-18 labeled choline (18

FCH) is a promising radiotracer due to its effective use in

prostate cancer diagnosis [1, 2, 3, 4]. Recently, 18

FCH have shown good results on detection

of many other tumor types such as, breast and brain neoplasm [5, 6]. Fluorocholine

commercialization is already approved in several countries of European Community.

Research in [18F] Fluorocholine synthesis was initiated in CDTN in 2010. Main parameters

of synthesis and quality control required for 18FCH production have been established in

Unidade de Pesquisa e Produção de Radiofármacos - UPPR/CDTN. However, in order to

obtain clinical trials approval, as well as to register 18

FCH at Agência Nacional de Vigilância

Sanitária (ANVISA), safety and efficacy preclinical tests were required[7, 8].

ANVISA provides guidance for conducting preclinical studies through its Guide for

Conduction of Non Clinical Studies of Security and Efficacy Necessary for Drug

INAC 2013, Recife, PE, Brazil.

Development [9]. The bacterial reverse mutation test is recommended by this guide to

evaluate genotoxicity of substances.

The bacterial reverse mutation test was developed in 1970s by Dr. Bruce Ames and

colleagues. It is a short term in vitro test for evaluation of possible mutagenic effects induced

by chemicals.

Fluorocholine is a choline (CH) analogue (Figure 1). Choline’s pharmacologic safety has

already been proven [10]. In vitro genotoxicity studies, including Ames test, have been

performed for this substance. Even at OECD’s maximum recommended doses (5 or 10 mg /

plate), CH didn't induced reversion rate increase [10]. However, studies evaluating FCH

genotoxicity are not known [11]. Despite the structural similarity to CH, it’s not possible to

say that FCH is not mutagenic for sure.

Figure 01 – Choline’s structural formula (A) and its analogous - fluorocholine (B).

The bacterial reverse mutation test for fluorocholine was performed in two steps. Initially, the

method was applied to "cold" fluorocholine (19

FCH) acquired from ABX ®. The aim was

analyzing the mutagenicity of fluorocholine molecule. Subsequently, the decayed 18

FCH

(decay time > 10.T1/2 of 18

F) resulting from radioactive fluorocholine synthesis at CDTN was

evaluated. The synthesis product, in addition of 18

FCH, includes possible contaminants

originating from the production process.

Several procedures for performing the bacterial reverse mutation test have been described.

Among those commonly used are the plate incorporation method and the preincubation

method [12]. The plate incorporation method [13] was utilized in this study to test

fluorocholine mutagenic potential.

The measurement of fluorocholine mutagenic activity can be performed through comparing

the number of revertant colonies on plates containing different fluorocholine concentrations

and the number of revertant colonies on the plates containing positive and negative controls.

2. MATERIALS AND METHODS

Bacterial cells in suspension were exposed to test substances directly into minimum agar

plates. Mutant Escherichia coli and Salmonella typhimurium bacterial strains were used.

(A) (B)


They are amino acid dependent. In the absence of an external source of amino acids such

bacteria cannot form colonies. When reverse mutation occurs, cells recover the ability to

grow in minimal culture medium. Spontaneous reversions occur naturally with these strains.

Mutagenic compounds tend to increase basal reversion rate causing an increase in the number

of revertant colonies.

Many carcinogen chemicals only become biologically active after liver metabolism. Bacteria

do not have metabolic systems compatible with vertebrates. Therefore, for detection of

metabolism activated substances, the use of an exogenous metabolic activation system

(rodent liver enzyme) is required [13].

The plate incorporation method [13] was employed in this study (Figure 02). Different

components (bacteria, the test substance and metabolic activation system or PBS) were added

to top agar containing biotin and histidine trace amounts and poured on minimal agar plates.

In E. coli WP2 uvrA case, top agar was supplemented with tryptophan traces. After top agar

solidification, plates were incubated at 37 ° C for 48 hours. After the incubation, the number

of revertant colonies on each plate was counted. The number of colonies counted on FCH

plates were compared with positive and negative controls.

Figure 02 – Diagram illustrating the steps involved in plate incorporation method for

bacterial reverse mutation test. Adapted [13]


2.1. Bacterial strains

Five distinct bacterial strains were utilized to assess 19

FCH and 18

FCH genotoxicity as

recommended by ANVISA [9]. Salmonella typhimurium TA-1535, TA-1537, TA-98 and

TA-100 were employed to detect point mutations at guanine-cytosine (GC) sites, as can be

seen in Table 01 [13]. Escherichia coli WP2 uvrA strain was used for detection of point

mutations at adenine-thymine (AT) sites [14].

Table 01 –S. typhimurium and E. coli mutation targets at DNA and reversion events.

Strain DNA target Reversion event

S. typhimurium TA-98 –C–G–C–G–C–G–C–G– Frameshifts

S. typhimurium TA-100

S. typhimurium TA-1535 –G–G–G– Base-pair substitution

S. typhimurium TA-1537 –C–C–C– Frameshifts

E. coli WP2-uvrA –T–A–A– Frameshifts

2.2. Controles negativos e positivos.

Positive and negative controls were included in all tests. OECD and FDA recommends the

usage of own vehicle or solvent employed in samples dissolution as negative control [12, 13,

15,16]. Thus, NaCl 0.9% w/v solution, the 18

FCH vehicle, was selected as a negative control.

Negative control should result in reversion rates similar to each strain spontaneous reversion

historical levels.

Table 02 – Positive controls for S. typhimurium and E. coli strains, including applied

mass per plate and proper solvent.

Without metabolic activation With metabolic activation (S9 mix) Strain

Control Chemical µµµµg/plate Solvent Control Chemical µµµµg/plate Solvent

TA-98 2-Nitrofluorene 10 DMSO 2-Aminoanthracene 5 DMSO

TA-100 Sodium azide 5 NaCl 0.9 % 2-Aminoanthracene 5 DMSO

TA-1535 Sodium azide 5 NaCl 0.9 % 2-Aminoanthracene 5 - 10 DMSO

TA-1537 9-Aminoacridine or

2-Nitrofluorene 50 or 10 NaCl 0.9 % 2-Aminoanthracene 5 - 10 DMSO

WP2-uvrA 4-nitroquinoline-N-oxide 5 DMSO 2-Aminoanthracene 15 DMSO


Positive controls for each strain utilized were based on OECD [12], FDA [15] by CETESB

[16] and Mortelmans [12] publications. Table 02 presents the selected positive controls for

each strain, applied mass per plate and solvent used, considering the tests with and without

metabolic activation (S9 Mix).

2.3. Exposure concentrations

Five exposure concentrations (or doses) of 19

FCH and decayed 18

FCH were established for

each strain according to OECD [12] and FDA [15] recommendations. Concentrations were

spaced by approximately a √10 factor. One positive and one negative control group per strain

were also included.

2.3.1.

19FCH doses

Doses of cold fluorocholine utilized in trials were established based on 18

FCH maximum dose

recommended for humans [17].

According to a calculation performed in a previous study (data not Showed), we estimated

the maximum fluorocholine mass to be injected into 70 Kg human. The approximate value

obtained was 1 µg of fluorocholine

A safety margin of 100 times the maximum fluorocholine mass to be injected into a 70 Kg

human, i.e. 100 µg (per plate), was adopted as 19

FCH maximum dose. Other dose levels

spaced by approximately √10 factors were obtained starting from this value. Table 03 shows

cold fluorocholine exposure concentrations.

Table 03 – Exposure Concentrations employed in Ames tests of 19

FCH and decayed 18

FCH.

Exposure

Concentrations Concentration of “cold”

Fluorcholine (mg/placa) Activity concentration of

18FCH

* (MBq/placa)

Dose 1 0,100 29,6 Dose 2 0,030 8,88 Dose 3 0,010 2,96 Dose 4 0,003 0,888 Dose 5 0,001 0,296

* Refers to activity concentrations obtained immediately after production. At the moment of testing

the product activity (decayed 18FCH) was near the "Background"

2.3.2. Decayed

18FCH doses

18

FCH was produced on the day preceding reverse mutation test. Fluorine-18 has a short half-

life (109.77 minutes [18]). Thus, at the moment of the test, radiation levels of the solution

were close to background level. A surface contamination meter was used to assess the

radiation level of the product.


The exposure concentrations employed in 18

FCH Ames tests were based on the activity

concentration usually obtained for the radiopharmaceutical solution at the end of synthesis.

In plate incorporation assays 0.1 ml of test substance were administered per plate (Figure 2).

It was found that the activity concentration normally achieved after synthesis is

approximately 296 MBq/ml (8 mCi/ml). The value of the activity is in 0.1 ml is 29.6 MBq.

This corresponds to the maximum product dose tested per plate.

Dilutions were prepared in order to obtain the other dose levels spaced by approximately √10

factors starting at maximum dose. Table 03 shows decayed 18

FCH dose values to be tested

taking into account the activity concentration of the solution usually obtained at the end of

synthesis.

2.4. Evaluated Parameters

The following parameters were evaluated in FCH Ames test: toxicity signs, precipitation

signs and reversion rate.

Plates were inspected in an inverted microscope (40x magnification) for the evaluation of

signs of toxicity and precipitation. Qualitative changes in micro-colonies density or

precipitate presence were reported.

Were also compared the tevertant rates of test groups with the negative control. A statistically

significant decrease in test group sreversion rates when compared to negative control may

indicate test substance toxicity.

Reversion rates were obtained for test groups, positive control and negative control for all

strains tested with and without metabolic activation. Mean number of revertant colonies per

plate (n = 3) of each group will be considered its reversion rate. Test group reversion rates

will be compared with those of control groups.

Negative result in bacterial reverse mutation test indicate that evaluate substance has no

mutagenic activity for tested strains under test conditions [16]. The result is considered

negative when no statistically significant differences between the negative control and test

groups were noted.

Positive result in bacterial reverse mutation test indicates that evaluated substance induces

mutations in tested strains genome. These mutations can be caused by a base pair

substitution, or a frame shift of the reading window. The result is considered positive when

test group mutation rate are higher (statistically significant) than negative control rates or

when reversion rate between a test group and negative control is greater than to two.

2.5 Statistical Analysis

Reversion rates means and standard deviations rates will be calculated for each group.

According to UK Environmental Mutagen Society recommendations [19], the Dunnett's t-test


method is recommended for bacterial reverse mutation test statistical evaluation. This test is

used to compare a particular group (negative control in this case) with each remaining

groups. In all cases, p < 0.05 was defined as the level of statistical significance.

3. RESULTS

Figure 3 shows, as an example, pictures of plates obtained for decayed 18

FCH Ames test with

TA-98 strain (without metabolic activation). Can be observed: a) positive control (2-

nitrofluorene - 10 µg/plate) - reversion rate> 1700 rev/plate; b) negative control (NaCl 0.9%

w/v) – normal basal reversion rate for strain (20<rev/plate<75); c) maximum dose of decayed 18

FCH (equivalent to 29.6 MBq/plate) - reversion rate statistically equal to the negative

control; and d) minimum dose of decayed 18

FCH (equivalent to 2.96 MBq/plate) - reversion

rate statistically similar to the negative control.

Figure 03 - Photographs showing reversion rate in Ames test plates for decayed 18

FCH,

with strain TA-98 without metabolic activation. A) Positive control, B) Negative control,

C) decayed 18

FCH maximum dose, D) decayed 18

FCH minimum dose.

(A) (B)

(C) (D)


Ames tests results for FCH with S. typhimurium strains are shown in Table 04. Results for E. coli strain were shown in Table 05.

Table 04 - 19

FCH e 18

FCH Ames test results for TA-98, TA-100, TA-1535 e TA-1537

Salmonella typhimurium strains, with and without metabolic activation.

Strain TA-98

18FCH without S9-mix 18FCH with S9-mix 19FCH without S9-mix 19FCH with S9-mix

Doses Mean

Rev/plate SD Ratio

Mean

Rev/plate SD Ratio

Mean

Rev/plate SD Ratio

Mean

Rev/plate SD Ratio

+ Control 1896 230 34.9 * 2035 141 38.2 * 1459 86 44.2 * 1392 179 43.5 *

Dose 1 55 9 1.01 60 10 1.13 33 5 1 36 5 1.11

Dose 2 60 2 1.1 57 6 1.06 31 10 0.95 35 5 1.08

Dose 3 54 7 0.99 57 7 1.06 29 6 0.88 33 8 1.04

Dose 4 50 4 0.92 50 6 0.93 28 2 0.83 28 6 0.86

Dose 5 51 3 0.94 62 6 1.16 27 5 0.83 35 4 1.09

- Control 54 6 1.00 53 5 1..00 33 4 1.00 32 4 1.00

Strain TA-100 18FCH without S9-mix 18FCH with S9-mix 19FCH without S9-mix 19FCH with S9-mix

Doses Mean

Rev/plate SD Ratio

Mean

Rev/plate SD Ratio

Mean

Rev/plate SD Ratio

Mean

Rev/plate SD Ratio

+ Control 2515 180 16.4 * 3139 262 19.6 * 2188 179 29.7 * 2283 150 26.8 *

Dose 1 184 6 1.20 150 11 0.94 67 5 0.90 90 17 1.05

Dose 2 174 19 1.13 148 19 0.93 70 8 0.95 82 15 0.96

Dose 3 148 6 0.96 151 5 0.94 58 3 0.79 85 6 0.99

Dose 4 147 4 0.96 144 11 0.90 67 6 0.91 90 13 1.05

Dose 5 137 6 0.89 157 9 0.98 71 5 0.96 78 9 0.91

- Control 154 12 1.00 160 16 1.00 74 9 1.00 85 9 1.00


Doses Mean

Rev/plate SD Ratio

Mean

Rev/plate SD Ratio

Mean

Rev/plate SD Ratio

Mean

Rev/plate SD Ratio

+ Control 1387 190 101.5* 110 16 9.46* 1803 283 108.2* 150 9 11.0*

Dose 1 12 3 0.85 14 3 1.17 15 3 0.92 12 3 0.88

Dose 2 16 4 1.15 12 5 1.03 16 3 0.96 13 2 0.98

Dose 3 16 4 1.17 13 3 1.11 13 2 0.80 15 3 1.10

Dose 4 16 4 1.15 13 2 1.14 12 1 0.70 13 1 0.95

Dose 5 14 5 1.05 11 3 0.94 14 4 0.86 13 4 0.95

- Control 14 4 1.00 12 2 1.00 17 4 1.00 14 2 1.00


Doses Mean

Rev/plate SD Ratio

Mean

Rev/plate SD Ratio

Mean

Rev/plate SD Ratio

Mean

Rev/plate SD Ratio

+ Control 48 4 6.26* 2097 119 217* 51 7 3.14* 391 64 24.0*

Dose 1 10 3 1.26 11 3 1.14 20 5 1.22 20 3 1.20

Dose 2 9 3 1.13 11 4 1.17 18 6 1.12 20 5 1.22

Dose 3 8 2 1.04 12 1 1.24 14 1 0.88 17 2 1.06

Dose 4 9 2 1.13 11 1 1.14 14 3 0.84 16 3 0.98

Dose 5 9 2 1.13 10 3 1.03 15 3 0.90 15 4 0.90

- Control 8 1 1.00 10 1 1.00 16 5 1.00 16 3 1.00

* Significant at Dunnett's Multiple Comparison t-test – p < 0,05


Table 05 - 19

FCH e 18

FCH Ames test results for WP2 uvrA de E. coli strain, with and

without metabolic activation.

Strain - WP2 uvrA

18FCH without S9-mix 18FCH with S9-mix 19FCH without S9-mix 19FCH with S9-mix

Doses Mean

Rev/plate SD Ratio

Mean

Rev/plate SD Ratio

Mean

Rev/plate SD Ratio

Mean

Rev/plate SD Ratio

+ Control 755 162 15.1* 427 28 9.34* 1469 160 22.3* 199 48 3.20*

Dose 1 45 5 0.90 37 4 0.81 61 7 0.92 70 4 1.13

Dose 2 52 4 1.03 40 9 0.87 61 9 0.92 60 8 0.97

Dose 3 45 4 0.89 38 11 0.84 62 9 0.93 51 7 0.82

Dose 4 44 2 0.89 44 16 0.97 53 9 0.80 58 10 0.94

Dose 5 44 3 0.88 41 8 0.91 72 3 1.09 54 3 0.87

- Control 50 7 1.00 46 12 1.00 66 3 1.00 62 11 1.00

* Significant at Dunnett's Multiple Comparison t-test – p < 0,05

Statistically significant increases in reversion rate for 19

FCH and 18

FCH have not been

observed at exposure concentrations tested for all strains when compared with their

respective negative controls.

Negative controls reversion rates were consistent with other laboratories historical averages.

Positive controls showed reversion rates significantly higher (Dunnett's t-test – p<0.05) than

those presented by the negative controls. This shows that test system was responsive to

mutagenic substances presence.

No precipitation signs were observed at tested exposure concentrations.

Low toxicity signs were observed when 2-nitrofluorene (10 µg/plate) was utilized as

TA-1537 strain positive control in the test without metabolic activation. Despite the mild

toxicity observed, reversion rates were significantly higher than the negative control group.

That fact ensures test effectiveness. The remaining strains did not show any signs of

citotoxicity at exposure concentrations tested, including positive and negative controls.

4. DISCUSSION

Ames tests results demonstrates that doses of 19

FCH per plate 100 times larger than the dose

prescribed for human administration produced no mutagenic response for any evaluated

strains. Considering de exposure concentrations tested, cold fluorocholine molecule not

revealed to be mutagenic. Further studies can be programmed to evaluate the mutagenic

potential of 19

FCH at higher concentrations (up to 5 mg/plate).

In the same way, decayed 18

FCH (t > 10.t1/2 18

F) resulting from radioactive fluorocholine

synthesis process in CDTN also showed no signs of mutagenicity for any tested strains. This

shows that the syntheses produced no mutagenic substances or contaminants in quantities

detectable by the Ames test.


Additionally, diagnostic radiopharmaceuticals are commonly administered in a single dose

and the injected mass is extremely reduced (micro to nanogram of radiotracer per

application). Cancer development is understood to be a multistep process time dependent.

There are currently three main phases recognized in carcinogenesis (initiation, promotion and

progression). Promotion phase, particularly, requires repeated stimuli for prolonged times.

Thus, it can be said that the probability of cancer induction by a single dose administration of

determined substance is low. Indeed, to support the clinical trials of single administration,

ANVISA does not require the complete genotoxicity test battery [9].

5. CONCLUSIONS

This study results demonstrate that 18

FCH produced in CDTN, as well as 19

FCH can be

considered non-mutagenic in Ames test, for concentrations of exposure evaluated.

This work is part of a preclinical studies set conducted to evaluate [18

F]-fluorocholine

produced at CDEN/CNEN. The purpose of these tests was providing to ANVISA information

concerning 18

FCH safety and efficacy enabling its administration in clinical trials and

subsequent registration. For that reason, it is expected that the results obtained in this work

may help in process of decision making regarding the use of 18

FCH produced in

CDTN/CNEN in humans.

19

FCH exposure to concentrations up to 5 mg/plate can be evaluated in future increasing test

safety.

ACKNOWLEDGMENTS

CDTN would like to thanks INCT - Molecular Medicine for partnership in development and

implementation of preclinical testing for evaluation of new radiopharmaceuticals safety and

efficacy. We also would like to thanks Prof. Miriam Teresa Paz Lopes of Pharmacology and

Antitumor Drug Laboratory at UFMG and her student Denise Regina Arão for training

CDTN personnel in the execution of the Ames test.

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PRECLINICAL TEST - BACTERIAL REVERSE MUTATION TEST