Track 1:
Toxicology
Approaches
Human Safety of free Hydroquinone
derived from Herbal medicinal products
containing Uvae ursi folium
Dr. Susana Garcia de Arriba
Scientific & Medical Expert - Regulatory Affairs
1
Content
1. Herbal Medicinal Product:
• HMPC – Monograph
2. Arctostaphylos uva-ursi folium
3. Pharmacokinetics – Elimination
4. Human Exposure to Free HQ
5. Exposure limits for use in risk identification
Permitted Daily Exposure of free HQ
6. Assessment Genotoxic Potential of Free HQ
• AMES
• Micronucleous in mice
7. Safety Data – Cystinol akut
8. Conclusions
2
Herbal Medicine Market
The Global Herbal Medicine Market is estimated* to be US$ 60 billion, which is
poised to grow to $5 trillon by the year 2050
Increase in herbal medicine sales 10 % per Year
Global Herbal Medicine Market
USA
National markets for Herbal medicines
7 US$
Japan
annual sales
per capita
consumption
Germany --
$2.2 billion ---
$36.55 million
Denmark --
$ 30.2 million --
$ 26.6 million
10 US$
*World Health Organization (WHO) / Secretariat of the convention on Biological Diversity (2008)
3
EU
28 US$
Asia
+ Australia
11 US$
(US$ billion)
1. Herbal Medicinal Products (HMPs)
 *HMPs are defined as any medicinal product, exclusively containing as
active ingredients one or more herbal substances or one or more herbal
preparations, or one or more such herbal substances in combination with
one or more such herbal preparations.
HERBAL SUBSTANCE (HS)
- dry /fresh: whole or parts
of plants, algae, fungi and
lichens.
HERBAL PREPARATION (HP)
- obtained by subjecting HS
to treatments such as
extraction, distillation,
expression,
fractionation,
purification, concentration
or fermentation.
4
• Require a Marketing Authorization or Registration
before placement on the market In the EU community
• Drug Law on pharmaceutical products for human use
- - -> *Directive 2001/83/EC applies to HMP
 Protect public health
 Harmonized authorization across the EU
 Facilitate the authorization/ registration
procedure of HMP
1. Herbal Medicinal Products
1.1 Market access for HMP in the EU
under European medicines legislation, HMPs must fall within one of the following 3
categories to reach the market:
1. Traditional use registration (simplified registration procedure) Directive
2004/24/EC amending Directive 2001/83/EC.
- HMP with sufficient evidence of medical use >30 years, >15 years within EU.
Sufficient safety data and plausible efficacy. THMP with Traditional medicinal use
based upon long-standing use.
2. Marketing authorisation (Bibliographic application)  HMP with wellestablished medicinal use on basis of recognized efficacy, at least one controlled
clinical study of good quality is required to substantiate efficacy, and an acceptable
level of safety.
3. Marketing authorisation – (full/ mixed application)  a product with safety and
efficacy data from the company’s own development (‘stand alone’) or a
combination of own studies and bibliographic data (‘mixed application’). As a result
the product is granted a marketing authorisation.
http://ec.europa.eu/health/human-use/herbal-medicines/index_en.htm
http://www.ema.europa.eu/ema/index.jsp?curl=pages/regulation/general/general_content_000208.jsp&mid=WC0b01ac05800240cf
5
1. Herbal Medicinal Products
1.2 EMA, HMPC and HMPs
Responsible for Agency's
opinions
on
herbal
medicines
Experts in the field of
herbal medicines. Advisers
28 EU Member States (+
Iceland and Norway)
Scientific evaluation of
non-clinical and clinical
data; safety and efficacy
data; quality of the herbal
substance
/preparations
intended for medicinal use
and license
Evaluation of
longstanding medicinal use in
the EU
Listing!!!*
2004/24/EC
HMPC Monograph
Committee on
Herbal Medicinal Products
HMPC
Medical Products human use
EMA
European Medical Agency
Sept. 2004
Regulation
(EC)
No
726/2004 and Directive
HS/HP complies
qualitative - quantitative
declaration, therapeutic
indication and daily
recommended dose
 efficacy and safety of
the HS or HP are ensured
• Marketing Autorisation
• Registration
*list of herbal substances, preparations and combinations thereof
for use in traditional herbal medicinal products
http://ec.europa.eu/health/human-use/herbal-medicines/index_en.htm
6
1. Herbal Medicinal Product
1.3 HMPs in the EU market
All HMPs in the EU market have to fulfill the demands of
HMPC and competent national authorities
 EU legislation on pharmaceutical products for human use
 Pharmaceutical quality – Eur. Pharmacopoeia/NfG
 Standardized production process: GMP (GDP, GACP)
 Drug Safety:
 Preclinical: Genotoxic/Mutagenic potential (GLP)
 Pharmacovigilance (GVP)
 Proved Well Established/ Traditional long-standing
medicinal use in the EU
http://ec.europa.eu/health/human-use/herbal-medicines/index_en.htm
http://www.ema.europa.eu/ema/index.jsp?curl=pages/regulation/general/general_content_000208.jsp&mid=WC0b01ac05800240cf
7
1. Herbal Medicinal Product
1.4 EMA - HMPC
8
http://www.ema.europa.eu/ema/index.jsp?curl=pages/regulation/general/general_content_000208.jsp&mid=WC0b01ac05800240cf
1. Herbal Medicinal Product
1.5 Committee on Herbal Medicinal Products
http://www.ema.europa.eu/docs/en_GB/document_library/Other/2009/12/WC500017724.pdf
9
1. Herbal Medicinal Product
1.6 HMPC Monograph
Latin name of the genus
Latin name of herbal
substance
Botanical name of plant
English common name of
herbal substance
Status
Date added to the inventory
Date added to priority list
Outcome of European
assessment
10
Arctostaphylos
Uvae ursi folium
Arctostaphylos uva-ursi (L.) Spreng.
Bearberry Leaf
F: Assessment finalised
23/11/2005
23/11/2005
Community herbal monograph
http://www.ema.europa.eu/ema/index.jsp?curl=pages/medicines/landing/herbal_medicines_search_landing_page.jsp&mid=
1. Herbal Medicinal Product
1.6 HMPC Monograph
Final
Assessment
report
Final
community
herbal
monograph
11
1. Herbal Medicinal Product
1.6 HMPC Monograph
12
1. Herbal Medicinal Product
1.7 Assessment Report - HMPC Monograph
13
2. Arctostaphylos uva-ursi folium (Cystinol® akut)
• HMP -
Cystinol® akut
• Herbal Preparation 
ethanolic (60% v/v) dry extract of
Arctostaphylos uva-ursi folium
• Composition: 1 Tablet
 238.7-297.5 mg ethanolic (60% v/v) dry extract
Arctostaphylos uva-ursi folium (DER 3.5 – 5.5:1)
 corresponding to 70 mg arbutin anhydrous
• Posology adults and children over 12 years
Take 2 coated tablets 3 times daily
• Recommended Daily Dose (RDD):
≈ 1.6 g Extract = 420 mg arbutin
• Traditionally used to treat symptoms of lower urinary tract infections
14
2.1
 COMPLIANT with the qualitative and quantitative
composition, posology as well as with the therapeutic area of
application
15
2.1 HMPC monograph
Arctostaphylos uva-ursi folium
Information about HMP on the market in the Member States Regulatory status overview
Country
Date – Regulatory
status
Formulation
1 Dose
Daily Dose, RDD
Powdered herbal substance in solid dosage forms
Germany
>1976 MA
coated tablets
500 mg
6 tablets 4 times - 12 g
Spain
>1991 R
capsule
270 mg
1 capsule 3 times - 810 mg
France
>1982 R
hard capsules
350 mg
2 capsules 2 times - 1.4 g
Germany, Poland, Spain,
Estonia, Slovenia
R/MA
Herbal Tea
1.5-3 g/150ml
3 - 6 times - 4.5 - 18 g
Herbal preparations - Dry extracts
Germany
> 1976 MA (39 y)
coated tablets
Poland
> 2000 R
film-coated
tablets
France
> 1992 R
hard capsules
238.7 – 297.5 mg
2 tablets 3 times - 1.6 g
215 mg of
4 tablets 3 times - 2.6 g
200 mg
1 capsule 2 times - 400 mg
MA - Marketing authorisation
R - Registration
http://www.ema.europa.eu/docs/en_GB/document_library/Herbal_-_HMPC_assessment_report/2011/07/WC500108750.pdf
16
2.2 Cystinol® akut - HMP Documentation Ethanolic Uvae ursi folium extract
Quality
of the extract
Eur. Pharmacopoeia
GMP, GDP, GACP
Safety Data
- preclinical -

Safety Data
- Human -
„Good Laboratory
Practice“ GLP
No systemic toxic
No genotoxic-
Pharmacovigilance
Data
mutagenic Risk
effects
Medicinal use
Therapeutical experience
Medicinal use
Therapeutical use
HMPC monograph
WHO, ESCOP, Comm. E
Monographs
Quality, DER, HP
HMPC Monograph
1. high pharmaceutical quality
2. widely documented drug safety
3. Medicinal use proven by HMPC /National-International Monographs
17
2.3 The therapeutic efficacy and safety
Uvae ursi folium extract - Cystinol® akut
long-standing medicinal use and experience  testified by bibliographic and expert evidence
 National and International herbal monographs on Uvae ursi folium
 Commission E, 1994; WHO, 20021; ESCOP2, 2012 and HMPC, 20123
 HMPC Monograph on Arctostaphylos uva-ursi (L.) Spreng, folium
 There is sufficient evidence of medicinal use throughout a period of at least 30
years, including at least 15 years in European Community
1World
Health Organization. Folium Uvae ursi. Monografie 2 (2002): 342-351
Scientific Cooperative on Phytotherapy (ESCOP) Uvae ursi folium, bearberry leaf. (2012), online series. www.escop.com
3HMPC Community herbal monograph on Arctostaphylos uva-ursi (L.) Spreng., folium (28.02.2012)
http://www.ema.europa.eu/docs/en_GB/document_library/Herbal_-_Community_herbal_monograph/2011/04/WC500105350.pdf
2European
18
2.4 International /national Monographs
Commission E
1994
WHO
(World Health Organisation)
2002
ESCOP
HMPC
(European Scientific
Cooperative on Phytotherapy)
(Herbal Medicinal Products
Committee of EMA)
2012
2012
Indication
Inflammatory diseases
of the lower urinary
tract
Moderate inflammatory
conditions of the urinary tract
and bladder, such as cystitis,
urethritis and dysuria
Uncomplicated infections of
the lower urinary tract, when
antibiotic treatment is not
considered essential
Treatment of symptoms of
mild recurrent lower urinary
tract infections (traditional
use)
Dosage
3 g drug or 400 - 840
mg hydroquinone
derivates, calculated as
arbutin, up to 4x daily
3 g drug or 400 - 850 mg
hydroquinone derivates,
calculated as arbutin, up to 4x
daily
400-800 mg of arbutin/day,
equivalent preparations
Single dose corresponding
to 100-210 mg
hydroquinone derivates
calculated as anhydrous
arbutin, 2-4x daily
Duration of
use
 Positive Benefit-Risk Profile
officially approved
Without medical advice: No long-term use, patients with
up to 5x per year, not
persistent symptoms should
longer than 1 week in
consult a physician
each case
Until complete disappearance
of symptoms (up to a
maximum of 1 week)
Not longer than 1 week
Pregnancy, lactation,
children < 12 years
Pregnancy, lactation, children,
patients with kidney disorders
Pregnancy, lactation, children
< 12 years
Hypersensitivity, kidney
disorders
Interactions
Urinary acidifying
substances*
Urinary acidifying substances
None reported
None reported
Side effects
Nausea and vomiting
possible in persons
with a sensitive
stomach
Nausea and vomiting may occur
due to stomach irritation from
the high tannin content
Nausea and vomiting may
occur due to stomach irritation
from the high tannin content
Nausea, vomiting, stomachache
Contraindictions
19
2.5 Relevant Constituents : Qualitative -Quantitative
Arbutin
HQ
Extract = active principle
Hydroquinon (HQ) derivatives:
arbutin to 23.5 - 29.3%
free HQ < 0.3%
Phenolic acids
gallic acid, Ellagic acid
Polyphenols (tannins)
gallotannins, ellagitannins
Flavonoids
hyperoside
Triterpenes
ursolic acid, uvaeol
Gallic acid
20
Ellagic acid
3. Pharmacokinetics
Elimination
Free HQ  hydroquinone
Total HQ = Free HQ + conjugated HQ
STUDY 1 - Pharmacokinetics
• 12 healthy volunteers
(6 males - 6 females)
• Dose 3 x 2 Tablets Cystinol® akut
1.6 g Extract/day
420 mg Arbutin (169 mg HQ)
• Urine samples collected after 36h
• Free and Total HQ quantified
in individual urine samples – HPLC
Siegers et al. 1997
21
3.1 Single Data Elimination
6/12 eliminate Free HQ
3/6 high levels free HQ at t=0
High Free HQ elimination no related
To increase of Metabolism/elimination
Free HQ (mg)
Individual urine samples n=12
Subject 3 had an extreme high level of
free HQ before HMP intake.
Time (hours)
Elimination of Free HQ and Total HQ
Time-dependent and
gender independent
22
n=11
109.4
0.99
Free HQ (mg)
Total HQ = Free HQ + HQ-Conjugate
Total HQ (mg)
HMP intake
3.2 Quantitative Elimination of Free and Total HQ
STUDY 1 - RESULTS
N= 11
• 0.6% Arbutin Dose as Free HQ
= 0.99 mg Free HQ
• 70% Arbutin Dose as conjugate HQ
to glucuronic and sulfuronic acid
Time (h)
= 109.4 mg total HQ
• Mean values ± SD
• Parallel and progressive elimination
23
3.3 Metabolism - Elimination
70% Total HQ
Major elimination:
HQ-glucuronide >> HQ-sulphate
(≈ 70% of total conj.)
0.6% Free HQ
Pharmacokinetic profile
HQ; Hydroquinone
HQ-Glu: HQ-glucuronide; HQ-Sulf: HQ-sulphate
24
3.4 Comparison with Bibliographic Data
Subjects,
urine
collection (h)
Arbutin
Daily Dose
% Arbutin Dose
Free HQ
Total HQ
Siegers et al. 1997
n=11, 36
420 mg
0.6%
70%
Paper et al. 1993
n=5, 24
100 mg
0
85%
Schindler et al. 2002
n=16, 36
210 mg
0.1%
66.7%
Quintus et al. 2005
n=3, 24
150 mg
0.6%
75%
* Detection limit (1µg/ml)
25
Arbutin MW 272.25
HQ MW 110.11
3.5 Bacterial deconjugation
STUDY 2 – Bacterial deconjugation
• 4 healthy volunteers
(2 males - 2 females)
100 µl/ml
Percloric acid
• Dose 3 x 2 Tablets Cystinol® akut
420 mg Arbutin
• Urine samples collected after 24h
>70%
• Free and Total HQ quantified
24h 37°C
HPLC quantification
20fold
0.6% HQ
HQ; Hydroquinone
HQ-Glu: HQ-glucuronide; HQ-Sulf: HQ-sulphate
Siegers et
26 al. 2003; Garcia et al. 2010; Garcia et al. 2013
3.6 Summary Elimination /deconjugation
The elimination of arbutin metabolites after oral intake of 420 mg arbutin
 Only 0.6 % of the administered arbutin-dose was excreted as free HQ in the urine
 In 6 out of 12 volunteers free HQ was not detected
 70% of the administered arbutin-dose was eliminated as HQ-conjugates (HQglucuronide; HQ-sulphate)
Free HQ deconjugation/accumulation inside of the bacteria
 Uropathogenic bacteria such as E. coli have the ability to deconjugate the HQconjugates eliminated in the human urine to a high extent
 20fold higher amount of free HQ was detected in bacteria sediments as compared
to the supernatant
 HQ-conjugates found in urine are ingested by the uropathogenic bacteria and
transformed into free HQ inside of the bacteria
 Intracellularly formed free HQ is responsible for the bactericide effect
Garcia et al. Int J Toxicol. 2013
27
3.7 Summary Elimination process
420 mg Arbutin
0.99 mg Free HQ
Recovered in Urine
28
4. Human exposure to Free HQ
• Elimination of Free HQ in 36 h: 0.99 mg
• Level of Free HQ in urine after 36 h:
0.6 – 0.5 µg/ml
• Corresponding to Human exposure level:
11 µg/Kg b.w. day *
* 60 kg body weight
*Garcia et al. Int J Toxicol. 2013
29
4.1 Food sources of Free HQ
Product
Arbutin
(µg/kg)
Free Hydroquinone
(HQ)
Per day
LEVEL OF EXPOSURE
µg free HQ/kg b.w./day*
20 - 90 µg /glass1
2-3
glass/day
0.66 - 4.5
Red wine
562 µg / glass1,2
1 glass/ day
9.3
non-filter
cigarettes
110 - 300 µg /cigar.3
18
cigar./day**
33 - 90
0.2 µg/g1
250 g/day
0.833
0.14 µg/g1
250 g/day
0.6
0.02 - 0.05 µg/g1
100 g/day
0.033 - 0.083
Coffee and Tea
Wheat products
0.015 0.13
Broccoli
Pears
0.037 0.18
High-HQ diet
(n=4)
Cystinol RDD
420
mg/day
4 mg/kg1
125 -2751
0.99 mg in urine5
11
*Body weight of 60 kg; 1Deisinger et al. 1996; 2IPCS, 1996; 3NTP, 2009; 4Carlson and Brewer (1953); 5Siegers et al. 1997.
**Mean value in EU.
30
Garcia et al. Int J Toxicol. 2013
5. Exposure limits for risk identification
Permitted Daily Exposure Definitions*
Estimates the dose below which there is a
negligible risk to human health
Represents a substance-specific dose that is unlikely
to cause an adverse effect if an individual is exposed
at or below this dose every day for a lifetime
*- Guideline on setting health based exposure limits for use in risk identification in the
manufacture of different medicinal products in shared Facilities EMA/CHMP/ CVMP/
SWP/169430/2012
*- Appendix 3 of ICH Q3C (R4) “Impurities: Guideline for Residual Solvents”
31
5.1 Calculation of PDE
Toxicological parameters of free HQ
CAS N.: 123-31-9 (IPCS, 1994; IPCS, 1996; OECD/
SIDS, 2002; Williams et al. 2007; NTP, 2009).
PDE for free HQ =
(25 mg/kg b.w./day x 50 kg b.w.)
(5 x 10 x 5)
PDE free HQ = 5 mg/day or 100 µg/kg b.w./ day
32
5.2 Results - PDA
PDE for Free HQ = 5 mg/day or 100 µg/kg b.w./ day
Exposure level of Free HQ derived from HMP = 11 µg/k.g. b.w./day
• PDE level is 9-times higher than the maximum exposure level to free HQ in
a “daily usual” scenario after ingestion of a therapeutic RDD (420 mg of
arbutin) of a herbal medicinal product containing Uvae ursi folium
• PDE level corresponds to 8.3-times RDD of Cystinol HMP (6 tables/day). A
patient should take 50 Tablets/day in order to be exposed to the PDE value
of free HQ
Accordingly,
the level of free HQ produced by the administration of a RDD of Uvae ursi folium has a
negligible probability of any toxicological risk to human health*
*Garcia et al. Int J Toxicol. 2013
33
5.3 Results - PDA
Supporting this low risk human exposure level, the HMPC monograph on Uvae ursi
folium concluded that concentrations reached in the human body by monographed
Uvae ursi folium-products are below the most conservative Thresholds of
Toxicological Concern (TTC) (HMPC, 2012).
In agreement with the above statement, the benefit-risk ratio of the medicinal use
of Uvae ursi folium extract has been considered favourable for the claimed
indication and dosage by all national and international current Monographs on
Uvae ursi folium.
• Commission E (1994)
• WHO monograph (2004).
• HMPC monograph (2012),
• ESCOP (2012)
34
6. Assessment of genotoxicity
• Guidelines for genotoxicity testing of pharmaceuticals have been established by
HMPC, OECD and ICH
• Testing of medicinal products involves a battery of genotoxicity tests, in which proand eukaryotic systems in in vitro and in vivo experimental setups with and without
metabolic activation are employed
•
EMEA/HMPC/107079/2007 into effect 01.12.2008 Guideline on the assessment of
genotoxicity of herbal substances / preparations
•
Note for guidance on genotoxicity: guidance on specific aspects of regulatory genotoxicity
tests for pharmaceuticals (CPMP/ICH/141/95) = ICH guideline S2 (R1)
•
OECD (1997). OECD Guidelines for the Testing of Chemicals No. 471: Bacterial Reverse
Mutation Test [the Ames test].
•
OECD (1997). OECD Guidelines for the Testing of Chemicals No. 474: Mammalian Erythrocyte
Micronucleus Test [in vivo micronucleus test].
35
6.1 Scheme Mutagenic-Genotoxic Studies
Risk????
In vitro
AMES test
STUDY 3 – AMES test
N= 12
Urine pool
RDD 6 tablets Cystinol/ day
1.6 g Uvae ursi folium extract =
total HQ: 36.00 µg/ml
free HQ: 0.512 µg/ml
7 mg/kg/day arbutin
STUDY 1 - Pharmacokinetics
In vivo
Mice Micronucleus test
STUDY 4 – Micronucleus test
36
6.2 AMES Test
 Salmonella typhimurium LT2 : Four different histidine auxotrophic strains.
Identify two basic classes of point mutations:
 base pair substitutions (TA 1535, TA 100)
 frameshift mutations (TA 1537, TA 98).
All strains contain GC base pairs at the site of the histidine mutation
 Fifth strain for detecting point mutations at adenine-thymine (AT)
 Do not comply with genotoxicity guidelines
 Investigation: Urine samples according to OECD-AMES guideline by two
independent experiments:
(I) the plate incorporation test
(II) the pre-incubation test
 Evaluation: Mutagenic Potential  it induces a > 2-fold increase in the number
of revertant colonies over negative control (spontaneous reversion rate)
37
6.2 AMES Test:
•
•
•
•
Preliminary Toxicity Test
Concentrations from 36.00/0.511 µg/ml to 0.240/0.0026 µg/ml of total HQ and free HQ
Bacterial strains TA 98 and TA100
Plate incorporation method with or without addition of S9 fraction.
Two independent assays (n=3).
• 4-nitro-o-phenylene-diamine (4-NOPD, 10 μg/plate)
• sodium azide (NaN3, 10 μg/plate)
• 2-aminoanthracene (2-AA, 2.5 μg/plate)
• control urine sample + 5 µg/plate free HQ
38
6.2 AMES Test:
Plate Incorporation Test
• sodium azide (NaN3, 10 μg/plate)
- Plate incorporation test with/without metabolic activation.
- Two independent assays (n=3).
39
• 2-aminoanthracene (2-AA, 2.5 μg/plate)
• control urine sample + 5 µg/plate free HQ
6.2 AMES Test:
Pre-incubation Test
• Pre-incubation test with /without metabolic activation.
• Two independent assays (n=3). Mean±SD
40
• sodium azide (NaN3, 10 μg/plate)
• 2-aminoanthracene (2-AA, 2.5 μg/plate)
• control urine sample + 5 µg/plate free HQ
6.3 Micronucleus Test: in bone marrow cells
Preliminary study on acute toxicity
• 4 NMRI mice (2 females and 2 males)
• Under identical conditions as in the in vivo micronucleus assay
concerning test item, animal strain, vehicle, route, frequency and
volume of administration.
• Administration undiluted pooled urine (D1) i.p., 10 ml/kg b.w.
• Examination of acute toxic symptoms (e.g. death, reduced spontaneous
activity, eyelid closure, apathy etc.) at intervals of 1, 6, 24 and 48 hours
RESULTS: No signs of toxic reactions after 1, 6, 24 and 48 h were observed
The undiluted urine D1 was considered as a suitable test item
41
6.3 Micronucleus Test: in bone marrow cells
Urine
1/10 D1
Control
0.9%
NaCl
Urine
½ D1
Urine
Undiluted
D1
Control
Urine +
free HQ
Control
Urine
Cellular target: Polychromatic
erythrocytes (PCE) in the bone
marrow mouse.
+
Control
CPA*
(10 ml/kg b.w.) i.p.
10 NMRI mice /test group
Each group: 5 male/5 female
42
24 – 48 h
Preparation of
the marrow
and cells
*cyclophosphamide (CPA) at 30 mg/kg b.w
6.3 Micronucleus Test
Evaluation:
 2000 polychromatic erythrocytes (PCEs, reticulocytes; immature
erythrocytes) were scored per animal for frequency of micronucleated
cells
 in 1000 erythrocytes the ratio of PCEs/normochromatic erythrocytes
(NCE) in the bone marrow is scored for each dose group as an indicator of
chemical-induced toxicity
• A test item is considered mutagenic, if it induces either a dose-related
increase in the number of micronucleated PCE or a statistically significant
positive response for at least one of the test points
• The cytotoxic effect of the test item due to the treatment was estimated
as the ratio between PCE and NCE and reported as the number of NCE
per 1000 PCE
• Statistical significance p< 0.05 was evaluated by means of non-parametric
Mann-Whitney test
43
6.3 Micronucleus Test: DATA -RESULTS
• Frequencies of micronucleated PCE in 2000 PCEs per animal
• Ratio PCEs/ NCE in 1000
Test Item
Control urine
Control urine + HQ
(50µg/ml)
0.9% NaCl-solution
Urine 1/10 D1 diluted
Urine ½ D1 diluted
Urine undiluted (D1)
Cyclophosphamide
Urine 1/10 D1 diluted
Urine ½ D1 diluted
Urine undiluted (D1)
Dose
(ml/kg b.w.)
10
10
Sampling
time (h)
24
24
PCE with
micronuclei (%)
0.13
0.12
Range
ratio PCE/NCE
0-4
0-2
1000/726
1000/745
10
10
10
10
30 mg/kg
b.w.
10
10
10
24
24
24
24
24
0.13
0.14
0.10
0.09
0.97*
0-4
0-4
0-3
0-3
4-25
1000/722
1000/736
1000/747
1000/790
1000/736
48
48
48
0.11
0.09
0.12
0-4
0-2
0-4
1000/718
1000/738
1000/756
*increase micronucleus frequency statistically significant (p<0.0001, non-parametric Mann-Whitney test
44
6.3 Micronucleus Test:
Results :
• NO dose-related increase in the number of micronucleated
PCE cells were observed
• NO clear increase in the number of micronucleated cells in a
single dose group at a single sampling time were observed
• NO Cytotoxicity = No change in the ratio polychromatic
erythrocytes (PCEs) /normochromatic erythrocytes (NCE) was
observed
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7. Safety
7.1 Cystinol acut – Medicinal use

A multicenter survey with 75 registered doctor’s offices located in Germany
was conducted in 1992-1993 reporting their clinical experience with Cystinol
mono (Cystinol akut since February 1996) for the treatment of diseases of the
lower urinary tract



A total of 186,122 patients, (117,282 women)
56 doctors reported experience with a total of 17,430 children and adolescents.
Physicians with >30 years of experience (average of 10 years)
 34.6%
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7. Safety
7.2 Cystinol akut– Medical use
Judgment of Therapy Effect by the
Doctors (%) Good - very Good
Prevention of recurrence
bacterial cystitis
Urinary tract infection
Irritable bladder
98.6%
Uncomplicated cystitis
85
90
95
100
105
Mean duration
Treatment (days)
Time to reach
Effect (days)
9
10
10
28
3
4
4
Uncomplicated cystitis
Irritable bladder
Urinary tract infection
Prevention of
recurrence bacterial
cystitis
Tolerability (%)
Urine alkalinization (%)
Prevention of recurrence
bacterial cystitis
Prevention of recurrence…
Urinary tract infection
Urinary tract infection
Irritable bladder
Irritable bladder
Uncomplicated cystitis
Uncomplicated cystitis
0
25
50
75
100
0
very Poor
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Poor
20
40
60
80
100
120
very Good -Good
7. Safety
7.2 Pharmacovigilance
Wide-spread OTC medicinal use  Pharmacovigilance Data
Post-marketing patient exposure
 Cystinol Akut from all over the world
 Cumulative period of 10 years  ≈ 7,000,000 patients
 In this period of time  A total of 161 cases (275 AEs)
SPC/PIL  Side effects, adverse reactions
≥ 1/10,000 to < 1/1,000
< 1/10,000
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gastrointestinal complaints
(nausea, stomachache and vomiting)
allergic reactions
Relevant beneficial Properties
 Antibacterial/anti-adherent /anti-inflammatory properties
 Broad spectrum of antibacterial activity
 NO development of antibiotic resistant bacteria
 Local antibacterial effect  Urine + Bladder
 NO effect on intestinal, vaginal flora/immuno defenses
 Safe and well-tolerated
Benefit – Risk Balance clearly positive
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8. Conclusions
 Free und conjugate HQ were detected and quantified in human urine
samples.
 Human exposure to Free HQ was below its Permitted Daily Exposure
 No risk for human health exposed at or below this dose every day
for a lifetime
 In vitro and in vivo mutagenic and genotoxic studies were negative
 Accordingly, urine samples obtained from healthy volunteers
receiving a regular recommended dose as proposed for therapeutic use
in the HMPC monograph bears no mutagenic risk.
 Pharmacovigilance Data from more than 30 years do not found AE
different as reported in SPC/PIL
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Thank you for your attention
Don’t worry…
We can eat him.
He took Uva ursi only!!
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